Improved methods for the production, recovery and secretion of hydrophobic compounds in fermentation - Patent Application 20070122999

Abstract

The present invention relates to an improved method for producing hydrophobic compounds, particularly hydrophobic compounds that are pheromones, such as insect pheromones, in a fermentation process involving the cultivation of a microorganism, such as yeast, which produces the hydrophobic compound, and the method facilitates recovery of the hydrophobic compound from the fermentation broth, increases the titer of the hydrophobic compound, and / or increases the secretion of the hydrophobic compound from the microorganism.

Description

[Technical Field]

[0001] The present invention relates to an improved method for producing hydrophobic compounds, particularly hydrophobic compounds that are pheromones, such as insect pheromones, in a fermentation process involving the cultivation of a microorganism, such as a yeast, which produces the hydrophobic compound, and the method facilitates recovery of the hydrophobic compound from the fermentation broth, increases the titer of the hydrophobic compound, and / or increases the secretion of the hydrophobic compound from the microorganism. [Background technology]

[0002] Living cells, particularly microbial cells, are now widely used for the biological production of many compounds. Examples of such compounds are fatty alcohols, fatty acyl acetates, and fatty aldehydes, such as insect pheromones, which can be produced, for example, in yeast cells. Such compounds have applications in agriculture, for example, as green pest repellents. Other useful compounds that can be produced by cells, such as genetically engineered cells, are terpenes and terpenoids. Terpenes are naturally produced by plants and have many industrial applications in the fields of food, medicine, cosmetics, and biotechnology. For example, they are used as part of natural agricultural insecticides. Terpenoids (also called isoprenoids) are modified terpenes containing additional groups, usually O-containing groups. They are often used as part of traditional herbal remedies due to their aromatic properties. Although terpenes and terpenoids are widespread, their extraction from natural sources is often problematic. As a result, they are typically produced by chemical synthesis, often from petrochemicals.

[0003] A common characteristic of these compounds is that they are hydrophobic or lipophilic. Their recovery from the fermentation broth usually requires the use of several organic solvents, which poses several challenges, including process safety, the need for multiple extraction steps, the need to remove solvent residues from the final product, and significant effort and cost (both financial and environmental).

[0004] Another challenge posed by fermentation methods involving recombinant microorganisms is that the compound of interest may be largely retained intracellularly. Limited secretion of the product from the cells into the fermentation broth therefore limits recovery of the compound of interest or, in the best case, requires an additional step, including cell lysis, to release the compound into the broth. Secretion of the product from the cells into the fermentation broth offers several advantages, including reduced product inhibition and degradation, reduced impact on host cells, higher titers, and easier and less expensive recovery processes (Borodina I., 2019). Certain process advantages can be achieved if the secreted lipophilic product can be recovered in a separate phase.

[0005] Therefore, there is a need for improved methods for recovering hydrophobic fermentation products in fermentation processes, as well as improved methods for increasing the secretion of hydrophobic compounds from cells. Summary of the Invention

[0006] The method of the present invention solves the above problems. Provided herein is a method for producing hydrophobic compounds, such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and / or terpenes, such as terpenoids, by fermentation, the method comprising providing a microorganism capable of producing the hydrophobic compound and culturing the microorganism in a medium under conditions that allow for the production of the hydrophobic compound, the medium comprising an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution, the extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C6H4. 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The polyethoxylated surfactant is selected from antifoam agents including alcohol-based antifoam agents and combinations thereof, and the method optionally further comprises recovering the hydrophobic compound. Accordingly, provided herein is a method for producing a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes by fermentation, the method comprising: providing yeast cells capable of producing the hydrophobic compound; and culturing the yeast cells in a medium under conditions allowing production of the hydrophobic compound, the culturing being carried out at a culture temperature; the medium comprising an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution such as the medium at the culture temperature; the extractant is a nonionic ethoxylated surfactant; and the method optionally further comprises recovering the hydrophobic compound.

[0007] Also provided herein is a method for increasing the titer of a hydrophobic compound, such as a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, a fatty aldehyde, and / or a terpene, such as a terpenoid, in a fermentation, the method comprising culturing a microorganism capable of producing the hydrophobic compound in a medium under conditions that allow for the production of the hydrophobic compound, the medium comprising an extractant in an amount equal to or greater than its cloud concentration in an aqueous solution, the extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C6H4. 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The polyethoxylated surfactant is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, thereby increasing the titer of the hydrophobic compound compared to fermentation carried out under similar conditions in the absence of an extractant or in the presence of an extractant in an amount less than its cloud concentration in aqueous solution. Also provided herein is a method for increasing the titer of a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and terpenes in fermentation, the method comprising culturing yeast cells capable of producing the hydrophobic compound in a medium under conditions that allow the production of the hydrophobic compound, the culturing step being carried out at a culture temperature, the medium containing an extractant in an amount equal to or greater than its cloud concentration in aqueous solution measured at the culture temperature, the extractant being a nonionic ethoxylated surfactant, thereby increasing the titer of the hydrophobic compound compared to fermentation carried out under the same conditions but in the absence of an extractant or in the presence of an extractant in an amount less than its cloud concentration in aqueous solution at the culture temperature.

[0008] Also provided herein is a method for enhancing secretion of hydrophobic compounds, such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and / or terpenes, such as terpenoids, from a microorganism capable of producing said hydrophobic compounds during fermentation, the method comprising culturing the microorganism in a medium under conditions that allow production of said hydrophobic compounds, the medium comprising an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution, the extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C6H4. 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The polyethoxylated surfactant is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, thereby increasing the secretion of hydrophobic compounds from microorganisms compared to fermentation carried out under similar conditions in the absence of an extractant or in the presence of an extractant in an amount less than its cloud concentration measured in aqueous solution. Also provided herein is a method for increasing the secretion of hydrophobic compounds selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and terpenes from yeast cells capable of producing the hydrophobic compounds in fermentation, the method comprising culturing the yeast cells in a medium under conditions that allow the production of the hydrophobic compounds, the culturing step being carried out at a culture temperature, the medium containing an extractant in an amount equal to or greater than its cloud concentration measured in aqueous solution at the culture temperature, the extractant being a nonionic ethoxylated surfactant, thereby increasing the secretion of hydrophobic compounds from yeast cells compared to fermentation carried out under the same conditions but in the absence of an extractant or in the presence of an extractant in an amount less than its cloud concentration in aqueous solution at the culture temperature.

[0009] Also provided herein are hydrophobic compounds obtainable by the methods disclosed herein, preferably the hydrophobic compounds are selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes. Also provided herein is a method of monitoring the presence of pests or disrupting pest mating, the method comprising: i) producing a hydrophobic compound by the methods described herein, wherein the hydrophobic compound is as defined herein; ii) formulating the unsaturated fatty alcohol, unsaturated fatty acyl acetate, and / or unsaturated fatty aldehyde as a pheromone composition; and iii) using the pheromone composition as an integrated pest management composition; Includes: [Brief explanation of the drawings]

[0010] [Figure 1] In situ extraction and recovery of fatty alcohols produced by fermentation. Antifoam A was added at concentrations of 0% vol / vol (A); 0.4% vol / vol (B); 2% vol / vol (C), or 5% vol / vol to the fermentation of a Yarrowia lipolytica strain capable of producing fatty alcohols. In the absence (A) or at 0.4% vol / vol (B) of Antifoam A, the fermentation broth after centrifugation consisted of two phases: a solid cell fraction and an aqueous phase. When Antifoam A was added at 2% vol / vol (C) or 5% vol / vol (D), an additional immiscible phase was observed. After centrifugation, the fermentation broth consisted of three phases: a solid cell phase, an aqueous phase, and a product phase containing antifoam and fatty alcohols. Thus, fatty alcohols were separated into this phase. [Figure 2] Antifoam and Oil as Extractants. Various antifoams were added at a concentration of 3% vol / vol to the fermentation of a Yarrowia lipolytica strain capable of producing fatty alcohols. (A) No antifoam; (B) Corn oil; (C) Oleic acid; (D) Antifoam A; (E) Kolliphor® P407; (F) A-204; (G) Simethicone; (H) Dodecane. After centrifugation, three phases, including a product phase, were observed in the fermentation broths supplemented with Antifoam A (D), Kolliphor® P407 (E), A-204 (F), or simethicone (G). DETAILED DESCRIPTION OF THE INVENTION

[0011] The present disclosure relates to the discovery that the fermentation of microorganisms, particularly yeast, capable of producing hydrophobic compounds can be improved in several ways by including a nonionic surfactant, particularly a nonionic ethoxylated surfactant, such as an antifoaming agent, in an aqueous system at a concentration equal to or greater than its cloud concentration. Under such conditions, the nonionic ethoxylated surfactant acts as an extractant, thereby facilitating the recovery of the hydrophobic compounds. In addition, the presence of the nonionic ethoxylated surfactant surprisingly increases the titer of the hydrophobic compounds in the fermentation and also increases the secretion of the hydrophobic compounds from the yeast cells, thereby further enhancing production and facilitating recovery.

[0012] definition Surfactant: This term refers to a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, defoamers, and dispersants. Surfactants are typically amphiphilic organic compounds, meaning they contain both hydrophobic groups (their tails) and hydrophilic groups (their heads). Thus, surfactants typically contain both water-insoluble (or oil-soluble) and water-soluble components. Most commonly, surfactants are classified according to the polar head group. Nonionic surfactants do not have an uncharged group in their head.

[0013] Extractant: As used herein, the term "extractant" refers to a non-ionic surfactant, more specifically a non-ionic ethoxylated surfactant, such as an agent that can also be used as an anti-foaming agent to facilitate the recovery of hydrophobic compounds produced in fermentation, particularly polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18It refers to an ethoxylated surfactant, such as a fatty alcohol alkoxylate or a polyethoxylated surfactant, selected from agents or antifoaming agents including alcohol-based antifoaming agents and combinations thereof. Nonionic ethoxylated surfactants are often also referred to as low-foaming antifoaming agents.

[0014] Polyethoxylated surfactant: As used herein, this term refers to polyethoxylated surfactants, i.e., ethoxylated surfactants that may be nonionic surfactants.

[0015] Ethoxylated and Propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers: This term refers to a group of polyethoxylated nonionic surfactants, which are 16 -C 18 ethoxylated and propoxylated alcohols of, for example, CAS No. 68002-96-0, comprising or consisting essentially of C 16 -C 18 Alkyl alcohol ethoxylate propoxylate or C 16 -C 18 Also referred to as alcohol ethoxylated propoxylated polymers. Some compounds in this group are commonly used as antifoam agents, while others are not, and therefore are generally referred to herein as "ethoxylated and propoxylated C 16 -C 18 These are called "alcohol-based drugs."

[0016] Polyethylene Polypropylene Glycol: This term refers to a class of polyethoxylated nonionic surfactants that comprise or consist primarily of PEG-PPG-PEG block copolymer antifoam agents, such as Kolliphor® P407 (CAS No. 9003-11-6), also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol).

[0017] Mixture of polyether dispersions: This term refers to a group of polyethoxylated nonionic surfactants that comprise or consist primarily of mixtures of polyether dispersions, such as Sigma Aldrich Organic Antifoam 204 (product numbers A6426 and A8311, MDL number MFCD00130523).

[0018] Simethicone: This term refers to a group of polyethoxylated nonionic surfactants that contain or consist primarily of simethicone, also known as simethicone (CAS No. 8050-81-5), dimethylpolysiloxane, or activated polymethylsiloxane. Simethicone is a silicone-based emulsion that also contains 1.2-1.6% polyethylene glycol monostearate.

[0019] Cloud point: The cloud point of a solution of a surfactant, particularly a nonionic solution, or a glycol solution, e.g., an aqueous solution, is the temperature at which a mixture of the surfactant and the solution, e.g., the aqueous solution, begins to phase separate, two phases appear, and the solution becomes cloudy. This behavior is characteristic of nonionic surfactants containing polyoxyethylene chains, which exhibit the opposite behavior of their solubility versus temperature in water, and therefore "become cloudy" at some point as the temperature increases. Glycols that exhibit this behavior are known as "cloud-point glycols." The cloud point is affected by salinity and is usually lower in saline fluids.

[0020] Cloud concentration: This term is used to refer to the concentration of a surfactant in a solution, particularly a nonionic or glycol solution, above which, at a given temperature, a mixture of the surfactant and the solution begins to phase separate, resulting in the appearance of two phases and thus becoming turbid. For example, the cloud concentration of a surfactant in an aqueous solution at a given temperature is the minimum concentration of the surfactant that, when mixed with the aqueous solution, results in two phases. The cloud concentration can be obtained from the surfactant manufacturer or can be determined experimentally by creating a dosage curve and determining the concentration at which the mixture phases separate. For example, the method used in Example 7 can be applied. The cloud concentration can be determined in an aqueous solution, such as the medium used in this method, at room temperature. It can also be determined at the incubation temperature, e.g., 30°C, at which the incubation step is performed. For surfactants that can be used as antifoaming agents, the cloud concentration is usually higher than the concentration recommended by the manufacturer for foam control.

[0021] Nonionic Ethoxylated Surfactants The method of the present invention relies on the use of a non-ionic ethoxylated surfactant, e.g., an antifoam agent, which acts essentially as an extractant in the fermentation broth, and the non-ionic ethoxylated surfactant is present in an amount equal to or greater than its cloud concentration as measured in aqueous solution, such that the hydrophobic compounds produced are produced at higher titers, are more easily secreted into the broth from the producer bacteria, particularly the yeast cells, and / or are more easily recovered, compared to fermentations carried out in the absence of the non-ionic ethoxylated surfactant, with an amount of the same non-ionic ethoxylated surfactant less than its cloud concentration as measured in aqueous solution.

[0022] Nonionic surfactants, including nonionic ethoxylated surfactants, particularly antifoaming agents, are routinely used in fermentation processes to prevent foam formation. However, the present inventors have discovered that when nonionic ethoxylated surfactants are included in the fermentation broth in an amount equal to or greater than their cloud concentration measured in aqueous solution, this results in increased titer and secretion, facilitating the recovery of hydrophobic compounds. The cloud concentration in aqueous solution is determined at a predetermined temperature, preferably room temperature, or the temperature at which fermentation will be carried out, e.g., 30°C. This temperature is referred to herein as the "culture temperature." As used herein, the term "extractant" refers to nonionic surfactants, more specifically nonionic ethoxylated surfactants, particularly antifoaming agents, that facilitate the recovery of hydrophobic compounds produced in fermentation. For example, nonionic surfactants include polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C. 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agents are fatty alcohol alkoxylate or polyethoxylated surfactants selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof.

[0023] Nonionic surfactants that are particularly useful in the present method are ethoxylated and polyethoxylated surfactants, some of which are also routinely used as antifoaming agents, although their use as antifoaming agents usually involves their use at concentrations lower than those described herein, i.e., lower than their cloud concentrations measured in aqueous solution. These include polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18Includes fatty alcohol alkoxylate or polyethoxylated surfactants, such as alcohol-based antifoamers, as well as antifoamers including combinations thereof.

[0024] Thus, in one embodiment, the nonionic surfactant acting as an extractant is an antifoaming agent. In some embodiments, the nonionic ethoxylated surfactant is a fatty alcohol alkoxylate. In some embodiments, the nonionic ethoxylated surfactant is a polyethoxylated surfactant. In some embodiments, the antifoaming agent is polyethylene polypropylene glycol. In another embodiment, the antifoaming agent is a mixture of polyether dispersions. In another embodiment, the antifoaming agent is an antifoaming agent comprising polyethylene glycol monostearate or simethicone. In another embodiment, the antifoaming agent is an ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 In some embodiments, the extractant is a mixture of the above agents or antifoam agents and / or nonionic ethoxylated surfactants.

[0025] In a preferred embodiment of the method, the nonionic surfactant is an ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 and nonionic ethoxylated surfactants such as antifoam agents comprising or consisting of alcohol-based antifoam agents. 16 -C 18Surfactants containing alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0), Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, Antifoam 204, polyethylene glycol monostearate and fatty alcohol alkoxylates, in particular the fatty alcohol alkoxylates listed below, have been found to be particularly advantageous. Thus, in one embodiment, the antifoaming agent is C 16 -C 18 It is an alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0).

[0026] In another embodiment of the method, the nonionic surfactant is a nonionic ethoxylated surfactant, such as an antifoam agent comprising or consisting of polyethylene polypropylene glycol. For example, the antifoam agent is Kolliphor® P407 (CAS No. 9003-11-6).

[0027] In another embodiment of the method, the nonionic surfactant is a nonionic ethoxylated surfactant, such as an antifoam agent comprising or consisting of a mixture of polyether dispersions, for example, the antifoam agent is Sigma Aldrich Antifoam 204 (product numbers A6426 or A8311).

[0028] In another embodiment of the method, the nonionic surfactant is a nonionic ethoxylated surfactant such as polyethylene glycol monostearate or simethicone (CAS No. 8050-81-5), preferably an antifoaming agent comprising or consisting of simethicone.

[0029] In another embodiment of the method, the nonionic surfactant is a nonionic ethoxylated surfactant, such as Agnique BP420 (CAS No. 68002-96-0).

[0030] In another embodiment of the method, the non-ionic surfactant is a non-ionic ethoxylated surfactant, such as Antifoam 204.

[0031] In some embodiments of the method, the nonionic ethoxylated surfactant is a fatty alcohol alkoxylate preferably selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574.

[0032] In one embodiment, the extractant is Plurafac® LF300 (CAS No. 196823-11-7). In another embodiment, the extractant is Plurafac® LF1300 (CAS No. 68002-96-0). In another embodiment, the extractant is Plurafac® SLF180 (CAS No. 196823-11-7). In another embodiment, the extractant is Dehipon® 2574 (CAS No. 68154-97-2). In another embodiment, the extractant is Imbentin SG / 251 (CAS No. 68002-96-0).

[0033] The inventors have found that the nonionic surfactants, particularly the nonionic ethoxylated surfactants, some of which are routinely used for foam control in fermentation, when added in amounts equal to or greater than their cloud concentration in aqueous solution, i.e., amounts higher than those required for foam control, result in increased titer and increased secretion and promote recovery of hydrophobic compounds in fermentation. The cloud concentration of a surfactant is the concentration of surfactant at which, when mixed in aqueous solution, the mixture begins to phase separate, two phases appear, and the mixture becomes turbid.

[0034] To determine the cloud concentration of a surfactant, and thus the minimum amount of surfactant to use in the present method, those skilled in the art will know how to perform a dosage curve in which the surfactant is added to a solution, preferably an aqueous solution, at a predetermined temperature to determine the concentration of surfactant at which the appearance of two phases in the mixture is observed. Cloud concentration can be measured at room temperature, i.e., 18-25°C, e.g., 19°C, 20°C, 21°C, 22°C, 23°C, or 24°C, or at a temperature suitable for the envisioned fermentation method, e.g., 30°C or 37°C. The temperature of the fermentation broth can be adjusted after fermentation to enhance phase separation, as described herein. Example 7 describes one method for determining the cloud concentration of a surfactant.

[0035] In some embodiments, the nonionic surfactant or nonionic ethoxylated surfactant is added in an amount greater than its cloud concentration measured in aqueous solution. In some embodiments, the ethoxylated surfactant, such as fatty alcohol alkoxylate or polyethoxylated surfactant, is added in an amount greater than its cloud concentration measured in aqueous solution. The cloud concentration may be measured at room temperature or at incubation temperature.

[0036] In some embodiments, the medium contains an extractant, i.e., a nonionic surfactant, particularly a nonionic ethoxylated surfactant, in an amount at least 50%, such as at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% greater than its cloud concentration, the cloud concentration preferably being measured in aqueous solution, for example at room temperature or incubation temperature. The extractant is preferably a fatty alcohol alkoxylate, or a mixture of polyethylene polypropylene glycol, polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 An ethoxylated surfactant, such as a polyethoxylated surfactant selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof.

[0037] In some embodiments, the medium contains extractants, namely, Agnique BP420 (CAS No. 68002-96-0), Antifoam 204, polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The composition may comprise a nonionic ethoxylated surfactant, such as a fatty alcohol alkoxylate or a polyethoxylated surfactant, selected from antifoam agents including alcohol-based antifoam agents and combinations thereof, in an amount at least 50%, such as at least 100%, for example at least 150%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such as at least 400%, for example at least 500%, such as at least 750%, for example at least 1000% or more greater than its cloud concentration, the cloud concentration preferably being measured in aqueous solution, e.g. at room temperature or incubation temperature.

[0038] In some embodiments, the medium contains extractants, namely, Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, of a nonionic ethoxylated surfactant such as a fatty alcohol alkoxylate selected from the group consisting of SG / 251 (CAS No. 68002-96-0), Plurafac® LF300 or Dehipon® 2574, and combinations thereof, in an amount at least 50%, such as at least 100%, for example at least 150%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such as at least 400%, for example at least 500%, for example at least 750%, such as at least 1000% or more greater than its cloud concentration, wherein the cloud concentration is preferably measured in aqueous solution, for example at room temperature or incubation temperature.

[0039] In another embodiment, the medium comprises an extractant, i.e., a non-ionic surfactant such as a fatty alcohol alkoxylate or a polyethoxylated surfactant, in particular a non-ionic ethoxylated surfactant, in an amount of at least 2 times the cloud concentration, for example at least 3 times the cloud concentration, for example at least 4 times the cloud concentration, for example at least 5 times the cloud concentration, for example at least 6 times the cloud concentration, for example at least 7 times the cloud concentration, for example at least 8 times the cloud concentration, for example at least 9 times the cloud concentration, for example at least 10 times the cloud concentration, for example at least 12.5 times the cloud concentration, for example at least 15 times the cloud concentration, for example at least 17.5 times the cloud concentration, for example at least 20 times the cloud concentration, for example at least 25 times the cloud concentration, for example at least 30 times the cloud concentration, wherein the cloud concentration is preferably measured in an aqueous solution, for example at room temperature or at culture temperature.

[0040] In other embodiments, the medium contains no extractants, namely Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin a nonionic ethoxylated surfactant such as a fatty alcohol alkoxylate selected from SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, in an amount of at least 2 times its cloud concentration, such as at least 3 times its cloud concentration, for example at least 4 times its cloud concentration, such as at least 5 times its cloud concentration, for example at least 6 times its cloud concentration, such as at least 7 times its cloud concentration, for example at least 8 times its cloud concentration, such as at least 9 times its cloud concentration, for example at least 10 times its cloud concentration, such as at least 12.5 times its cloud concentration, for example at least 15 times its cloud concentration, such as at least 17.5 times its cloud concentration, for example at least 20 times its cloud concentration, for example at least 25 times its cloud concentration, for example at least 30 times its cloud concentration, wherein the cloud concentration is preferably measured in aqueous solution, for example at room temperature or incubation temperature.

[0041] In some embodiments, the medium contains an extractant, namely polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18a nonionic ethoxylated surfactant, such as a fatty alcohol alkoxylate or a polyethoxylated surfactant, selected from antifoam agents including alcohol-based antifoam agents and combinations thereof, in an amount of at least 2 times the cloud concentration, such as at least 3 times the cloud concentration, for example at least 4 times the cloud concentration, such as at least 5 times the cloud concentration, for example at least 6 times the cloud concentration, such as at least 7 times the cloud concentration, for example at least 8 times the cloud concentration, such as at least 9 times the cloud concentration, for example at least 10 times the cloud concentration, such as at least 12.5 times the cloud concentration, for example at least 15 times the cloud concentration, such as at least 17.5 times the cloud concentration, for example at least 20 times the cloud concentration, such as at least 25 times the cloud concentration, for example at least 30 times the cloud concentration, wherein the cloud concentration is preferably measured in aqueous solution, for example at room temperature or incubation temperature.

[0042] In some embodiments, the medium contains extractants, namely, Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin a nonionic ethoxylated surfactant such as a fatty alcohol alkoxylate selected from SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, in an amount of at least 2 times its cloud concentration, such as at least 3 times its cloud concentration, for example at least 4 times its cloud concentration, such as at least 5 times its cloud concentration, for example at least 6 times its cloud concentration, such as at least 7 times its cloud concentration, for example at least 8 times its cloud concentration, such as at least 9 times its cloud concentration, for example at least 10 times its cloud concentration, such as at least 12.5 times its cloud concentration, for example at least 15 times its cloud concentration, such as at least 17.5 times its cloud concentration, for example at least 20 times its cloud concentration, for example at least 25 times its cloud concentration, for example at least 30 times its cloud concentration, wherein the cloud concentration is preferably measured in aqueous solution, for example at room temperature or incubation temperature.

[0043] In some embodiments the medium comprises at least 1% vol / vol extractant, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol extractant, wherein the extractant is a non-ionic surfactant. In some embodiments, the medium contains at least 1% vol / vol of extractant, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 2 0%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol of extractant, such as Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin nonionic ethoxylated surfactants such as fatty alcohol alkoxylates, such as fatty alcohol alkoxylates selected from SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, or polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Polyethoxylated surfactants such as those selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof.

[0044] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, e.g., C 16 -C 18 The antifoaming agent comprises or consists of alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0). The cloud concentration of C16-C18 alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0) is about 1% vol / vol at room temperature. Therefore, when this antifoaming agent is used, the medium preferably contains at least 1% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates propoxylates, for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, for example at least 27.5%, such as at least 30% vol / vol of C 16 -C 18 Includes alkyl alcohol ethoxylates and propoxylates.

[0045] In some embodiments, the nonionic surfactant is a defoamer that comprises or consists of a nonionic ethoxylated surfactant, particularly a polyethylene polypropylene glycol, such as Kolliphor® P407 (CAS No. 9003-11-6), also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). Kolliphor® P407 has a cloud concentration of 10% at temperatures above 100° C. Thus, when polyethylene polypropylene glycol such as Kolliphor® P407 is used the culture medium preferably comprises at least 10% vol / vol of polyethylene polypropylene glycol such as Kolliphor® P407, such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol such as Kolliphor® P407.

[0046] In some embodiments, the nonionic surfactant is a defoamer comprising or consisting of a nonionic ethoxylated surfactant, particularly a polyether dispersion such as Antifoam 204 (Sigma Aldrich product numbers A6426 or A8311). The cloud concentration of Antifoam 204 is 1% in aqueous solution at a temperature of 18.0-21.0°C. Thus, when a mixture of polyether dispersions such as Antifoam 204 is used, the medium preferably comprises at least 1% vol / vol of the mixture of polyether dispersions such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of the mixture of polyether dispersions such as Antifoam 204.

[0047] In some embodiments, the nonionic surfactant comprises or consists of a nonionic ethoxylated surfactant, particularly the antifoaming agent comprising polyethylene glycol monostearate or simethicone.Simethicone comprises polyethylene glycol monostearate, which, without being bound by theory, appears to be an important compound due to simethicone's ability to act as an extractant.Polyethylene glycol monostearate has a cloud point of 1% at 5°C in aqueous solution. Thus, when a surfactant comprising polyethylene glycol monostearate is used as the antifoaming agent, the medium preferably comprises at least 1% vol / vol polyethylene glycol monostearate or simethicone, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more polyethylene glycol monostearate or simethicone.

[0048] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly Agnique BP420 (CAS No. 68002-96-0). Agnique BP420 (CAS No. 68002-96-0) has a cloud point of 1% in aqueous solution at room temperature. Thus, when Agnique BP420 (CAS No. 68002-96-0) is used as an antifoaming agent, the medium preferably contains at least 1% vol / vol Agnique BP420 (CAS No. 68002-96-0), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Agnique BP420 (CAS No. 68002-96-0).

[0049] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly a fatty alcohol alkoxylate such as Plurafac® LF300 (CAS No. 196823-11-7), which has a cloud concentration of about 1% vol / vol at room temperature. Thus, when Plurafac® LF300 (CAS No. 196823-11-7) is used, the culture medium preferably comprises at least 1% vol / vol Plurafac® LF300 (CAS No. 196823-11-7), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol Plurafac® LF300 (CAS No. 196823-11-7).

[0050] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly a fatty alcohol alkoxylate such as Plurafac® LF1300 (CAS No. 68002-96-0), which has a cloud concentration of about 1% vol / vol at room temperature. Thus, when Plurafac® LF1300 (CAS No. 68002-96-0) is used, the culture medium preferably comprises at least 1% vol / vol Plurafac® LF1300 (CAS No. 68002-96-0), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Plurafac® LF1300 (CAS No. 68002-96-0).

[0051] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly a fatty alcohol alkoxylate such as Plurafac® SLF180 (CAS No. 196823-11-7), which has a cloud concentration of about 1% vol / vol at room temperature. Thus, when Plurafac® SLF180 (CAS No. 196823-11-7) is used, the culture medium preferably comprises at least 1% vol / vol Plurafac® SLF180 (CAS No. 196823-11-7), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, such as at least 20%, for example at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Plurafac® SLF180 (CAS No. 196823-11-7).

[0052] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly a fatty alcohol alkoxylate such as Dehipon® 2574 (CAS No. 68154-97-2), which has a cloud concentration of about 1% vol / vol at room temperature. Thus, when Dehypon® 2574 (CAS No. 68154-97-2) is used, the culture medium preferably comprises at least 1% vol / vol of Dehypon® 2574 (CAS No. 68154-97-2), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of Dehypon® 2574 (CAS No. 68154-97-2).

[0053] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, particularly a fatty alcohol alkoxylate such as Imbentin® SG / 251 (CAS No. 68002-96-0), which has a cloud concentration of about 1% vol / vol at room temperature. Thus, when Imbentin® SG / 251 (CAS No. 68002-96-0) is used the culture medium preferably comprises at least 1% vol / vol Imbentin SG / 251 (CAS No. 68002-96-0), such as at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, such as at least 8%, for example at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, such as at least 20%, for example at least 22.5%, such as at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Imbentin® SG / 251 (CAS No. 68002-96-0).

[0054] Hydrophobic compounds The methods disclosed herein are useful for facilitating the recovery of hydrophobic compounds from fermentation broth, for increasing the titer of hydrophobic compounds in fermentation, and for increasing the secretion of hydrophobic compounds from producer bacteria.

[0055] The hydrophobic compound may be any hydrophobic compound produced by a microorganism during fermentation. In a preferred embodiment, the microorganism is a yeast cell. In particular, the hydrophobic compound is selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes, such as terpenoids. The hydrophobic compound may be one or more mixtures of several hydrophobic compounds, for example, at least one fatty alcohol, at least one fatty acyl acetate, at least one fatty aldehyde, and at least one terpene, such as at least one terpenoid. In some embodiments, the hydrophobic compound is a mixture of one or more fatty alcohols, one or more fatty acyl acetates, and / or one or more fatty aldehydes. In some embodiments, the hydrophobic compound is a mixture of one or more terpenes, such as a mixture of one or more terpenoids, or a mixture of one or more terpenes and one or more terpenoids. The methods of the present invention are useful for producing and recovering hydrophobic compounds that are pheromones, particularly insect pheromones.

[0056] fatty alcohols The fatty alcohol may be a saturated fatty alcohol, an unsaturated fatty alcohol, or a mixture thereof. In one embodiment, the fatty alcohol has a chain length of 8. In another embodiment, the fatty alcohol has a chain length of 9. In another embodiment, the fatty alcohol has a chain length of 10. In another embodiment, the fatty alcohol has a chain length of 11. In another embodiment, the fatty alcohol has a chain length of 12. In another embodiment, the fatty alcohol has a chain length of 13. In another embodiment, the fatty alcohol has a chain length of 14. In another embodiment, the fatty alcohol has a chain length of 15. In another embodiment, the fatty alcohol has a chain length of 16. In another embodiment, the fatty alcohol has a chain length of 17. In another embodiment, the fatty alcohol has a chain length of 18. In another embodiment, the fatty alcohol has a chain length of 19. In another embodiment, the fatty alcohol has a chain length of 20. In another embodiment, the fatty alcohol has a chain length of 21. In another embodiment, the fatty alcohol has a chain length of 22.

[0057] In some embodiments, the fatty alcohol is an unsaturated fatty alcohol. Such compounds are naturally produced, for example, by insect cells, and they act as pheromones. The unsaturated fatty alcohol can be: (Z)-Δ3 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ3 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ5 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ5 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ6 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ6 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ7 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ7 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ8 unsaturated fatty alcohols with a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ8 unsaturated fatty alcohols with a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ9 unsaturated fatty alcohols having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ9 unsaturated fatty alcohols with a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ10 unsaturated fatty alcohols having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ10 unsaturated fatty alcohols having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ11 unsaturated fatty alcohols having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ11 unsaturated fatty alcohols having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ12 unsaturated fatty alcohols with a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ12 unsaturated fatty alcohols with a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ13 unsaturated fatty alcohols having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22; and (E)-Δ13 unsaturated fatty alcohols having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21 or 22.

[0058] In some embodiments, the fatty alcohol is an unsaturated fatty alcohol having a carbon chain length of 14, such as: (Z)-Δ5 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ5 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ6 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ6 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ7 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ7 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ8 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ8 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ9 unsaturated fatty alcohols with a carbon chain length of −14; (E)-Δ9 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ10 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ10 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ11 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ11 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ12 unsaturated fatty alcohols with a carbon chain length of -14; (E)-Δ12 unsaturated fatty alcohols with a carbon chain length of -14; (Z)-Δ13 unsaturated fatty alcohols having a carbon chain length of -14; and An (E)-Δ13 unsaturated fatty alcohol with a carbon chain length of −14.

[0059] In some embodiments, the fatty alcohol is an unsaturated fatty alcohol having a carbon chain length of 16, such as: (Z)-Δ5 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ5 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ6 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ6 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ7 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ7 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ8 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ8 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ9 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ9 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ10 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ10 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ11 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ11 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ12 unsaturated fatty alcohols with a carbon chain length of -16; (E)-Δ12 unsaturated fatty alcohols with a carbon chain length of -16; (Z)-Δ13 unsaturated fatty alcohols having a carbon chain length of -16; and An (E)-Δ13 unsaturated fatty alcohol with a carbon chain length of −16.

[0060] The unsaturated fatty alcohol may be desaturated in more than one position. The unsaturated fatty alcohol may be desaturated in at least two positions, such as at least three positions, such as at least four positions. For example, the fatty alcohol is an (E)7, (Z)9 unsaturated fatty alcohol having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the fatty alcohol is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, e.g., 14. In some embodiments, the fatty alcohol is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22. In other embodiments, the fatty alcohol is an (E)7, (Z)9 unsaturated fatty alcohol having a carbon chain length of 14. In another embodiment, the unsaturated fatty alcohol is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol having a carbon chain length of 14. In another embodiment, the unsaturated fatty alcohol is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol having a carbon chain length of 14. For example, the fatty alcohol is an (E)7, (Z)9 unsaturated fatty alcohol having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol is an (E)8, (E)10 unsaturated fatty alcohol having a carbon chain length of 12. In certain embodiments, the fatty alcohol is (Z)-11-hexadecen-1-ol or (Z)-9-tetradecen-1-ol.

[0061] fatty alcohol esters The fatty alcohol ester may be a saturated fatty alcohol ester, an unsaturated fatty alcohol ester, or a mixture thereof. In one embodiment, the fatty alcohol ester has a chain length of 8. In another embodiment, the fatty alcohol ester has a chain length of 9. In another embodiment, the fatty alcohol ester has a chain length of 10. In another embodiment, the fatty alcohol ester has a chain length of 11. In another embodiment, the fatty alcohol ester has a chain length of 12. In another embodiment, the fatty alcohol ester has a chain length of 13. In another embodiment, the fatty alcohol ester has a chain length of 14. In another embodiment, the fatty alcohol ester has a chain length of 15. In another embodiment, the fatty alcohol ester has a chain length of 16. In another embodiment, the fatty alcohol ester has a chain length of 17. In another embodiment, the fatty alcohol ester has a chain length of 18. In another embodiment, the fatty alcohol ester has a chain length of 19. In another embodiment, the fatty alcohol ester has a chain length of 20. In another embodiment, the fatty alcohol ester has a chain length of 21. In another embodiment, the fatty alcohol ester has a chain length of 22.

[0062] In some embodiments, the fatty alcohol ester is an unsaturated fatty alcohol ester. Such compounds are naturally produced, for example, by insect cells, and they act as pheromones. The unsaturated fatty alcohol ester can be: (Z)-Δ3 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ3 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ5 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ5 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ6 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ6 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ7 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ7 unsaturated fatty alcohol esters having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ8 unsaturated fatty alcohol esters having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ8 unsaturated fatty alcohol esters having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ9 unsaturated fatty alcohol esters having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ9 unsaturated fatty alcohol esters having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ10 unsaturated fatty alcohol esters having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ10 unsaturated fatty alcohol esters having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ11 unsaturated fatty alcohol esters having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ11 unsaturated fatty alcohol esters having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ12 unsaturated fatty alcohol esters having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ12 unsaturated fatty alcohol esters having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ13 unsaturated fatty alcohol esters having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22; and (E)-Δ13 unsaturated fatty alcohol esters having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21 or 22.

[0063] In some embodiments, the fatty alcohol ester is an unsaturated fatty alcohol ester having a carbon chain length of 14, such as: (Z)-Δ5 unsaturated fatty alcohol esters with a carbon chain length of -14; (E)-Δ5 unsaturated fatty alcohol esters with a carbon chain length of -14; (Z)-Δ6 unsaturated fatty alcohol esters with a carbon chain length of -14; (E)-Δ6 unsaturated fatty alcohol esters with a carbon chain length of -14; (Z)-Δ7 unsaturated fatty alcohol esters with a carbon chain length of -14; (E)-Δ7 unsaturated fatty alcohol esters with a carbon chain length of -14; (Z)-Δ8 unsaturated fatty alcohol esters with a carbon chain length of -14; (E)-Δ8 unsaturated fatty alcohol esters with a carbon chain length of -14; (Z)-Δ9 unsaturated fatty alcohol esters with a carbon chain length of -14; (E)-Δ9 unsaturated fatty alcohol esters with a carbon chain length of −14; (Z)-Δ10 unsaturated fatty alcohol esters having a carbon chain length of -14; (E)-Δ10 unsaturated fatty alcohol esters having a carbon chain length of -14; (Z)-Δ11 unsaturated fatty alcohol esters having a carbon chain length of -14; (E)-Δ11 unsaturated fatty alcohol esters having a carbon chain length of -14; (Z)-Δ12 unsaturated fatty alcohol esters having a carbon chain length of -14; (E)-Δ12 unsaturated fatty alcohol esters having a carbon chain length of -14; (Z)-Δ13 unsaturated fatty alcohol esters having a carbon chain length of -14; and (E)-Δ13 unsaturated fatty alcohol ester with a carbon chain length of −14.

[0064] In some embodiments, the fatty alcohol ester is an unsaturated fatty alcohol ester having a carbon chain length of 16, such as: (Z)-Δ5 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ5 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ6 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ6 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ7 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ7 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ8 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ8 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ9 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ9 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ10 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ10 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ11 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ11 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ12 unsaturated fatty alcohol esters with a carbon chain length of -16; (E)-Δ12 unsaturated fatty alcohol esters with a carbon chain length of -16; (Z)-Δ13 unsaturated fatty alcohol esters having a carbon chain length of -16; and (E)-Δ13 unsaturated fatty alcohol esters with a carbon chain length of −16.

[0065] The unsaturated fatty alcohol ester can be desaturated in two or more positions. The unsaturated fatty alcohol ester can be desaturated in at least two positions, such as at least three positions, such as at least four positions. For example, the fatty alcohol ester is an (E)7, (Z)9 unsaturated fatty alcohol ester having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the fatty alcohol ester is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol ester having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, e.g., 14. In some embodiments, the fatty alcohol ester is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol ester having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22. In other embodiments, the fatty alcohol ester is an (E)7, (Z)9 unsaturated fatty alcohol ester having a carbon chain length of 14. In another embodiment, the unsaturated fatty alcohol ester is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol ester having a carbon chain length of 14. In another embodiment, the unsaturated fatty alcohol ester is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol ester having a carbon chain length of 14. For example, the fatty alcohol ester is an (E)7, (Z)9 unsaturated fatty alcohol ester having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol ester is an (E)3, (Z)8, (Z)11 unsaturated fatty alcohol ester having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol ester is a (Z)9, (E)11, (E)13 unsaturated fatty alcohol ester having a carbon chain length of 12. In another embodiment, the unsaturated fatty alcohol ester is an (E)8, (E)10 unsaturated fatty alcohol ester having a carbon chain length of 12. In certain embodiments, the fatty alcohol ester is a (Z)-11-hexadecen-1-ol ester or a (Z)-9-tetradecen-1-ol ester. The fatty alcohol ester may be a fatty alcohol acetate ester.

[0066] fatty acyl acetate The fatty acyl acetate may be saturated or unsaturated, or a mixture thereof. Fatty acyl acetates, particularly unsaturated fatty acyl acetates, are also naturally found in pheromones, particularly those produced by species belonging to the order Lepidoptera.

[0067] In one embodiment, the fatty acyl acetate has a chain length of 8. In another embodiment, the fatty acyl acetate has a chain length of 9. In another embodiment, the fatty acyl acetate has a chain length of 10. In another embodiment, the fatty acyl acetate has a chain length of 11. In another embodiment, the fatty acyl acetate has a chain length of 12. In another embodiment, the fatty acyl acetate has a chain length of 13. In another embodiment, the fatty acyl acetate has a chain length of 14. In another embodiment, the fatty acyl acetate has a chain length of 15. In another embodiment, the fatty acyl acetate has a chain length of 16. In another embodiment, the fatty acyl acetate has a chain length of 17. In another embodiment, the fatty acyl acetate has a chain length of 18. In another embodiment, the fatty acyl acetate has a chain length of 19. In another embodiment, the fatty acyl acetate has a chain length of 20. In another embodiment, the fatty acyl acetate has a chain length of 21. In another embodiment, the fatty acyl acetate has a chain length of 22.

[0068] In some embodiments, the fatty acyl acetate is an unsaturated fatty acyl acetate. The unsaturated fatty acyl acetate can be an unsaturated fatty acyl acetate having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, such as: (Z)-Δ3 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ3 unsaturated fatty alcohols with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ5 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ5 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ6 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ6 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ7 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ7 unsaturated fatty acyl acetates having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ8 unsaturated fatty acyl acetates having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ8 unsaturated fatty acyl acetates having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ9 unsaturated fatty acyl acetates having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (E)-Δ9 unsaturated fatty acyl acetates having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ10 unsaturated fatty acyl acetates having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (E)-Δ10 unsaturated fatty acyl acetates having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ11 unsaturated fatty acyl acetates having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ11 unsaturated fatty acyl acetates having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ12 unsaturated fatty acyl acetates having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; -(E)-Δ12 unsaturated fatty acyl acetates having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ13 unsaturated fatty acyl acetates having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22; and (E)-Δ13 unsaturated fatty acyl acetates having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21 or 22.

[0069] In some embodiments, the fatty acyl acetate is an unsaturated fatty acyl acetate having a carbon chain length of 14, such as: (Z)-Δ5 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ5 unsaturated fatty acyl acetates with a carbon chain length of −14; (Z)-Δ6 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ6 unsaturated fatty acyl acetates with a carbon chain length of -14; (Z)-Δ7 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ7 unsaturated fatty acyl acetates with a carbon chain length of -14; (Z)-Δ8 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ8 unsaturated fatty acyl acetate with a carbon chain length of -14; (Z)-Δ9 unsaturated fatty acyl acetates with a carbon chain length of −14; (E)-Δ9 unsaturated fatty acyl acetates with a carbon chain length of −14; (Z)-Δ10 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ10 unsaturated fatty acyl acetates with a carbon chain length of -14; (Z)-Δ11 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ11 unsaturated fatty acyl acetates with a carbon chain length of -14; (Z)-Δ12 unsaturated fatty acyl acetates with a carbon chain length of -14; (E)-Δ12 unsaturated fatty acyl acetates with a carbon chain length of -14; (Z)-Δ13 unsaturated fatty acyl acetates having a carbon chain length of -14; and (E)-Δ13 unsaturated fatty acyl acetate with a carbon chain length of −14.

[0070] In some embodiments, the fatty acyl acetate is an unsaturated fatty acyl acetate having a carbon chain length of 16, such as: (Z)-Δ5 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ5 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ6 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ6 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ7 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ7 unsaturated fatty acyl acetates with a carbon chain length of −16; (Z)-Δ8 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ8 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ9 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ9 unsaturated fatty acyl acetates with a carbon chain length of −16; (Z)-Δ10 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ10 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ11 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ11 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ12 unsaturated fatty acyl acetates with a carbon chain length of -16; (E)-Δ12 unsaturated fatty acyl acetates with a carbon chain length of -16; (Z)-Δ13 unsaturated fatty acyl acetates having a carbon chain length of -14; and (E)-Δ13 unsaturated fatty acyl acetate with a carbon chain length of −16.

[0071] The unsaturated fatty acyl acetate can be desaturated in two or more positions. The unsaturated fatty acyl acetate can be desaturated in at least two positions, such as at least three positions, such as at least four positions. For example, the fatty acyl acetate is an (E)7, (Z)9 unsaturated fatty acyl acetate having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the fatty acyl acetate is an (E)3, (Z)8, (Z)11 unsaturated fatty acyl acetate having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22. In some embodiments, the fatty acyl acetate is a (Z)9, (E)11, (E)13 unsaturated fatty acyl acetate having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22. In other embodiments, the fatty acyl acetate is an (E)7, (Z)9 unsaturated fatty acyl acetate having a carbon chain length of 14. In some embodiments, the fatty acyl acetate is an (E)3, (Z)8, (Z)11 unsaturated fatty acyl acetate having a carbon chain length of 14. In some embodiments, the fatty acyl acetate is a (Z)9, (E)11, (E)13 unsaturated fatty acyl acetate having a carbon chain length of 14. In other embodiments, the fatty acyl acetate is an (E)7, (Z)9 unsaturated fatty acyl acetate having a carbon chain length of 12. In some embodiments, the fatty acyl acetate is an (E)3, (Z)8, (Z)11 unsaturated fatty acyl acetate having a carbon chain length of 12. In certain embodiments, the fatty acyl acetate is (Z)-11-hexadecen-1-yl acetate or (Z)-9-tetradecen-1-yl acetate. Fatty acyl acetates are produced by microorganisms in fermentation, for example, microorganisms can convert fatty alcohols to the corresponding fatty acyl acetates, or they can be obtained by chemical conversions known in the art.

[0072] fatty aldehydes The fatty aldehyde may be a saturated fatty aldehyde or an unsaturated fatty aldehyde or a mixture thereof. Fatty aldehydes, particularly unsaturated fatty aldehydes, are also naturally found in pheromones, particularly insect pheromones.

[0073] In one embodiment, the fatty aldehyde has a chain length of 8. In another embodiment, the fatty aldehyde has a chain length of 9. In another embodiment, the fatty aldehyde has a chain length of 10. In another embodiment, the fatty aldehyde has a chain length of 11. In another embodiment, the fatty aldehyde has a chain length of 12. In another embodiment, the fatty aldehyde has a chain length of 13. In another embodiment, the fatty aldehyde has a chain length of 14. In another embodiment, the fatty aldehyde has a chain length of 15. In another embodiment, the fatty aldehyde has a chain length of 16. In another embodiment, the fatty aldehyde has a chain length of 17. In another embodiment, the fatty aldehyde has a chain length of 18. In another embodiment, the fatty aldehyde has a chain length of 19. In another embodiment, the fatty aldehyde has a chain length of 20. In another embodiment, the fatty aldehyde has a chain length of 21. In another embodiment, the fatty aldehyde has a chain length of 22.

[0074] In some embodiments, the fatty aldehyde is an unsaturated fatty aldehyde. The unsaturated fatty aldehyde can have a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, such as: (Z)-Δ3 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ3 unsaturated fatty aldehydes with a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ5 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ5 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ6 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ6 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ7 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ7 unsaturated fatty aldehydes having a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ8 unsaturated fatty aldehydes having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ8 unsaturated fatty aldehydes having a carbon chain length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ9 unsaturated fatty aldehydes having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ9 unsaturated fatty aldehydes having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ10 unsaturated fatty aldehydes having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ10 unsaturated fatty aldehydes having a carbon chain length of 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22; (Z)-Δ11 unsaturated fatty aldehydes having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (E)-Δ11 unsaturated fatty aldehydes having a carbon chain length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ12 unsaturated fatty aldehydes having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; -(E)-Δ12 unsaturated fatty aldehydes having a carbon chain length of 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22; (Z)-Δ13 unsaturated fatty aldehydes having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22; and -(E)-Δ13 unsaturated fatty aldehydes having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21 or 22.

[0075] In some embodiments, the fatty aldehyde is an unsaturated fatty aldehyde having a carbon chain length of 14, such as: (Z)-Δ5 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ5 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ6 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ6 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ7 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ7 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ8 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ8 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ9 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ9 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ10 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ10 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ11 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ11 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ12 unsaturated fatty aldehydes with a carbon chain length of -14; (E)-Δ12 unsaturated fatty aldehydes with a carbon chain length of -14; (Z)-Δ13 unsaturated fatty aldehydes having a carbon chain length of -14; and An (E)-Δ13 unsaturated fatty aldehyde with a carbon chain length of -14.

[0076] In some embodiments, the fatty aldehyde is an unsaturated fatty aldehyde having a carbon chain length of 16, such as: (Z)-Δ5 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ5 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ6 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ6 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ7 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ7 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ8 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ8 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ9 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ9 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ10 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ10 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ11 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ11 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ12 unsaturated fatty aldehydes with a carbon chain length of -16; (E)-Δ12 unsaturated fatty aldehydes with a carbon chain length of -16; (Z)-Δ13 unsaturated fatty aldehydes having a carbon chain length of -16; and An (E)-Δ13 unsaturated fatty aldehyde with a carbon chain length of -16.

[0077] The unsaturated fatty aldehyde produced can be desaturated at two or more positions. The unsaturated fatty aldehyde can be desaturated at at least two positions, such as at least three positions, such as at least four positions. For example, the fatty aldehyde is an (E)7, (Z)9 unsaturated fatty aldehyde having a carbon chain length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22, e.g., 14. In some embodiments, the fatty aldehyde is an (E)3, (Z)8, (Z)11 unsaturated fatty aldehyde having a carbon chain length of 14. In some embodiments, the fatty aldehyde is a (Z)9, (E)11, (E)13 unsaturated fatty aldehyde having a carbon chain length of 14, 15, 16, 17, 18, 19, 20, 21, or 22, such as 14. In some embodiments, the unsaturated fatty aldehyde is an (E)7, (Z)9 unsaturated fatty aldehyde having a carbon chain length of 12. In other embodiments, the fatty aldehyde is an (E)3, (Z)8, (Z)11 unsaturated fatty aldehyde having a carbon chain length of 12. In some embodiments, the fatty aldehyde is a (Z)9, (E)11, (E)13 unsaturated fatty aldehyde having a carbon chain length of 12. In certain embodiments, the fatty aldehyde is (Z)-11-hexadecenal. Fatty aldehydes can be produced by microorganisms in fermentation, for example, microorganisms can convert fatty alcohols to the corresponding fatty aldehydes, or they can be obtained by chemical conversions known in the art.

[0078] Terpenes and Terpenoids Terpenes are naturally produced by plants and have many industrial uses in the fields of food, medicine, cosmetics, and biotechnology. They are used, for example, as part of natural agricultural pesticides. Terpenoids (also called isoprenoids) are modified terpenes that contain additional groups, usually O-containing groups. They are often used for their aromatic properties and as part of traditional herbal remedies.

[0079] In some embodiments of the method, the hydrophobic compound is a terpene, such as a hemiterpene, a monoterpene, a sesquiterpene, a diesterterpene, a triterpene, a sesquaterpene, a tetraterpene, or a polyterpene. In some embodiments, the terpene is a monoterpene, such as geraniol, terpineol, limonene, myrcene, linalool, pinene, or menthol. In some embodiments, the terpene is a sesquiterpene, such as humulene, farnesene, or farnesol. In some embodiments, the terpene is a triterpene, such as squalene. In some embodiments, the terpene is a tetraterpene, such as lycopene, or a carotene, such as α-carotene, β-carotene, and γ-carotene.

[0080] In some embodiments, the hydrophobic compound is a terpene, such as a terpenoid, such as a hemiterpenoid, monoterpenoid, sesquiterpenoid, diesterterpenoid, triterpenoid, sesquaterpenoid, tetraterpenoid, or polyterpenoid. In some embodiments, the terpenoid is a monocyclic monoterpenoid, such as menthol, thymol, or carvacrol, or a bicyclic monoterpenoid, such as camphor, borneol, or eucalyptol. In some embodiments, the terpenoid is a sesquiterpenoid, such as geosmin, vetiverzulene, guaiazulene, or farnesol. In some embodiments, the terpenoid is a diterpenoid, such as a taxane, retinol, or phytol. In some embodiments, the terpenoid is a triterpenoid, such as a steroid, such as a sterol, or a cucurbitacin. In some embodiments, the terpenoid is a tetraterpenoid, such as a carotenoid.

[0081] microorganisms The method is useful for recovering hydrophobic compounds produced in fermentation by a microorganism, and / or for increasing the titer of a hydrophobic compound, and / or for increasing the secretion of a hydrophobic compound from a microorganism, preferably a yeast. The microorganism may be a bacterium or a eukaryote, hi some embodiments, the microorganism is a yeast cell.

[0082] In some embodiments, microorganisms or yeast cells are modified at the genome level, for example, by gene editing in the genome.Cells can also be modified by inserting at least one nucleic acid construct, such as at least one vector.Vector can be designed to allow nucleic acid sequence to be integrated into genome, or to allow polypeptides encoded by the nucleic acid sequence contained in the vector to be expressed without genome integration, as known to those skilled in the art.In other embodiments, microorganisms are naturally producing cells of desired hydrophobic compounds, such as yeast that naturally produce fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and terpenes.

[0083] Certain embodiments of the present disclosure employ yeast or fungi from genera including, but not limited to, Blakeslea, Candida, Cryptococcus, Kuningamella, Lipomyces, Mortierella, Mucor, Phycomyces, Phycium, Rhodosporidium, Rhodotorula, Trichosporon, Saccharomyces, and Yarrowia. In certain embodiments, Blakeslea trispora, Candida pulcherrima, C. rebkaufii, C. tropicalis, Cryptococcus culbatus, Kuningamella echinulata, C. elegans, C. japonica, Lipomyces starchii (oleaginous yeast), L. lipoferrus, Mortierella alpina, M. isabellina, M. lamaniana, M. vinacea, and Mucor circinelloides. Phycomyces brachesleanus, Pythium irregulare, Rhodosporidium toruloides (oleaginous yeast), Rhodotorula glutinis (red yeast), R. gracilis, R. graminis, R. mucilaginosa, R. pinicola, Trichosporon planus, T. cutaneum, Saccharomyces cerevisiae (budding yeast), and Yarrowia lipolytica (alkane-utilizing yeast) are used. In a preferred embodiment, the microorganism is a yeast, particularly Yarrowia lipolytica (alkane-utilizing yeast) or Saccharomyces cerevisiae (budding yeast).

[0084] Several microorganisms, particularly yeast cells, have been reported to produce hydrophobic compounds, particularly fatty alcohols, fatty acyl acetates, and fatty aldehydes, which can be incorporated into pheromone compositions and used as pest repellents. The methods of the present invention employ such yeast cells in fermentation processes to produce such compounds, facilitate their recovery, increase their titer, and / or enhance their secretion from the cells. Such yeast cells and resulting products are described in detail, for example, in International Publication Nos. WO 2016 / 207339, WO 2018 / 109163, and WO 2018 / 109167, as well as in International Application No. PCT / EP2020 / 053306, filed December 20, 2019, and European Patent Application No. 19218703.7, entitled "Yeast cells and methods for production of E8, E10-dodecadienyl coenzyme A, codlemone, and derivatives thereof."

[0085] Generally, yeast cells useful for the production of such compounds rely on the expression of several enzymes, particularly heterologous enzymes, such as desaturases such as Δ11 desaturases (EC 1.14.19.5), fatty acyl reductases (FAR) (EC 1.2.1.84), fatty acyl-CoA synthetases (FAA) (EC 2.3.1.86), acetyltransferases (EC 2.3.1.84) or acyl-CoA oxidases (EC 1.3.3.6). Hereinafter, some specific embodiments are described.

[0086] Unsaturated fatty alcohols In some embodiments, the microorganism is a yeast cell, such as an oleaginous yeast cell, and the hydrophobic compound is an unsaturated fatty alcohol. A yeast cell capable of producing an unsaturated fatty alcohol, e.g., a Yarrowia cell, such as a Yarrowia lipolytica (Yarrowia lipolytica) cell, can be: - expressing at least one heterologous desaturase capable of introducing at least one double bond into fatty acyl-CoA; and - expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol; and - a mutation resulting in a reduced activity of Fao1 (SEQ ID NO: 11) and a mutation resulting in a reduced activity of at least one protein of Hfd1 (SEQ ID NO: 12), Hfd4 (SEQ ID NO: 13), Pex10 (SEQ ID NO: 14) and GPAT (SEQ ID NO: 15), or a mutation resulting in a reduced activity of at least one protein having at least 90% homology to Fao1 (SEQ ID NO: 11), and a mutation resulting in a reduced activity of at least one protein of Hfd1 (SEQ ID NO: 12), Hfd4 (SEQ ID NO: 13), Pex10 (SEQ ID NO: 14) and GPAT (SEQ ID NO: 15); and has mutations resulting in reduced activity, such as at least 91% homology, for example at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, for example at least 96% homology, such as at least 97% homology, for example at least 98% homology, for example at least 99% homology, to at least one of Fao1 (SEQ ID NO: 11) and Hfd1 (SEQ ID NO: 12), Hfd4 (SEQ ID NO: 13), Pex10 (SEQ ID NO: 14) and GPAT (SEQ ID NO: 15).

[0087] Throughout this disclosure, mutations resulting in reduced activity of a protein or enzyme are preferably mutations in the gene encoding said protein or enzyme, the mutation preferably being in the promoter of the gene, or in the coding sequence of the gene, or both.

[0088] The desaturase is preferably selected from the group consisting of Δ3 desaturase, Δ5 desaturase, Δ6 desaturase, Δ7 desaturase, Δ8 desaturase, Δ9 desaturase, Δ10 desaturase, Δ11 desaturase, Δ12 desaturase, Δ13 desaturase and Δ14 desaturase, preferably the desaturase is from an insect, such as from the order Lepidoptera, and preferably the desaturase is a Δ11 desaturase having at least 60% identity to the Δ11 desaturase from Amielois transitella (walnut moth) set forth in SEQ ID NO: 1 or a Δ9 desaturase having at least 60% identity to the Δ9 desaturase from Drosophila melanogaster (Drosophila melanogaster) set forth in SEQ ID NO: 16. In some embodiments, the fatty acyl reductase is: i) a FAR having at least 80% homology to the FAR from Helicoverpa armigera (Helicoverpa armigera) set forth in SEQ ID NO: 5; ii) a FAR having at least 80% homology with FAR from Helicoverpa assulta (tobacco budworm) as set forth in SEQ ID NO: 7; iii) a FAR having at least 80% homology to the FAR from Heliothis subflexa (the nightshade moth) set forth in SEQ ID NO: 6; and iv) FAR having at least 80% homology with FAR from Bicyclus annina (Satyridae) as set forth in SEQ ID NO: 17; Preferably, the FAR has at least 80% homology to a FAR from Helicoverpa armigera (helicobacterium armigera) or a FAR from Heliothis subflexa (ground cherry moth).

[0089] Such yeast cells are suitable for the production of hydrophobic compounds as defined herein, in particular unsaturated fatty alcohols, fatty acyl acetates and fatty aldehydes, and are described in detail in WO 2016 / 207339.

[0090] In some embodiments, the microorganism is a Yarrowia cell, e.g., a yeast cell such as Yarrowia lipolytica (Yeast) capable of producing the unsaturated fatty alcohol, wherein the yeast cell expresses: at least one heterologous desaturase capable of introducing at least one double bond into a fatty acyl-CoA having a carbon chain length of −14; and - at least one heterologous fatty acyl-CoA reductase (FAR) capable of converting at least a portion of said unsaturated fatty acyl-CoAs into unsaturated fatty alcohols.

[0091] Preferably, in such embodiments, the desaturase has a higher specificity for tetradecanoyl-CoA than for hexadecanoyl-CoA and / or the fatty acyl-CoA reductase has a higher specificity for unsaturated tetradecanoyl-CoA than for unsaturated hexadecanoyl-CoA. Such yeast cells are suitable for the production of unsaturated fatty alcohols with a chain length of 14 carbons and are described in detail in WO 2018 / 109167.

[0092] In such embodiments, the at least one heterologous desaturase may be from an organism selected from Pelargonium holtorum (geranium), Ricinus communis (castor bean), Drosophila melanogaster (Drosophila melanogaster), Spodoptera litura (spodoptera litura) and Tribolium castanium (red flour beetle); preferably the desaturase is from Drosophila melanogaster (Drosophila melanogaster); and preferably the at least one heterologous desaturase is selected from the group consisting of: i) a Δ9 desaturase having at least 60% homology to the Δ9 desaturase from Drosophila melanogaster (Drosophila melanogaster) set forth in SEQ ID NO: 16; ii) a Δ9 desaturase having at least 60% homology to the Δ9 desaturase from Spodoptera litura (common cutworm) set forth in SEQ ID NO: 18; iii) a desaturase having at least 60% homology to the desaturase from Lobesia botlana (grass leaf moth) set forth in SEQ ID NO: 43; iv) a desaturase having at least 60% identity to the desaturase from Drosophila grimshawii (Hawaiian fruit fly) set forth in SEQ ID NO: 44; v) a desaturase having at least 60% homology to the desaturase from Drosophila virilis (fruit fly) set forth in SEQ ID NO: 45; vi) a Δ11 desaturase having at least 60% identity to the Δ11 desaturase from Choristoneura parallela (Lepidoptera: Tortricidae) set forth in SEQ ID NO: 42; vii) A Δ11 desaturase having at least 60% homology to the Δ11 desaturase from Choristoneura rosacea (lotus band moth) set forth in SEQ ID NO:35.

[0093] A desaturase having at least 60% homology to a given desaturase has at least 60% homology, such as at least 61% homology, for example at least 62% homology, such as at least 63% homology, for example at least 64% homology, such as at least 65% homology, for example at least 66% homology, such as at least 67% homology, for example at least 68% homology, such as at least 69% homology, for example at least 70% homology, such as at least 71% homology, for example at least 72% homology, such as at least 73% homology, for example at least 74% homology, such as at least 75% homology, for example at least 76% homology, such as at least 77% homology, for example at least 78% homology, e.g. The sequence may have at least 79% homology, such as at least 80% homology, for example at least 81% homology, such as at least 82% homology, for example at least 83% homology, such as at least 84% homology, for example at least 85% homology, such as at least 86% homology, for example at least 87% homology, such as at least 88% homology, for example at least 89% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology.

[0094] Fatty acyl reductases are: i) a FAR having at least 80% homology to the FAR from Helicoverpa armigera (Helicoverpa armigera) set forth in SEQ ID NO: 5; ii) a FAR having at least 80% homology with FAR from Helicoverpa assulta (tobacco budworm) as set forth in SEQ ID NO: 7; iii) a FAR having at least 80% homology to the FAR from Heliothis subflexa (the nightshade moth) set forth in SEQ ID NO: 6; and iv) FAR having at least 80% homology with FAR from Bicyclus annina (Satyridae) as set forth in SEQ ID NO: 17; Preferably, the FAR is a FAR having at least 80% homology to the FAR from Helicoverpa armigera (Helicoverpa armigera) set forth in SEQ ID NO:5.

[0095] A FAR having at least 80% homology to a given FAR has at least 80% homology, such as at least 81% homology, for example at least 82% homology, such as at least 83% homology, for example at least 84% homology, such as at least 85% homology, for example at least 86% homology, such as at least 87% homology, for example at least 88% homology, such as at least 89% homology, for example at least 90% homology, such as at least 91% homology, for example at least 92% homology, such as at least 93% homology, for example at least 94% homology, such as at least 95% homology, for example at least 96% homology, such as at least 97% homology, for example at least 98% homology, such as at least 99% homology.

[0096] In some embodiments, the hydrophobic compound is an unsaturated fatty alcohol and the microorganism is a yeast cell capable of producing said unsaturated fatty alcohol, wherein the yeast cell comprises: -having one or more mutations that result in reduced activity of one or more native acyl-CoA oxidases; expressing at least one first group of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing a fatty acyl-CoA, wherein the first group of enzymes is capable of shortening a fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', where X'≦X-2; - expressing at least one heterologous desaturase capable of introducing at least one double bond into said fatty acyl-CoA and / or said truncated fatty acyl-CoA; and expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of said unsaturated fatty acyl-CoAs into unsaturated fatty alcohols.

[0097] Such yeast cells are described in detail in WO 2020 / 169389.

[0098] The native acyl-CoA oxidase and / or heterologous acyl-CoA oxidase can be a peroxisomal acyl-CoA oxidase. In some embodiments, at least one acyl-CoA oxidase of the first group of enzymes is a native acyl-CoA oxidase or a heterologous acyl-CoA oxidase, which may be overexpressed relative to a reference yeast strain that does not express the at least one first group of enzymes. In some embodiments, at least one acyl-CoA oxidase of the first group of enzymes is a heterologous acyl-CoA oxidase. In some embodiments, the at least one first group of enzymes comprises an acyl-CoA oxidase from an organism selected from the genera Yarrowia, Agrotis, Arabidopsis, Aspergillus, Cucurbita, Homo, Penaltrobacter, and Rattus. Preferably, the at least one first group enzyme comprises an acyl-CoA oxidase from Yarrowia lipolytica (alkane-utilizing yeast), Agrotis segetum (turnip moth), Arabidopsis thaliana (Arabidopsis thaliana), Aspergillus nidulans (pseudo-infesting fungus), Cucurbita maxima (chestnut squash), Homo sapiens (human), Penarthrobacter ureafaciens (nylon-eating bacterium), or Rattus norvegicus (brown rat).In certain embodiments, preferably, the at least one acyl-CoA oxidase of the first group of enzymes is selected from the group consisting of Yli_POX1 (SEQ ID NO: 19), Yli_POX2 (SEQ ID NO: 20), Yli_POX3 (SEQ ID NO: 21), Yli_POX4 (SEQ ID NO: 22), Yli_POX5 (SEQ ID NO: 23), Yli_POX6 (SEQ ID NO: 24), Ase_POX (SEQ ID NO: 25), Ath_POX1 (SEQ ID NO: 26), Ath_POX2 (SEQ ID NO: 27), Ani_POX (SEQ ID NO: 28), Cma_POX (SEQ ID NO: 29), Hsa_POX1-2 (SEQ ID NO: 30), Pur_POX (SEQ ID NO: 31), and Rno_POX2 (SEQ ID NO: 32), or a group having at least 60% homology thereto, such as at least 6 5%, such as at least 70%, for example at least 75%, such as at least 80%, for example at least 81%, such as at least 82%, for example at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% homology thereto.

[0099] In some embodiments, the at least one heterologous desaturase is selected from the group consisting of a Δ3 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ7 desaturase, a Δ8 desaturase, a Δ9 desaturase, a Δ10 desaturase, a Δ11 desaturase, a Δ12 desaturase, a Δ13 desaturase and a Δ14 desaturase, and / or the desaturase is from a yeast, such as Saccharomyces or Yarrowia, such as Saccharomyces cerevisiae (budding yeast) or Yarrowia lipolytica (alkane-utilizing yeast), or from Drosophila melanogaster (Drosophila melanogaster), Amielois transitella (walnut moth), Choristoneura roretzi (Chrysanthemum moth), or from a yeast, such as Saccharomyces cerevisiae (budding yeast) or Yarrowia lipolytica (alkane-utilizing yeast), or from a yeast, such as ... and insects from the orders Diptera, Coleoptera, or Lepidoptera, such as Amielois, Choristoneura, Drosophila, Ostrinia, Thaumetpoea, Dendrophilus, Grahorita, Scydia, Epiphias, or Spodoptera, such as Sakeana (lotus band moth), Ostrinia nubilalis (European corn borer), Thaumetpoea piciocampa (pine borer), Dendrophilus punctatus, Grahorita molesta (pear fruit moth), Scydia pomonella (codling moth), Epiphias postificittana (apple leaf moth), Spodoptera littoralis (African armyworm), or Choristoneura parallella (Lepidoptera: Tortricidae). For example, desaturases include Δ Z9 -desaturase, such as Sce_OLE1 (SEQ ID NO: 33), Yli_OLE1 (SEQ ID NO: 34) or Dme_D9 (SEQ ID NO: 16), Δ Z11 -desaturases, such as Atr_D11 (SEQ ID NO: 1), Cro_Z11 (SEQ ID NO: 35), Onu_11 (SEQ ID NO: 36), Tpi_D13 (SEQ ID NO: 37), Δ E9 -desaturases, such as Dpu_E9-14 (SEQ ID NO: 38), Δ Z / E10a desaturase, such as Gmo_CPRQ (SEQ ID NO: 39), or a desaturase, such as Epo_E11 (SEQ ID NO: 40), Sls_ZE11 (SEQ ID NO: 41), Lbo_PPTQ (SEQ ID NO: 43), Dgd9 (SEQ ID NO: 44), Dvd9 (SEQ ID NO: 45) or Cpa_E11 (SEQ ID NO: 42), or at least 60% homology thereto, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, For example a functional variant thereof having at least 83%, such as at least 84%, for example at least 85%, such as at least 86%, for example at least 87%, such as at least 88%, for example at least 89%, such as at least 90%, for example at least 91%, such as at least 92%, for example at least 93%, such as at least 94%, for example at least 95%, such as at least 96%, for example at least 97%, such as at least 98%, for example at least 99% homology thereto.

[0100] In some embodiments, the fatty acyl-CoA reductase is from an insect, such as a Lepidoptera insect, such as a member of the genus Helicoverpa, Heliothis, or Bicyclus, preferably the fatty acyl-CoA reductase is a fatty acyl-CoA reductase specific to Helicoverpa armigera (helicobacterium armigera), Helicoverpa assulta, Heliothis subflexa (ground cherry moth), Bicyclus annina (oriental butterfly), or a functional variant thereof, preferably the fatty acyl-CoA reductase is selected from the group consisting of fatty acyl-CoA reductases having at least 80% homology to Har_FAR (SEQ ID NO: 5), Has_FAR (SEQ ID NO: 7), Ban_FAR (SEQ ID NO: 17), or Hs_FAR (SEQ ID NO: 6).

[0101] The yeast cell producing unsaturated fatty alcohols may further express a fatty acyl synthetase (FAA) such as Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9) or a variant thereof having at least 75% homology to Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology.

[0102] The microorganism can be further modified to express an acetyltransferase, such as a heterologous acetyltransferase (AcT), or to overexpress a native acetyltransferase, which can convert at least a portion of the produced unsaturated fatty alcohols to the corresponding fatty acyl acetate. In some embodiments, the acetyltransferase is Sc_Atf1 (SEQ ID NO: 10) or a variant thereof having at least 75% homology to Sc_Atf1 (SEQ ID NO: 10), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, for example at least 98% homology, such as at least 99% homology, for example 100% homology.

[0103] Unsaturated fatty alcohols can also be converted to the corresponding fatty acyl acetates by chemical conversion, for example, by performing an acetylation reaction using the unsaturated fatty alcohol produced by the cell as a substrate.

[0104] (Z)-11-Hexadecen-1-ol In some embodiments, the hydrophobic compound is (Z)-11-hexadecen-1-ol. In some embodiments, the microorganism is a yeast cell capable of producing (Z)-11-hexadecen-1-ol at a titer of at least 0.2 mg / L. The yeast cell is: a Δ11-desaturase selected from the group consisting of Amielois transitella (walnut moth) Δ11-desaturase (Atr_Δ11; SEQ ID NO: 1), Spodoptera littoralis (African armyworm) Δ11-desaturase (Sl_Δ11; SEQ ID NO: 2), Agrotis segetum (turnip cutworm moth) Δ11-desaturase (As_Δ11; SEQ ID NO: 3), and Trichoprasia ni (nettle looper) Δ11-desaturase (Tni_Δ11; SEQ ID NO: 4), or SEQ ID NO:2), As_Δ11 (SEQ ID NO:3), or Tni_Δ11 (SEQ ID NO:4), and variants thereof having at least 65% homology, such as at least 70% homology, for example at least 71% homology, such as at least 72% homology, for example at least 73% homology, such as at least 74% homology, for example at least 75% homology, such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 95% homology, such as 100% homology, and an alcohol-forming fatty acyl-CoA reductase (FAR) selected from the group consisting of Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), and Has_FAR (SEQ ID NO: 7), or a variant thereof having at least 80% homology with Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), or Has_FAR (SEQ ID NO: 7), such as at least 85% homology, for example at least 90% homology, such as at least 95% homology, for example 100% homology; thereby expressing -Δ11-desaturase capable of converting at least a portion of the hexadecanoyl-CoA to (Z)11-hexadecenoyl-CoA; and -FAR can convert at least a portion of the (Z)11-hexadecenoyl-CoA to (Z)11-hexadecenol. In some embodiments, the yeast cell is a Saccharomyces cerevisiae (budding yeast) cell.

[0105] The yeast cell producing (Z)-11-hexadecen-1-ol may further express a fatty acyl synthetase (FAA) such as Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9), or a variant thereof having at least 75% homology to Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology.

[0106] The microorganism can be further modified to express an acetyltransferase, such as a heterologous acetyltransferase (AcT), or to overexpress a native acetyltransferase, which is capable of converting at least a portion of the (Z)-11-hexadecen-1-ol to (Z)11-hexadecen-1-yl acetate. In some embodiments the acetyltransferase is Sc_Atf1 (SEQ ID NO: 10), or a variant thereof having at least 75% homology to Sc_Atf1 (SEQ ID NO: 10), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology.

[0107] (Z)-11-hexadecen-1-ol can also be converted to the corresponding (Z)11-hexadecen-1-yl acetate by chemical conversion, for example, by performing an acetylation reaction using (Z)11-hexadecen-1-ol produced by the cell as a substrate. Such yeast cells are suitable for the production of hydrophobic compounds as defined herein, in particular unsaturated fatty alcohols, fatty acyl acetates and fatty aldehydes, and are described in detail in WO 2016 / 207339.

[0108] Codremon In some embodiments, the hydrophobic compound is codlemone (E8, E10-dodecadien-1-ol), or one or more of its derivatives E8, E10-dodecadienyl acetate and / or E8, E10-dodecadienal. The yeast cell capable of producing codlemone or one or more derivatives thereof preferably expresses at least one heterologous desaturase capable of introducing one or more double bonds into fatty acyl-CoAs having a carbon chain length of 12, thereby converting said fatty acyl-CoAs to unsaturated fatty acyl-CoAs, at least a portion of which are E8, E10-dodecadienyl coenzyme A (E8, E10-C12:CoA), and further expresses at least one unsaturated fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of said unsaturated fatty acyl-CoAs to unsaturated fatty alcohols, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of said E8, E10-dodecadienicoenzyme A (E8, E10-C12:CoA) to E8, E10-dodecadien-1-ol. Such yeast cells are described in detail in EP Application No. 19218703.7, filed on December 20, 2019, by the same applicant as the present application, entitled "Yeast cells and methods for production of E8, E10-dodecadienyl coenzyme A, codlemone and derivatives thereof." This application describes desaturases and fatty acyl-CoA reductases that are particularly useful for the production of codlemone and its derivatives, in particular in the section entitled "Desaturases" (pp. 12-16 of EP19218703.7) and the section entitled "Fatty acyl-CoA reductases (EC 1.2.1.84)" (pp. 16-20 of EP19218703.7). Codlemone can be further converted to E8, E10-dodecadienyl acetate, which can be performed ex vivo, for example by chemical conversion, as known in the art, or it can be performed in vivo by the action of an acetyltransferase (EC 2.3.1.84) that can convert at least a portion of the E8, E10-dodecadien-1-ol to E8, E10-dodecadienyl acetate, as described in the section entitled "Production of E8, E10-dodecadienyl acetate" (EP 19218703.7, pp. 37-38).It may also be of interest to further convert at least a portion of the E8, E10-dodecadien-1-ol produced by the cells to E8, E10-dodecadienal, which can be achieved by chemical conversion or by further genetic modification of the yeast cells, for example, as described in the section entitled "Production of E8, E10-dodecadienal" (pp. 39-40 of EP19218703.7).

[0109] Methods for producing hydrophobic compounds Disclosed herein is a method for producing a hydrophobic compound, which may be any of the hydrophobic compounds described herein above. The method includes providing a microorganism capable of producing the hydrophobic compound and culturing the microorganism in a medium under conditions that allow production of the hydrophobic compound, the medium preferably containing an extractant at the culture temperature or at room temperature in an amount equal to or greater than the cloud concentration of the extractant measured in aqueous solution. As detailed above, such agents are routinely used in fermentation for foam control, but when used as an antifoaming agent, the agent is used at a concentration lower than the cloud concentration measured in aqueous solution. Preferably, the microorganism is yeast. The extractant is a nonionic surfactant, in particular a nonionic ethoxylated surfactant, such as a fatty alcohol alkoxylate, preferably selected from: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF1300 or Dehipon® 2574, and combinations thereof, or polyethoxylated surfactants such as antifoaming agents, for example polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 A polyethoxylated surfactant selected from antifoam agents, including alcohol-based antifoam agents, and combinations thereof. The method may also further include recovering the hydrophobic compounds from the fermentation broth.

[0110] The methods of the present invention are particularly useful for facilitating the recovery of hydrophobic compounds produced by fermentation of microorganisms capable of producing these compounds, such as any of the microorganisms described above in the section "Microorganisms." The hydrophobic compounds can be any of the compounds described above in the section "Hydrophobic Compounds," particularly fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and / or terpenes, such as terpenoids.

[0111] The inventors have found that when a nonionic surfactant, particularly a nonionic ethoxylated surfactant, preferably a fatty alcohol alkoxylate or polyethoxylated surfactant, such as an antifoam agent, particularly any of the nonionic surfactants and antifoam agents described in the section above, "Nonionic Ethoxylated Surfactants," is included in the culture medium or fermentation broth, preferably at or below its cloud concentration measured in aqueous solution at the cultivation temperature, the nonionic surfactant acts as an in situ extractant and facilitates the recovery of hydrophobic compounds from the fermentation broth. Accordingly, provided herein is a method for producing hydrophobic compounds, such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and / or terpenes, such as terpenoids, by fermentation, the method comprising the steps of providing a microorganism capable of producing the hydrophobic compound, and culturing the microorganism in a medium under conditions that allow for the production of the hydrophobic compound, the medium comprising an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution, the extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C6H4. 16 -C 18Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The polyethoxylated surfactant is selected from antifoam agents including alcohol-based antifoam agents and combinations thereof, and the method optionally further comprises recovering the hydrophobic compound from the fermentation broth. Accordingly, provided herein is a method for producing a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes by fermentation, the method comprising: providing yeast cells capable of producing the hydrophobic compound; and culturing the yeast cells in a medium under conditions allowing production of the hydrophobic compound, the culturing step being carried out at a culture temperature; the medium comprising an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution such as the medium at the culture temperature; the extractant is a nonionic ethoxylated surfactant; and the method optionally further comprises recovering the hydrophobic compound.

[0112] In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, or a fatty aldehyde as described herein. In other embodiments, the hydrophobic compound is a terpene, such as a terpenoid, as described herein. In some embodiments, the hydrophobic compound is a mixture of hydrophobic compounds, such as a fatty alcohol, a fatty acyl acetate, a fatty aldehyde, and / or a terpene, such as a terpenoid, as described herein. In certain embodiments, the hydrophobic compound is an unsaturated fatty alcohol, an unsaturated fatty acyl acetate, or an unsaturated fatty aldehyde as described herein.

[0113] In some embodiments, the nonionic surfactant is a nonionic ethoxylated surfactant, such as a fatty alcohol alkoxylate, preferably selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, or a nonionic polyethoxylated surfactant, such as an antifoaming agent. The antifoaming agent is preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Polyethoxylated surfactants, such as antifoam agents including alcohol-based antifoam agents and combinations thereof.

[0114] In some embodiments, the nonionic surfactant is added in an amount greater than its cloud concentration measured in aqueous solution. In some embodiments, the nonionic ethoxylated surfactant is added in an amount greater than its cloud concentration measured in aqueous solution. In some embodiments, the polyethoxylated surfactant is added in an amount greater than its cloud concentration measured in aqueous solution. In some embodiments, the fatty alcohol alkoxylate is added in an amount greater than its cloud concentration measured in aqueous solution. The cloud concentration may be determined in the medium at room temperature or culture temperature, for example, as described in detail elsewhere herein.

[0115] In some embodiments, the non-ionic surfactant is present in an amount at least 50%, such as at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% greater than its cloud concentration. Preferably, the cloud concentration is determined in the medium, for example at room temperature or culture temperature.

[0116] In some embodiments, the non-ionic surfactant is a non-ionic ethoxylated surfactant that is present at or above its cloud concentration, at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000%.Preferably, the cloud concentration is determined in the medium, for example at room temperature or culture temperature.

[0117] In some embodiments, the non-ionic surfactant is a fatty alcohol alkoxylate present at or above its cloud concentration, at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000%.Preferably, the cloud concentration is determined in the medium, for example at room temperature or culture temperature. In some embodiments, the fatty alcohol alkoxylate is selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof.

[0118] In some embodiments, the nonionic surfactant is a polyethoxylated surfactant, and its cloud concentration is at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% more than its cloud concentration.Preferably, the cloud concentration is determined in culture medium, for example at room temperature or culture temperature.In some embodiments, the polyethoxylated surfactant is polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The antifoaming agent is selected from the group consisting of alcohol-based antifoaming agents and combinations thereof.

[0119] In some embodiments, the amount of nonionic surfactant (extractant) is at least 2 times its cloud concentration, for example, at least 3 times its cloud concentration, for example, at least 4 times its cloud concentration, for example, at least 5 times its cloud concentration, for example, at least 6 times its cloud concentration, for example, at least 7 times its cloud concentration, for example, at least 8 times its cloud concentration, for example, at least 9 times its cloud concentration, for example, at least 10 times its cloud concentration, for example, at least 12.5 times its cloud concentration, for example, at least 15 times its cloud concentration, for example, at least 17.5 times its cloud concentration, for example, at least 20 times its cloud concentration, for example, at least 25 times its cloud concentration, for example, at least 30 times its cloud concentration.In some embodiments, the polyethoxylated surfactant is polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Antifoaming agents are selected from alcohol-based antifoaming agents, as well as combinations thereof. Preferably, the cloud concentration is determined in the medium, for example, at room temperature or incubation temperature.

[0120] In some embodiments, the amount of nonionic ethoxylated surfactant (extractant) is at least 2 times its cloud concentration, for example, at least 3 times its cloud concentration, for example, at least 4 times its cloud concentration, for example, at least 5 times its cloud concentration, for example, at least 6 times its cloud concentration, for example, at least 7 times its cloud concentration, for example, at least 8 times its cloud concentration, for example, at least 9 times its cloud concentration, for example, at least 10 times its cloud concentration, for example, at least 12.5 times its cloud concentration, for example, at least 15 times its cloud concentration, for example, at least 17.5 times its cloud concentration, for example, at least 20 times its cloud concentration, for example, at least 25 times its cloud concentration, for example, at least 30 times its cloud concentration.In some embodiments, the ethoxylated surfactant is fatty alcohol alkoxylate.Preferably, cloud concentration is determined in culture medium, for example, at room temperature or culture temperature.

[0121] In some embodiments, nonionic surfactant is polyethoxylated surfactant, and the amount of polyethoxylated surfactant (extractant) is at least 2 times its cloud concentration, for example, at least 3 times its cloud concentration, for example, at least 4 times its cloud concentration, for example, at least 5 times its cloud concentration, for example, at least 6 times its cloud concentration, for example, at least 7 times its cloud concentration, for example, at least 8 times its cloud concentration, for example, at least 9 times its cloud concentration, for example, at least 10 times its cloud concentration, for example, at least 12.5 times its cloud concentration, for example, at least 15 times its cloud concentration, for example, at least 17.5 times its cloud concentration, for example, at least 20 times its cloud concentration, for example, at least 25 times its cloud concentration, for example, at least 30 times its cloud concentration.In some embodiments, polyethoxylated surfactant is polyethylene polypropylene glycol, mixture of polyether dispersion, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Antifoaming agents are selected from alcohol-based antifoaming agents, as well as combinations thereof. Preferably, the cloud concentration is determined in the medium, for example, at room temperature or incubation temperature.

[0122] In some embodiments, the nonionic surfactant is a fatty alcohol alkoxylate, and the amount of fatty alcohol alkoxylate (extractant) is at least 2 times the cloud concentration, such as at least 3 times the cloud concentration, for example at least 4 times the cloud concentration, such as at least 5 times the cloud concentration, for example at least 6 times the cloud concentration, such as at least 7 times the cloud concentration, for example at least 8 times the cloud concentration, such as at least 9 times the cloud concentration, for example at least 10 times the cloud concentration, such as at least 12.5 times the cloud concentration, for example at least 15 times the cloud concentration, such as at least 17.5 times the cloud concentration, for example at least 20 times the cloud concentration, for example at least 25 times the cloud concentration, for example at least 30 times the cloud concentration. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. Preferably, the cloud concentration is determined in the medium, for example, at room temperature or culture temperature.

[0123] In some embodiments, the medium comprises at least 1% vol / vol of extractant, for example at least 1.5%, for example at least 2%, for example at least 2.5%, for example at least 3%, for example at least 3.5%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10%, for example at least 12.5%, for example at least 15%, for example at least 17.5%, for example at least 20%, for example at least 22.5%, for example at least 25%, for example at least 27.5%, for example at least 30% vol / vol of extractant, wherein the extractant is a non-ionic surfactant, preferably a non-ionic ethoxylated surfactant, for example a fatty alcohol alkoxylate or a non-ionic polyethoxylated surfactant. In some embodiments, the non-ionic surfactant is a polyethoxylated surfactant, for example polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agent is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof.

[0124] In some embodiments, the nonionic ethoxylated surfactant is an ethoxylated and propoxylated C 16 -C 18Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, e.g., C 16 -C 18 Alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0). 16 -C 18 The cloud concentration of alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0) is about 1% vol / vol at room temperature. Therefore, when this antifoaming agent is used, the medium preferably contains at least 1% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates propoxylates, for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, such as at least 8%, for example at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, such as at least 20%, for example at least 22.5%, such as at least 25%, for example at least 27.5%, such as at least 30% vol / vol or more of C 16 -C 18 Includes alkyl alcohol ethoxylates and propoxylates.

[0125] In some embodiments, the nonionic ethoxylated surfactant is a polyethylene polypropylene glycol, such as Kolliphor® P407 (CAS No. 9003-11-6), also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). Kolliphor® P407 has a cloud concentration of 10% at temperatures above 100° C. Thus, when polyethylene polypropylene glycol such as Kolliphor® P407 is used the culture medium preferably comprises at least 10% vol / vol of polyethylene polypropylene glycol such as Kolliphor® P407, such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol such as Kolliphor® P407.

[0126] In some embodiments, the nonionic ethoxylated surfactant is a mixture of polyether dispersions such as Antifoam 204 (Sigma Aldrich product numbers A6426 or A8311). Antifoam 204 has a cloud concentration of 1% in aqueous solution at temperatures between 18.0 and 21.0°C. Thus, when a mixture of polyether dispersions such as Antifoam 204 is used, the medium preferably comprises at least 1% vol / vol of the mixture of polyether dispersions such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of the mixture of polyether dispersions such as Antifoam 204.

[0127] In some embodiments, the nonionic ethoxylated surfactant is Agnique BP420 (CAS No. 68002-96-0). The cloud concentration of Agnique BP420 (CAS No. 68002-96-0) is 1% in aqueous solution at a temperature of 18.0 to 21.0°C. Thus, when a mixture of polyether dispersions such as Antifoam 204 is used, the medium preferably contains at least 1% vol / vol Agnique BP420 (CAS No. 68002-96-0), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Agnique BP420 (CAS No. 68002-96-0).

[0128] In some embodiments, the nonionic ethoxylated surfactant is an antifoaming agent, comprising polyethylene glycol monostearate or simethicone.Simethicone comprises polyethylene glycol monostearate, which, without being bound by theory, appears to be an important compound due to the ability of simethicone to act as an extractant.Polyethylene glycol monostearate has a cloud point of 1% in aqueous solution at 5°C. Thus, when a surfactant comprising polyethylene glycol monostearate is used, the medium preferably comprises at least 1% vol / vol polyethylene glycol monostearate or simethicone, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more polyethylene glycol monostearate or simethicone.

[0129] In some embodiments, the nonionic surfactant is a fatty alcohol alkoxylate such as Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (CAS No. 68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574. The cloud concentration of these surfactants is about 1% vol / vol at room temperature.Thus, Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (CAS number 68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2) or Imbentin When SG / 251 (CAS No. 68002-96-0) is used, the medium preferably contains at least 1% vol / vol of Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (CAS No. 68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, such as at least 8%, for example at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, for example at least 20%, for example at least 22.5%, such as at least 25%, for example at least 27.5%, such as at least 30% vol / vol of or more Plurafac® SLF300 (CAS No. 196823-11-7), Plurafac® LF1300 (CAS No. 68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0).

[0130] The fermentation itself can be carried out as known in the art. In some embodiments, the fermentation is carried out in a bioreactor. The fermentation is carried out under conditions that allow the microorganisms present in the fermentation to produce the hydrophobic compound of interest.

[0131] The addition of an extractant, i.e., a nonionic ethoxylated surfactant, preferably a fatty alcohol alkoxylate or polyethoxylated surfactant such as any of the antifoam agents described herein, results in the formation of an emulsion in the fermentation broth, and the hydrophobic compounds produced by the microorganism, preferably yeast cells, are present in the emulsion. Therefore, the method may also include a step of breaking the emulsion and recovering a product phase containing the extractant and hydrophobic compounds. Once the emulsion is broken, the fermentation broth separates into three phases: an aqueous phase containing primarily water and aqueous compounds, a phase containing cells and necrotic cell debris, and a product phase containing primarily the extractant and hydrophobic compounds. Thus, the resulting composition consists of three phases. In a preferred embodiment, most of the hydrophobic compounds in the fermentation broth are present in the product phase. For example, at least 50% of the hydrophobic compounds are present in the product phase, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, such as 100% of the hydrophobic compounds are present in the product phase. In some embodiments, the product phase comprises at least 50% of the hydrophobic compounds initially present in the fermentation broth, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, for example at least 75%, such as at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, such as at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, such as 100% of the hydrophobic compounds initially present in the fermentation broth.

[0132] Breaking the emulsion can be carried out as known in the art, for example, by subjecting the emulsion to a phase separation step known in the art. In some embodiments, the phase separation step is, for example, centrifugation at 10,000 g for 5 minutes. In some embodiments, the centrifugation is carried out for 1 minute or more, for example, 2 minutes or more, for example, 3 minutes or more, for example, 4 minutes or more, for example, 5 minutes or more, for example, 6 minutes or more, for example, 7 minutes or more, for example, 8 minutes or more, for example, 9 minutes or more, for example, 10 minutes or more. In some embodiments, centrifugation is carried out at 3000g or more, such as 4000g or more, for example 5000g or more, such as 6000g or more, for example 7000g or more, such as 8000g or more, for example 9000g or more, such as 10000g or more, for example 11000g or more, such as 12000g or more, for example 13000g or more, such as 14000g or more, for example 15000g or more, such as 17500g or more, for example 20000g or more.

[0133] After the emulsion-breaking step, a product phase comprising the extractant and the hydrophobic compounds can be recovered from the composition. In such embodiments, the method can further comprise separating the hydrophobic compounds from the extractant. This can be accomplished by methods known in the art, such as distillation, e.g., distillation under reduced pressure, or column purification. The extractant can be recycled, e.g., recycled back into the fermentation.

[0134] In some embodiments, the method includes culturing a microorganism capable of producing a fatty alcohol, such as an unsaturated fatty alcohol or a mixture of (saturated and / or unsaturated) fatty alcohols. In some embodiments, the method includes culturing a yeast cell capable of producing a fatty alcohol, such as an unsaturated fatty alcohol or a mixture of (saturated and / or unsaturated) fatty alcohols. The unsaturated fatty alcohol may be recovered as described above. In such embodiments, the method may further include recovering the produced fatty alcohol and chemically converting at least a portion thereof to the corresponding fatty acyl acetate and / or corresponding fatty aldehyde. The term "corresponding" as used herein refers to a compound, fatty acyl acetate, or fatty aldehyde having the same carbon chain length and double bond position as the fatty alcohol from which it is derived.

[0135] Therefore, when a microorganism such as a yeast cell produces a fatty alcohol, the method may further include, for example, recovering the fatty alcohol as described above and chemically converting at least a portion of the fatty alcohol to the corresponding fatty acyl acetate. This can be done by performing an acetylation reaction as known in the art, for example, as described in Fritz et al., 1959, or Mattson et al., 1964. The method may additionally or alternatively include chemically converting at least a portion of the fatty alcohol to the corresponding fatty aldehyde. This can be done by performing an oxidation reaction as known in the art, for example, as described in Steves et al., 2013. The resulting fatty acyl acetate and / or fatty aldehyde can then be recovered.

[0136] For example, acetylation can be carried out with acetic anhydride using pyridine as a catalyst. The resulting acetate is then extracted from the reaction mixture with an organic solvent, which is removed by evaporation.

[0137] The oxidation can be carried out using known procedures for the oxidation of primary alcohols, including, but not limited to, those published by Hoover et al., 2011, using the tetrakisacetonitrile copper(I) triflate / TEMPO catalyst system, Omura et al. (1978), Corey et al. (1972), Ratcliffe et al. (1970), Ley et al. (1994), or Anelli et al. (1987). The resulting fatty aldehyde is then extracted from the reaction mixture with an organic solvent, the solvent can be removed by evaporation, and the aldehyde is purified using distillation or column chromatography.

[0138] Method for increasing the titer of hydrophobic compounds in fermentation Disclosed herein is a method for increasing the titer of a hydrophobic compound in a fermentation. The method comprises culturing a microorganism capable of producing the hydrophobic compound in a medium under conditions that allow the production of the hydrophobic compound, the medium comprising an extractant in an amount equal to or greater than its cloud concentration measured in an aqueous solution. Preferably, the microorganism is a yeast cell. Preferably, the cloud concentration is determined at room temperature or at the incubation temperature. The extractant is preferably Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2), or Imbentin SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, antifoaming agents such as polyethoxylated surfactants, for example polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The nonionic surfactant, particularly a nonionic ethoxylated surfactant, is selected from a polyethoxylated nonionic surfactant, preferably selected from a fatty alcohol alkoxylate, selected from antifoam agents including alcohol-based antifoam agents and combinations thereof. The method may also further comprise recovering the hydrophobic compound from the fermentation broth.

[0139] The methods of the present invention are particularly useful for increasing the titer of hydrophobic compounds produced by fermentation of microorganisms capable of producing these compounds, such as yeast cells, for example, any of the microorganisms described above in the section "Microorganisms." The hydrophobic compounds can be any of the compounds described above in the section "Hydrophobic Compounds," particularly fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes, such as terpenoids. The presence in the culture medium of a non-ionic surfactant, in particular a non-ionic ethoxylated surfactant, preferably selected from fatty alcohol alkoxylates such as Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2) or Imbentin SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, or an antifoaming agent, in particular a polyethoxylated surfactant, results in an increase in the titer of hydrophobic compounds compared to the titer obtained in a fermentation carried out under similar conditions but with an amount of non-ionic surfactant lower than the cloud concentration. Thus, the method is useful for increasing the titer of hydrophobic compounds compared to fermentations carried out under the same conditions but in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution at culture temperature or room temperature.

[0140] In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, or a fatty aldehyde as described herein.In other embodiments, the hydrophobic compound is a terpene, such as a terpenoid, as described herein.In some embodiments, the hydrophobic compound is a mixture of hydrophobic compounds, such as a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, a fatty aldehyde, and a terpene, such as a terpenoid, as described herein.In certain embodiments, the hydrophobic compound is an unsaturated fatty alcohol, an unsaturated fatty alcohol ester, an unsaturated fatty acyl acetate, or an unsaturated fatty aldehyde as described herein.

[0141] The nonionic surfactants are preferably nonionic ethoxylated surfactants or fatty alcohol alkoxylates, preferably selected from Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2) or Imbentin SG / 251 (CAS number 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, or mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agent is selected from antifoaming agents including alcohol-based antifoaming agents or combinations thereof, such as polyethoxylated surfactants.

[0142] In some embodiments, the nonionic surfactant or nonionic ethoxylated surfactant is added in an amount greater than its cloud concentration measured in aqueous solution, preferably at room temperature or incubation temperature. In some embodiments, the nonionic ethoxylated surfactant, preferably a fatty alcohol alkoxylate or polyethoxylated surfactant, is added in an amount greater than its cloud concentration measured in aqueous solution, preferably at room temperature or incubation temperature.

[0143] In some embodiments, the non-ionic surfactant is present in an amount at least 50%, such as at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% greater than its cloud concentration. Cloud concentration may be determined in culture medium, for example at room temperature or culture temperature.

[0144] In some embodiments, nonionic surfactant is antifoaming agent such as polyethoxylated surfactant.Polyethoxylated surfactant is therefore preferably present in an amount at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% greater than its cloud concentration.In some embodiments, polyethoxylated surfactant is polyethylene polypropylene glycol, mixture of polyether dispersion, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18Antifoaming agents are selected from alcohol-based antifoaming agents, including combinations thereof. Cloud concentration may be determined in the medium, for example, at room temperature or incubation temperature.

[0145] In some embodiments, the nonionic surfactant is a fatty alcohol alkoxylate such as Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574. The fatty alcohol alkoxylate is therefore preferably present in an amount at least 50%, such as at least 100%, for example at least 150%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such as at least 400%, for example at least 500%, for example at least 750%, such as at least 1000% greater than its cloud concentration or more. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. The cloud concentration may be determined in the medium, for example, at room temperature or culture temperature.

[0146] In some embodiments, the amount of non-ionic surfactant (extractant) is at least 2 times the cloud concentration, for example, at least 3 times the cloud concentration, for example, at least 4 times the cloud concentration, for example, at least 5 times the cloud concentration, for example, at least 6 times the cloud concentration, for example, at least 7 times the cloud concentration, for example, at least 8 times the cloud concentration, for example, at least 9 times the cloud concentration, for example, at least 10 times the cloud concentration, for example, at least 12.5 times the cloud concentration, for example, at least 15 times the cloud concentration, for example, at least 17.5 times the cloud concentration, for example, at least 20 times the cloud concentration, for example, at least 25 times the cloud concentration, for example, at least 30 times the cloud concentration. The cloud concentration may be determined in the medium, for example, at room temperature or culture temperature.

[0147] In some embodiments, nonionic surfactant is polyethoxylated surfactant.In some embodiments, the amount of polyethoxylated surfactant (extractant) is at least 2 times its cloud concentration, for example, at least 3 times its cloud concentration, for example, at least 4 times its cloud concentration, for example, at least 5 times its cloud concentration, for example, at least 6 times its cloud concentration, for example, at least 7 times its cloud concentration, for example, at least 8 times its cloud concentration, for example, at least 9 times its cloud concentration, for example, at least 10 times its cloud concentration, for example, at least 12.5 times its cloud concentration, for example, at least 15 times its cloud concentration, for example, at least 17.5 times its cloud concentration, for example, at least 20 times its cloud concentration, for example, at least 25 times its cloud concentration, for example, at least 30 times its cloud concentration.In some embodiments, polyethoxylated surfactant is polyethylene polypropylene glycol, mixture of polyether dispersion, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18Antifoaming agents are selected from alcohol-based antifoaming agents, including combinations thereof. Cloud concentration may be determined in the medium, for example, at room temperature or incubation temperature.

[0148] In some embodiments, nonionic surfactant is fatty alcohol alkoxylate.In some embodiments, the amount of fatty alcohol alkoxylate (extractant) is at least 2 times its cloud concentration, for example at least 3 times its cloud concentration, for example at least 4 times its cloud concentration, for example at least 5 times its cloud concentration, for example at least 6 times its cloud concentration, for example at least 7 times its cloud concentration, for example at least 8 times its cloud concentration, for example at least 9 times its cloud concentration, for example at least 10 times its cloud concentration, for example at least 12.5 times its cloud concentration, for example at least 15 times its cloud concentration, for example at least 17.5 times its cloud concentration, for example at least 20 times its cloud concentration, for example at least 25 times its cloud concentration, for example at least 30 times its cloud concentration. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. The cloud concentration may be determined in the medium, for example, at room temperature or culture temperature.

[0149] In some embodiments, the medium comprises at least 1% vol / vol of extractant, for example at least 1.5%, for example at least 2%, for example at least 2.5%, for example at least 3%, for example at least 3.5%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10%, for example at least 12.5%, for example at least 15%, for example at least 17.5%, for example at least 20%, for example at least 22.5%, for example at least 25%, for example at least 27.5%, for example at least 30% vol / vol of extractant, and the extractant is a non-ionic surfactant.In some embodiments, the non-ionic surfactant is a non-ionic ethoxylated surfactant, such as a polyethoxylated surfactant or a fatty alcohol alkoxylate.In some embodiments, the polyethoxylated surfactant is polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agent is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof.

[0150] In some embodiments, the nonionic surfactant is an antifoaming agent. In some embodiments, the antifoaming agent is an ethoxylated and propoxylated C 16-C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, e.g. C 16 -C 18 The cloud concentration of C16-C18 alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0) is about 1% vol / vol at room temperature. Therefore, when this antifoaming agent is used, the medium preferably contains at least 1% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates propoxylates, for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, such as at least 8%, for example at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, such as at least 20%, for example at least 22.5%, such as at least 25%, for example at least 27.5%, such as at least 30% vol / vol or more of C 16 -C 18 Includes alkyl alcohol ethoxylates and propoxylates.

[0151] In some embodiments, the antifoaming agent is a polyethylene polypropylene glycol, such as Kolliphor® P407 (CAS No. 9003-11-6), also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). Kolliphor® P407 has a cloud concentration of 10% at temperatures above 100° C. Thus, when polyethylene polypropylene glycol such as Kolliphor® P407 is used the culture medium preferably comprises at least 10% vol / vol of polyethylene polypropylene glycol such as Kolliphor® P407, such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol such as Kolliphor® P407.

[0152] In some embodiments, the antifoaming agent is a mixture of polyether dispersions such as Antifoam 204 (Sigma Aldrich product numbers A6426 or A8311). The cloud concentration of Antifoam 204 is 1% in aqueous solution at temperatures between 18.0 and 21.0°C. Thus, when a mixture of polyether dispersions such as Antifoam 204 is used, the medium preferably comprises at least 1% vol / vol of the mixture of polyether dispersions such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of the mixture of polyether dispersions such as Antifoam 204.

[0153] In some embodiments, the antifoaming agent is Agnique BP420 (CAS No. 68002-96-0), which has a cloud concentration of 1% in an aqueous solution at a temperature of 18.0 to 21.0°C. Thus, when Agnique BP420 (CAS No. 68002-96-0) is used, the medium preferably comprises at least 1% vol / vol of a mixture of a polyether dispersion such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of Agnique BP420 (CAS No. 68002-96-0).

[0154] In some embodiments, the antifoaming agent comprises polyethylene glycol monostearate or simethicone.Simethicone comprises polyethylene glycol monostearate, which, without being bound by theory, appears to be an important compound due to simethicone's ability to act as an extractant.Polyethylene glycol monostearate has a cloud point of 1% in aqueous solution at 5°C. Thus, when a surfactant comprising polyethylene glycol monostearate is used, the medium preferably comprises at least 1% vol / vol polyethylene glycol monostearate or simethicone, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more polyethylene glycol monostearate or simethicone.

[0155] In some embodiments, the extractant is a fatty alcohol alkoxylate, preferably selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. These fatty alcohol alkoxylates have a cloud point of 1% in aqueous solution at room temperature.Thus, preferably Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, when a fatty alcohol alkoxylate is used, the medium preferably contains at least 1% vol / vol of said fatty alcohol alkoxylate, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, for example at least 9%, For example, it comprises at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol of fatty alcohol alkoxylate, preferably selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof.

[0156] The microorganisms used in the present method, preferably yeast cells, may already be genetically modified or selected to produce high titers of hydrophobic compounds. The method of the present invention can further increase the titer. In some embodiments, the titer of the hydrophobic compound is increased by at least 5%, such as at least 10%, for example at least 15%, for example at least 20%, for example at least 25%, for example at least 30%, for example at least 35%, for example at least 40%, for example at least 45%, for example at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, for example at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the titer obtained in fermentation carried out under similar conditions in the absence of extractant or in the presence of an amount of extractant lower than its cloud concentration in aqueous solution. The term "similar conditions" as used herein refers to fermentation of the same microorganism or yeast cell under the same conditions but in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution at culture temperature or room temperature. In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, and / or a fatty aldehyde. In some embodiments, the hydrophobic compound is a terpene, such as a terpenoid.

[0157] Therefore, when a microorganism such as a yeast cell produces fatty alcohols, the method may further include recovering the fatty alcohols and chemically converting at least a portion of the fatty alcohols to the corresponding fatty acyl acetates, for example, as described above. This can be done by performing an acetylation reaction known in the art, for example, as described in Fritz et al., 1959, or Mattson et al., 1964. The method may additionally or alternatively include chemically converting at least a portion of the fatty alcohols to the corresponding fatty aldehydes, for example, by performing an oxidation reaction known in the art, for example, as described in Steves et al., 2013. The resulting fatty acyl acetates and / or fatty aldehydes can then be recovered.

[0158] Acetylation can be carried out with acetic anhydride using pyridine as a catalyst. The resulting acetate is then extracted from the reaction mixture with an organic solvent, which is removed by evaporation.

[0159] The oxidation can be carried out using known procedures for the oxidation of primary alcohols, including, but not limited to, those published by Hoover et al., 2011, using the tetrakisacetonitrile copper(I) triflate / TEMPO catalyst system, Omura et al. (1978), Corey et al. (1972), Ratcliffe et al. (1970), Ley et al. (1994), or Anelli et al. (1987). The resulting fatty aldehyde can then be extracted from the reaction mixture with an organic solvent, the solvent removed by evaporation, and the aldehyde purified using distillation or column chromatography.

[0160] Method for enhancing secretion of hydrophobic compounds in fermentation - Patent Application 20070122997 Disclosed herein is a method for enhancing secretion of hydrophobic compounds during fermentation. The method comprises culturing a microorganism capable of producing the hydrophobic compound in a medium under conditions that allow production of the hydrophobic compound, the medium comprising an extractant in an amount equal to or greater than the cloud concentration of the hydrophobic compound measured in aqueous solution. The method thus preferably comprises culturing yeast cells in a medium under conditions that allow production of the hydrophobic compound, the culturing step being carried out at a culture temperature, the medium comprising an extractant in an amount equal to or greater than the cloud concentration of the hydrophobic compound measured in aqueous solution at the culture temperature, the extractant being a non-ionic ethoxylated surfactant, thereby enhancing secretion of the hydrophobic compound from the yeast cells compared to fermentation carried out under the same conditions but in the absence of the extractant or in the presence of an extractant in an amount less than the cloud concentration of the hydrophobic compound in aqueous solution at the culture temperature. The extractant is a non-ionic surfactant, preferably a fatty alcohol alkoxylate, preferably selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin. SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, or nonionic ethoxylated surfactants such as antifoaming agents, for example Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The polyethoxylated surfactant is selected from antifoaming agents, including alcohol-based antifoaming agents, and combinations thereof. The method may also further include recovering the hydrophobic compound from the fermentation broth. The method thus results in increased secretion compared to fermentation carried out under similar conditions in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution. The term "similar conditions" as used herein refers to fermentation of the same microorganism or yeast cell under the same conditions, but in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution at culture temperature or room temperature.

[0161] The method of the present invention is particularly useful for enhancing the secretion of hydrophobic compounds produced by fermentation of microorganisms capable of producing these compounds, preferably yeast cells, or any of the microorganisms described in the "Microorganisms" section above. The hydrophobic compound can be any of the compounds described in the "Hydrophobic Compounds" section above, particularly fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes, such as terpenoids. The presence of a nonionic surfactant, particularly a nonionic ethoxylated surfactant such as an antifoaming agent, preferably a fatty alcohol alkoxylate or polyethoxylated surfactant, in the medium results in increased secretion of hydrophobic compounds from the microorganism compared to the secretion observed in fermentation carried out under similar conditions but with an amount of nonionic surfactant lower than the cloud concentration.

[0162] In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, or a fatty aldehyde as described herein. In other embodiments, the hydrophobic compound is a terpene, such as a terpenoid, as described herein. In some embodiments, the hydrophobic compound is a mixture of hydrophobic compounds, such as a fatty alcohol, a fatty acyl acetate, a fatty aldehyde, and / or a terpene, such as a terpenoid, as described herein. In certain embodiments, the hydrophobic compound is an unsaturated fatty alcohol, an unsaturated fatty acyl acetate, or an unsaturated fatty aldehyde as described herein.

[0163] The nonionic surfactant is a nonionic ethoxylated surfactant, which may be an antifoaming agent. The antifoaming agent is preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Polyethoxylated nonionic surfactants, such as antifoam agents including alcohol-based antifoam agents and combinations thereof.

[0164] In some embodiments, the nonionic surfactant is preferably a nonionic ethoxylated surfactant selected from fatty alcohol alkoxylates and polyethoxylated surfactants, and is added in an amount greater than its cloud concentration measured in aqueous solution.In some embodiments, the nonionic ethoxylated surfactant is a polyethoxylated surfactant, and is added in an amount greater than its cloud concentration measured in aqueous solution.Cloud concentration can be determined in a medium, for example, at room temperature or culture temperature.

[0165] In some embodiments, the non-ionic surfactant is a non-ionic ethoxylated surfactant that is present at or above its cloud concentration at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000%.Cloud concentration can be determined in culture medium, for example at room temperature or culture temperature.

[0166] In some embodiments, the nonionic surfactant is preferably a nonionic ethoxylated surfactant selected from fatty alcohol alkoxylates and polyethoxylated surfactants, and is present in an amount at least 50%, for example at least 100%, for example at least 150%, for example at least 200%, for example at least 250%, for example at least 300%, for example at least 350%, for example at least 400%, for example at least 500%, for example at least 750%, for example at least 1000% greater than the cloud concentration. In some embodiments, the polyethoxylated surfactant is selected from Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agent is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. The cloud concentration can be determined in the medium, for example, at room temperature or culture temperature.

[0167] In some embodiments, the amount of nonionic ethoxylated surfactant is at least 2 times its cloud concentration, for example, at least 3 times its cloud concentration, for example, at least 4 times its cloud concentration, for example, at least 5 times its cloud concentration, for example, at least 6 times its cloud concentration, for example, at least 7 times its cloud concentration, for example, at least 8 times its cloud concentration, for example, at least 9 times its cloud concentration, for example, at least 10 times its cloud concentration, for example, at least 12.5 times its cloud concentration, for example, at least 15 times its cloud concentration, for example, at least 17.5 times its cloud concentration, for example, at least 20 times its cloud concentration, for example, at least 25 times its cloud concentration, for example, at least 30 times its cloud concentration.Cloud concentration can be determined in culture medium, for example, at room temperature or culture temperature.

[0168] In some embodiments, nonionic surfactant is polyethoxylated surfactant.In some embodiments, the amount of polyethoxylated surfactant (extractant) is at least 2 times its cloud concentration, for example at least 3 times its cloud concentration, for example at least 4 times its cloud concentration, for example at least 5 times its cloud concentration, for example at least 6 times its cloud concentration, for example at least 7 times its cloud concentration, for example at least 8 times its cloud concentration, for example at least 9 times its cloud concentration, for example at least 10 times its cloud concentration, for example at least 12.5 times its cloud concentration, for example at least 15 times its cloud concentration, for example at least 17.5 times its cloud concentration, for example at least 20 times its cloud concentration, for example at least 25 times its cloud concentration, for example at least 30 times its cloud concentration. In some embodiments, the polyethoxylated surfactants include Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C16 -C 18 Antifoaming agents are selected from alcohol-based antifoaming agents, including combinations thereof. Cloud concentration may be determined in the medium, for example, at room temperature or incubation temperature.

[0169] In some embodiments, nonionic surfactant is fatty alcohol alkoxylate.In some embodiments, the amount of fatty alcohol alkoxylate (extractant) is at least 2 times its cloud concentration, for example at least 3 times its cloud concentration, for example at least 4 times its cloud concentration, for example at least 5 times its cloud concentration, for example at least 6 times its cloud concentration, for example at least 7 times its cloud concentration, for example at least 8 times its cloud concentration, for example at least 9 times its cloud concentration, for example at least 10 times its cloud concentration, for example at least 12.5 times its cloud concentration, for example at least 15 times its cloud concentration, for example at least 17.5 times its cloud concentration, for example at least 20 times its cloud concentration, for example at least 25 times its cloud concentration, for example at least 30 times its cloud concentration. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. The cloud concentration may be determined in the medium, for example, at room temperature or culture temperature.

[0170] In some embodiments, the medium comprises at least 1 vol / vol of extractant, for example at least 1.5%, for example at least 2%, for example at least 2.5%, for example at least 3%, for example at least 3.5%, for example at least 4%, for example at least 5%, for example at least 6%, for example at least 7%, for example at least 8%, for example at least 9%, for example at least 10%, for example at least 12.5%, for example at least 15%, for example at least 17.5%, for example at least 20%, for example at least 22.5%, for example at least 25%, for example at least 27.5%, for example at least 30% vol / vol of extractant, wherein the extractant is a non-ionic ethoxylated surfactant such as a fatty alcohol alkoxylate or a polyethoxylated surfactant. In some embodiments, the polyethoxylated surfactant is Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The antifoaming agent is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof. In some embodiments, the fatty alcohol alkoxylate is selected from the following: Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof. The cloud concentration may be determined in the medium, for example, at room temperature or culture temperature.

[0171] In some embodiments, the nonionic surfactant is an antifoaming agent. In some embodiments, the antifoaming agent is an ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, e.g. C 16 -C 18 Alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0). 16 -C 18 The cloud concentration of alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0) is about 1% vol / vol at room temperature. Therefore, when this antifoaming agent is used, the medium preferably contains at least 1% vol / vol of C 16 -C 18 Alkyl alcohol ethoxylates propoxylates, for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, for example at least 22.5%, such as at least 25%, for example at least 27.5%, for example at least 30% vol / vol of C 16 -C 18 Includes alkyl alcohol ethoxylates and propoxylates.

[0172] In some embodiments, the antifoaming agent is a polyethylene polypropylene glycol, such as Kolliphor® P407 (CAS No. 9003-11-6), also known as poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol). Kolliphor® P407 has a cloud concentration of 10% at temperatures above 100° C. Thus, when polyethylene polypropylene glycol such as Kolliphor® P407 is used the culture medium preferably comprises at least 10% vol / vol of polyethylene polypropylene glycol such as Kolliphor® P407, such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol such as Kolliphor® P407.

[0173] In some embodiments, the nonionic surfactant is an antifoaming agent. In some embodiments, the antifoaming agent is Agnique BP420 (CAS No. 68002-96-0). The cloud concentration of Agnique BP420 (CAS No. 68002-96-0) is about 1% vol / vol at room temperature. Thus, when this antifoaming agent is used, the medium preferably comprises at least 1% vol / vol Agnique BP420 (CAS No. 68002-96-0), such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more Agnique BP420 (CAS No. 68002-96-0).

[0174] In some embodiments, the antifoaming agent is a mixture of polyether dispersions, such as Antifoam 204 (Sigma Aldrich product numbers A6426 or A8311). The cloud concentration of Antifoam 204 is 1% in aqueous solution at temperatures between 18.0 and 21.0°C. Thus, when a mixture of polyether dispersions such as Antifoam 204 is used, the medium preferably comprises at least 1% vol / vol of the mixture of polyether dispersions such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of the mixture of polyether dispersions such as Antifoam 204.

[0175] In some embodiments, the antifoaming agent comprises polyethylene glycol monostearate or simethicone.Simethicone comprises polyethylene glycol monostearate, which, without being bound by theory, appears to be an important compound due to simethicone's ability to act as an extractant.Polyethylene glycol monostearate has a cloud point of 1% in aqueous solution at 5°C. Thus, when a surfactant comprising polyethylene glycol monostearate is used, the medium preferably comprises at least 1% vol / vol polyethylene glycol monostearate or simethicone, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more polyethylene glycol monostearate or simethicone.

[0176] In some embodiments, the extractant is a fatty alcohol alkoxylate preferably selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574. These fatty alcohol alkoxylates have a cloud point of 1% in aqueous solution at room temperature.Thus, preferably Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, when a fatty alcohol alkoxylate is used, the medium preferably contains at least 1% vol / vol of said fatty alcohol alkoxylate, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, for example at least 9%, For example, it comprises at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol of a fatty alcohol alkoxylate, preferably selected from Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2) and Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof.

[0177] The microorganisms used in the method may already be genetically modified or selected to produce hydrophobic compounds. Preferably, the microorganism is a yeast cell. The method of the present invention can further enhance the secretion of hydrophobic compounds. In some embodiments, the secretion of hydrophobic compounds from cells is increased by at least 5%, for example, at least 5%, for example, at least 7.5%, for example, at least 10%, for example, at least 12.5%, for example, at least 15%, for example, at least 20%, for example, at least 25%, for example, at least 30%, for example, at least 35%, for example, at least 36%, for example, at least 37%, for example, at least 38%, for example, at least 39%, for example, at least 40%, or more, compared to the secretion observed in fermentation carried out under similar conditions in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution. The term "similar conditions" as used herein refers to fermentation of the same microorganism or yeast cell under the same conditions, but in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution at culture temperature or room temperature.

[0178] In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, and / or a fatty aldehyde. In some embodiments, the hydrophobic compound is a terpene, such as a terpenoid. Therefore, when a microorganism, particularly a yeast, produces fatty alcohols, the method may further comprise recovering the fatty alcohols and chemically converting at least a portion of the fatty alcohols into the corresponding fatty acyl acetates, for example, as described above. This can be achieved by performing an acetylation reaction known in the art, for example, as described in Fritz et al., 1959, or Mattson et al., 1964. The method may additionally or alternatively comprise chemically converting at least a portion of the fatty alcohols into the corresponding fatty aldehydes, for example, as described in Steves et al., 2013, by performing an oxidation reaction known in the art. The resulting fatty acyl acetates and / or fatty aldehydes can then be recovered. For example, acetylation can be carried out with acetic anhydride using pyridine as a catalyst. The resulting fatty acetate is then extracted from the reaction mixture with an organic solvent, and the solvent is removed by evaporation.

[0179] The oxidation can be carried out using known procedures for the oxidation of primary alcohols, including, but not limited to, those published by Hoover et al., 2011, using the tetrakisacetonitrile copper(I) triflate / TEMPO catalyst system, Omura et al. (1978), Corey et al. (1972), Ratcliffe et al. (1970), Ley et al. (1994), or Anelli et al. (1987). The resulting fatty aldehyde is then extracted from the reaction mixture with an organic solvent, the solvent is removed by evaporation, and the aldehyde is purified using distillation or column chromatography.

[0180] Product phase containing hydrophobic compounds ** Fermentation itself can be carried out as known in the art. In some embodiments, fermentation is carried out in a bioreactor. Fermentation is carried out under conditions that allow the microorganisms present in the fermentation to produce the hydrophobic compound of interest. Suitable conditions for the production of hydrophobic compounds such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and / or terpenes such as terpenoids by yeast cells are readily available to those skilled in the art. Suitable microorganisms, particularly yeast cells, are known in the art; for example, such yeast cells are described in WO 2016 / 207339, WO 2018 / 109163, WO 2018 / 109167, International Application No. PCT / EP2020 / 053306, and European Patent Application No. 19218703.7, and are also described herein.

[0181] Addition of an extractant, i.e., a nonionic surfactant, particularly a nonionic ethoxylated surfactant preferably selected from fatty alcohol alkoxylates or polyethoxylated surfactants, such as any of the nonionic surfactants, nonionic ethoxylated surfactants, antifoaming agents, or polyethoxylated surfactants described herein, results in the formation of an emulsion in the fermentation broth, and the hydrophobic compounds produced by the microorganisms are present in the emulsion. Similarly, addition of such surfactants, for example at incubation temperature or room temperature, at a concentration equal to or greater than their cloud concentration measured in aqueous solution, to a fermentation in which the microorganism is a yeast cell, also results in the formation of an emulsion in the fermentation broth, which contains the hydrophobic compounds. The method may also include a step of breaking the emulsion and recovering a product phase containing the extractant and the hydrophobic compounds. Once the emulsion is broken, the fermentation broth separates into three phases: an aqueous phase containing primarily water and aqueous compounds, a phase containing cells and necrotic cell debris, and a product phase containing primarily the extractant and hydrophobic compounds. Thus, a composition consisting of three phases is obtained.

[0182] Thus, in some embodiments, the method is for the production of a hydrophobic compound and comprises the steps of providing a microorganism, preferably a yeast cell, capable of producing said hydrophobic compound, and culturing said microorganism in a medium under conditions allowing the production of said hydrophobic compound, said medium being preferably selected from the group consisting of non-ionic surfactants, more particularly preferably Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin Nonionic ethoxylated surfactants, preferably selected from fatty alcohol alkoxylates, selected from SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, and Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 and a polyethoxylated surfactant, such as an antifoaming agent selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, preferably in an amount equal to or greater than its cloud concentration measured in aqueous solution at the culture temperature or room temperature, whereby a three-phase composition is obtained in the fermentation broth, the method further comprising recovering the product phase.

[0183] In some embodiments, a method is for increasing the titer of a hydrophobic compound in a fermentation as described herein, comprising the steps of providing a microorganism, preferably a yeast cell, capable of producing said hydrophobic compound, and culturing said microorganism or yeast cell in a medium under conditions that allow the production of said hydrophobic compound, wherein the medium is preferably selected from the group consisting of a non-ionic surfactant, more specifically, preferably Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin Nonionic ethoxylated surfactants selected from SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, preferably selected from fatty alcohol alkoxylates, and Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 and a polyethoxylated surfactant, such as an antifoam selected from alcohol-based antifoamers and combinations thereof, preferably in an amount equal to or greater than its cloud concentration as measured in aqueous solution at the culture temperature or room temperature, whereby a three-phase composition is obtained in the fermentation broth, the method further comprising recovering the product phase.

[0184] In some embodiments, a method is for enhancing secretion of a hydrophobic compound in a fermentation as described herein, comprising the steps of providing a microorganism, preferably a yeast cell, capable of producing said hydrophobic compound, and culturing said microorganism or yeast cell in a medium under conditions allowing production of said hydrophobic compound, said medium comprising a non-ionic surfactant, more particularly preferably Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), and Imbentin Nonionic ethoxylated surfactants selected from SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and combinations thereof, preferably selected from fatty alcohol alkoxylates, and Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, mixtures of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 and a polyethoxylated surfactant, such as an antifoam selected from alcohol-based antifoamers and combinations thereof, preferably in an amount equal to or greater than its cloud concentration as measured in aqueous solution at the culture temperature or room temperature, whereby a three-phase composition is obtained in the fermentation broth, the method further comprising recovering the product phase.

[0185] In a preferred embodiment, most of the hydrophobic compounds in the fermentation broth are present in the product phase. For example, at least 50% of the hydrophobic compounds are present in the product phase, such as at least 55%, for example at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% of the hydrophobic compounds present in the product phase. In some embodiments, the product phase comprises at least 50% of the hydrophobic compounds initially present in the fermentation broth, such as at least 55%, for example at least 60%, for example at least 65%, such as at least 70%, for example at least 75%, for example at least 80%, for example at least 85%, for example at least 90%, for example at least 95%, for example at least 96%, for example at least 97%, for example at least 98%, for example at least 99%, for example 100% of the hydrophobic compounds initially present in the fermentation broth. In some embodiments, the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, and / or a fatty aldehyde. In some embodiments, the hydrophobic compound is a terpene, such as a terpenoid.

[0186] Breaking the emulsion can be carried out as known in the art, for example, by subjecting the emulsion to a phase separation step known in the art. In some embodiments, the phase separation step is a centrifugation step, for example at 10,000 g for 5 minutes. In some embodiments, the centrifugation is carried out for 1 minute or more, for example, 2 minutes or more, for example, 3 minutes or more, for example, 4 minutes or more, for example, 5 minutes or more, for example, 6 minutes or more, for example, 7 minutes or more, for example, 8 minutes or more, for example, 9 minutes or more, for example, 10 minutes or more. In some embodiments, centrifugation is carried out at 3000g or more, such as 4000g or more, for example 5000g or more, such as 6000g or more, for example 7000g or more, such as 8000g or more, for example 9000g or more, such as 10000g or more, for example 11000g or more, such as 12000g or more, for example 13000g or more, such as 14000g or more, for example 15000g or more, such as 17500g or more, for example 20000g or more.

[0187] After the emulsion-breaking step, a product phase containing the extractant and the hydrophobic compound can be recovered from the composition. In such embodiments, the method can further include separating the hydrophobic compound from the extractant. This can be done by methods known in the art, such as distillation, e.g., under reduced pressure, or by column purification, or any other suitable method. The extractant can be recycled to the fermentor or bioreactor.

[0188] In some embodiments, the method includes culturing a microorganism, preferably a yeast cell, capable of producing a fatty alcohol, e.g., an unsaturated fatty alcohol or a mixture of (saturated and / or unsaturated) fatty alcohols. The unsaturated fatty alcohol may be recovered as described above. In such embodiments, the method may further include recovering the produced fatty alcohol and chemically converting at least a portion thereof to the corresponding fatty acyl acetate and / or corresponding fatty aldehyde. The term "corresponding" as used herein refers to a compound, fatty acyl acetate, or fatty aldehyde, having the same carbon chain length as the fatty alcohol from which it was derived. Therefore, when the microorganism, preferably yeast cell, produces fatty alcohols, the method may further comprise recovering the fatty alcohols and chemically converting at least a portion of the fatty alcohols into the corresponding fatty acyl acetates, for example, as described above. This can be achieved by performing an acetylation reaction known in the art, for example, as described in Fritz et al., 1959, or Mattson et al., 1964. The method may additionally or alternatively comprise chemically converting at least a portion of the fatty alcohols into the corresponding fatty aldehydes, for example, as described in Steves et al., 2013, by performing an oxidation reaction known in the art. The resulting fatty acyl acetates and / or fatty aldehydes can then be recovered.

[0189] Acetylation can be carried out, for example, with acetic anhydride using pyridine as a catalyst. The resulting fatty acetate is then extracted from the reaction mixture with an organic solvent, which is then removed by evaporation.

[0190] The oxidation can be carried out, for example, according to the Stahl protocol using the tetrakisacetonitrile copper(I) triflate / TEMPO catalyst system. The resulting fatty aldehyde can then be extracted from the reaction mixture with an organic solvent, which can be removed by evaporation.

[0191] Hydrophobic compounds obtainable by the method of the present invention The present disclosure also provides hydrophobic compounds obtainable by the methods described herein. In particular, fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes, such as terpenoids, obtainable by the methods are disclosed.

[0192] In some embodiments, the hydrophobic compound obtainable by this method is a fatty alcohol. The fatty alcohol may be saturated or unsaturated. Biological production of fatty alcohols from microorganisms such as yeast cells, particularly microorganisms and yeast cells genetically modified to produce the desired fatty alcohol, may produce a mixture of fatty alcohols containing odd-numbered chain fatty alcohols, in contrast to what is observed in chemical synthesis processes that only produce even-numbered chain fatty alcohols. Thus, in some embodiments, the hydrophobic compound obtainable by this method comprises or consists of a mixture of fatty alcohols containing odd-numbered chain fatty alcohols in addition to even-numbered chain fatty alcohols. The term "odd-numbered chain" fatty alcohol refers to a fatty alcohol with a carbon chain length of an odd number of carbon atoms, such as 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms. The term "even-numbered chain" fatty alcohol refers to a fatty alcohol with a carbon chain length of an even number of carbon atoms, such as 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms.

[0193] The method of the present invention allows for the recovery of different fatty alcohols produced by microorganisms, preferably yeast cells, in a single process. When expressing insect desaturases and reductases, the resulting fatty alcohol mixture produced by the microorganisms typically has a composition similar to that produced in the insect's pheromone gland. This allows for the production of pheromone mixtures suitable for a variety of insects, rather than producing individual pheromone components in separate processes that then need to be mixed in the appropriate ratios. Nevertheless, as shown in Example 5, the resulting fatty alcohol mixture may contain by-products characteristic of biological production. Thus, in some embodiments where desired unsaturated fatty alcohols are produced, the produced fatty alcohols comprise at least 1%, such as at least 2%, for example at least 3%, such as at least 4%, for example at least 5%, for example at least 10%, for example at least 15%, for example at least 20% unsaturated fatty alcohols having desaturation at a position other than that of the desired fatty alcohol and / or at least 1%, such as at least 2%, for example at least 3%, such as at least 4%, for example at least 5%, for example at least 10%, for example at least 15%, for example at least 20% of the corresponding saturated fatty alcohols. When the fatty alcohol mixture recovered from the fermentation broth is chemically oxidized to aldehydes or acetylated to acetate, the corresponding aldehyde and acetate mixture is produced.

[0194] In some embodiments, the hydrophobic compound obtained by the method is a fatty alcohol ester, which may be saturated or unsaturated. In some embodiments, the hydrophobic compound obtained by the method comprises or consists of a mixture of fatty alcohol esters, including odd-numbered chain fatty alcohol esters in addition to even-numbered chain fatty alcohol esters. The term "odd-numbered chain" alcohol ester refers to a fatty alcohol ester having an odd number of carbon atoms in its carbon chain length, such as 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms. The term "even-numbered chain" fatty alcohol ester refers to a fatty alcohol ester having an even number of carbon atoms in its carbon chain length, such as 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms.

[0195] In some embodiments, the hydrophobic compound obtained by this method is a fatty aldehyde, which may be saturated or unsaturated. In some embodiments, the hydrophobic compound obtained by this method comprises or consists of a mixture of fatty aldehydes, including odd-chain fatty aldehydes in addition to even-chain fatty aldehydes. The term "odd-chain" fatty aldehyde refers to a fatty aldehyde with a carbon chain length of an odd number of carbon atoms, such as 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms. The term "even-chain" fatty aldehyde refers to a fatty aldehyde with a carbon chain length of an even number of carbon atoms, such as 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms.

[0196] In some embodiments, the hydrophobic compound obtained by this method is a fatty acyl acetate, which may be saturated or unsaturated.In some embodiments, the hydrophobic compound obtained by this method comprises or consists of a mixture of fatty acyl acetates, including odd-numbered fatty acyl acetates in addition to even-numbered fatty acyl acetates.The term "odd-numbered chain" fatty acyl acetate refers to fatty acyl acetates with a carbon chain length of an odd number of carbon atoms, such as 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, or 23 carbon atoms.The term "even-numbered chain" fatty acyl acetate refers to fatty acyl acetates with a carbon chain length of an even number of carbon atoms, such as 8, 10, 12, 14, 16, 18, 20, or 22 carbon atoms. In some embodiments, the hydrophobic compounds obtained by the present methods are terpenes, such as, for example, the terpenoids described herein above.

[0197] Pheromone Composition Also disclosed herein are pheromone compositions comprising an unsaturated fatty alcohol, an unsaturated fatty acyl acetate, and / or an unsaturated fatty aldehyde. For example, the pheromone composition may comprise (Z)11-hexadecenol, (Z)-11-hexadecenal, and / or (Z)-11-hexadecen-1-yl acetate. For example, the pheromone composition may comprise codlemone (E8,E10-dodecadien-1-ol), E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal. At least one of the unsaturated fatty alcohol, unsaturated fatty acyl acetate, and unsaturated fatty aldehyde is preferably obtainable by the method disclosed herein above.

[0198] In some embodiments of the present disclosure, the pheromone composition comprises (Z)11-hexadecenol, (Z)-11-hexadecenal, and (Z)-11-hexadecen-1-yl acetate, wherein at least one of (Z)11-hexadecenol, (Z)-11-hexadecenal, or (Z)-11-hexadecen-1-yl acetate is obtainable by the methods disclosed hereinabove. In other embodiments, the pheromone composition comprises codlemone (E8,E10-dodecadien-1-ol), E8,E10-dodecadienyl acetate, and / or E8,E10-dodecadienal. Thus, the method may further include formulating the recovered unsaturated fatty alcohol, unsaturated fatty acyl acetate, or unsaturated fatty aldehyde into a pheromone composition. The pheromone compositions of the present invention may be used as incorporated pest management products, which can be used in methods of monitoring the presence of pests or in methods of disrupting pest mating.

[0199] The pheromone compositions disclosed herein can be used as biopesticides. Such compositions can be sprayed or dispensed onto cultivated plants in farms or orchards. They can also be impregnated onto rubber stoppers or mixed with other ingredients, as known in the art. This can cause mating disruption, thereby preventing the reproduction of pest insects, or it can be used in combination with traps to capture pest insects. Non-limiting examples of pests for which the pheromone compositions of the present invention can be used include the cotton bollworm (Helicoverpa armigera), rice stem borer (Chloro suppressalis), diamondback moth (Plutella xylostella), armyworm (Mamestra brassicae), large cabbage-heart caterpillar (Chlorochidromia binotalis), European cornstalk borer (Sesamia nonagrioides), corn earworm (Synantedon cypriformis), and artichoke plume moth (Platyptilia carjuidactyla). Thus, application of the compositions to cultivated crops can result in increased crop yields with substantially no environmental impact.

[0200] The relative amounts of the different compounds in the pheromone compositions of the present invention may vary depending on the nature of the crop and / or pest to be controlled; geographic variations may also exist. Determining the optimal relative amounts may therefore require periodic optimization. Examples of compositions used as repellents can be found in Kehat & Dunkelblum, 1993 for H. armigera; Alfaro et al., 2009 for C. spressalis; Eizaguirre et al., 2002 for S. nonagrioides; Wu et al., 2012 for P. xylostella; and Bari et al., 2003 for P. carjuidactyla.

[0201] In some embodiments of the present disclosure, the pheromone composition may further comprise one or more additional compounds, such as a liquid or solid carrier or substrate. For example, suitable carriers or substrates include vegetable oils, refined mineral oils or fractions thereof, rubbers, plastics, silica, diatomite, wax matrices, and cellulose powders.

[0202] The pheromone composition may be formulated as known in the art. For example, it may be in the form of a solution, gel, or powder. The pheromone composition may be formulated for easy administration as known in the art.

[0203] Example All strains are listed in Table 6. Example 1 - In situ extraction of fatty alcohols produced by fermentation Genetically modified Yarrowia lipolytica (alkane-utilizing yeast) strains ST8327 and ST8762 are capable of producing fatty alcohols (saturated and unsaturated). Strain ST8327 was genetically modified to produce (Z)11-hexadecen-1-ol. It also produces lesser amounts of (Z)9-hexadecen-1-ol and hexadecanol. This strain expresses the Δ11 desaturase (SEQ ID NO: 1) from Amielois transitella (walnut moth) and the fatty acyl-CoA reductase (SEQ ID NO: 5) from Helicoverpa armigera (helicobacterium armigera). Strain ST8762 was genetically modified to produce (Z)9-tetradecen-1-ol. This strain expresses the Δ9 desaturase (SEQ ID NO: 16) from Drosophila melanogaster (Drosophila melanogaster) and the fatty acyl-CoA reductase (SEQ ID NO: 5) from Helicoverpa armigera (Helicoverpa armigera). Both strains have additional modifications that reduce fatty alcohol degradation and improve fatty acid biosynthesis. The strains grow at an initial OD of 0.1-0.2 on YPD agar plates (10 g / L yeast extract, 10 g / L peptone, 20 g / L glucose, 15 g / L agar). 600The cells were inoculated into 2.5 mL of YPG medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glycerol) in a 24-well plate. The plate was incubated at 28°C, 300 rpm for 22 hours. The well plates were centrifuged at 3,500 g for 5 min at 4°C, the medium was removed, and the cells were resuspended in 1.25 mL of production medium (50 g / L glycerol, 5 g / L yeast extract, 4 g / L KH2PO4, 1.5 g / L MgSO4, 0.2 g / L NaCl, 0.265 g / L CaCl2·2H2O, 2 mL / L trace element solution: 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L N2SO4·4H2O). The sample was resuspended in 0.3 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, and 15 g / L EDTA. At the same time, 95 μL of Antifoam A (ethoxylated and propoxylated C) was added, corresponding to approximately 7% v / v (i.e., exceeding the 0.1% v / v recommended dosage for foam control and cloud concentration). 16 -C 18 Alcohol (CAS number 68002-96-0) was added. Plates were incubated at 28° C. and 300 rpm for 28 hours. Each experiment was performed in biological triplicate.

[0204] Intracellular and extracellular concentrations of fatty alcohols were assessed as follows: 1000 μL of culture broth was transferred to a 4 mL airtight glass extraction vial. The sample was centrifuged at 3,500 g for 5 minutes at room temperature. The supernatant was transferred to a new glass vial containing 990 μL of hexane and 10 μL of internal standard (IS) solution (20 mg / L methyl Z10-heptadecenoate in ethyl acetate). The vial was vortexed for 10 seconds and centrifuged as described above. 250 μL of the upper hexane phase was transferred to a GC vial for GC-MS analysis of extracellular fatty alcohol concentrations. The pellet remaining after removal of the supernatant from the centrifuged culture broth was resuspended in 990 μL of a solvent mixture (EtOAc and EtOH) and 10 μL of the above-mentioned IS solution. The sample was incubated for 1 hour with periodic mixing. 300 μL of water was added, and the vial was centrifuged at 3,500 g for 5 minutes at room temperature. 250 μL of the upper organic phase was transferred to a GC vial for GC-MS analysis of intracellular fatty alcohols. GC-MS analysis was performed on an Agilent 7820A GC coupled to an Agilent 5977B mass-selective detector. The GC was equipped with a DB Fatwax column (30 m x 0.25 mm x 0.25 μm) and helium was used as the carrier gas. The MS was operated in electron impact mode (70 eV), scanning between m / z 30 and 400, with the injector set to 200°C in split mode (20:1). The oven temperature was set to 80°C for 1 min, then ramped to 210°C at a rate of 20°C / min, held at 210°C for 7 min, and then ramped to 230°C at a rate of 20°C / min. Compounds were identified by comparison of retention time and mass spectrum with those of authentic compounds. Compounds were quantified by the m / z ion 55.1. The data were analyzed using Agilent Masshunter software. The concentrations of fatty alcohols were calculated based on a standard calibration curve generated with reference standards.

[0205] The results are shown in Table 1. Standard deviations were calculated from three biological replicates. The addition of antifoam had two beneficial effects: first, it increased the total fatty alcohol titer; and second, it increased the extracellular portion of the total fatty alcohols produced (from 2-8% to 70-73%). These effects simplify downstream processing and improve the overall economics of the process.

[0206] This example therefore demonstrates that the addition of extractant in amounts greater than the cloud concentration increases the titer of both saturated and unsaturated fatty alcohols, as well as their extracellular concentrations, regardless of the strain genotype, as this was observed in two different strains and confirmed in other strains (see Example 12). [Table 1]

[0207] Example 2 - In situ extraction and recovery of fatty alcohols produced by fermentation. Here, we investigated different amounts of antifoam added and their effect on the recovery of fatty alcohols in the separated phase. Substances such as Antifoam A, which are often used as antifoams in microbial fermentation, are known as emulsifiers. The cloud concentration of Antifoam A was experimentally determined to be approximately 1 v / v% antifoam in aqueous solution. The dosage recommended by the manufacturer for foam control is 0.1 v / v%.

[0208] Experiments were performed using the genetically modified Y. lipolytica strain ST8881 according to the procedure set forth in Example 1. Strain ST8881 is similar to ST8327 but has several additional genetic modifications that further enhance fatty acid biosynthesis. Antiform A was added at 0, 0.4, 2, or 5% v / v concentrations. The results are shown in Table 2 and Figure 1.

[0209] When Antifoam A was added at 0.4% v / v, below its cloud concentration in aqueous systems, it acted as an emulsifier in fermentation cultures (Figure 1B). In this case, the secretion of the target hydrophobic compound was 14.5%, with most of the product remaining intracellular. Application of centrifugation at 16,000 g for 5 min at room temperature resulted in separation of the solid cell fraction from the liquid phase. However, centrifugation at 16,000 g for 5 min at room temperature did not result in successful emulsion breaking, suggesting the need for complex recovery of the hydrophobic target compound using organic solvents and cell disruption.

[0210] When Antifoam A was added at 2 and 5% v / v, exceeding its cloud concentration in aqueous systems, it constituted a separate, immiscible light phase (Figures 1C and 1D). This separate, immiscible oily phase apparently acted as an in situ extractant, resulting in the secretion of 66.5% and 78.0% of the target hydrophobic compounds. Centrifugation at 16,000 g for 5 min at room temperature successfully separated the three existing phases, resulting in the isolation of the hydrophobic target compounds in the oily phase without the need for expensive cell disruption techniques and organic solvent extraction for product recovery. [Table 2]

[0211] Example 3 - In situ extraction and recovery of fatty alcohols produced by fermentation using various antifoam agents Here, we investigated the effect of different antifoam oils and agents on the recovery of fatty alcohols in the separating phase. The experiments were carried out using the genetically modified Y. lipolytica strain ST8881 according to the procedure shown in Example 1. The following commonly used antifoam oils and agents were tested at 3 v / v%; -corn oil, -oleic acid, -Antifoam A, Kolliphor® (poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol)), -A-204 (mixture of organic polyether dispersions), -Simethicone (silicone emulsion), and -Dodecane.

[0212] The suppliers are listed in Table 3. Control experiments were performed without any addition of antifoam oil or agents. Antifoam oil and agents were added to the culture broth at 3 vol / vol%, which is significantly lower than the concentration in the organic phase in classical two-phase fermentation known in the art (approximately 5-10 v / v%).

[0213] The in situ extraction performance of commonly used antifoam oils and agents is shown in Table 3. Corn oil, Antifoam A, Kolliphor® P407, A-204, and simethicone showed increased concentrations of fatty alcohols compared to the control cultures. Corn oil, Antifoam A, Kolliphor® P407, A-204, and simethicone also showed higher fatty alcohol secretion rates compared to the control cultures.

[0214] In the case of oleic acid, Antifoam A, Kolliphor® P407, A-204, and simethicone, application of centrifugation at 10,000 g for 5 min at 30°C resulted in the separation of the solid cell fraction from the liquid phase (Figures 2C, 2D, 2E, 2F, and 2G, respectively). In the case of corn oil and dodecane, the solid cell fraction constituted a dispersion with the organic upper phase (Figures 2B and 2H, respectively).

[0215] For Antifoam A, Kolliphor® P407, A-204 and simethicone, centrifugation at 10,000 g for 5 minutes at 30° C. resulted in good water-oil emulsion breakdown. [Table 3]

[0216] Example 4 - In situ extraction and recovery of other lipophilic / hydrophobic compounds Microorganisms capable of producing various lipophilic compounds, such as microorganisms genetically modified to produce free fatty acids, fatty acyl acetates, fatty aldehydes, and terpenes such as terpenoids, are cultured in the presence of antifoaming agents such as polyethoxylated surfactants at concentrations equal to or greater than their cloud concentration.

[0217] The resulting fermentation broth is centrifuged, and a light phase containing nonionic surfactants and the product is separated by centrifugation. The light phase is further subjected to distillation, optionally under reduced pressure, to separate the product from the nonionic surfactants and other nonvolatile impurities. The distilled product can be, for example, a mixture of fatty alcohols. The fatty alcohol mixture can be acetylated to the corresponding fatty alcohol acetate or oxidized to the corresponding fatty aldehyde.

[0218] Acetylation is carried out with acetic anhydride using pyridine as a catalyst. The resulting fatty alcohol acetate is then extracted from the reaction mixture with an organic solvent, and the solvent is removed by evaporation. Oxidation is carried out according to the Stahl protocol using a tetrakisacetonitrile copper(I) triflate / TEMPO catalyst system. The resulting fatty aldehyde is then extracted from the reaction mixture with an organic solvent, and the solvent is removed by evaporation. The resulting fatty alcohol acetate or fatty aldehyde is formulated and used for plant protection against insects.

[0219] Example 5 - Biological activity of pheromone preparations obtained by fermentation For example, (Z)11-hexadecen-1-ol (Z11-16:OH) produced by fermentation of Yarrowia lipolytica (a lipase-utilizing yeast) usually co-occurs with (Z)9-hexadecen-1-ol (Z9-16:OH), which is produced due to the action of the native Yarrowia lipolytica desaturase OLE1. In genetically engineered strains, Z9-16:OH was also produced in amounts 5–20% of the amount of Z11-16:OH. Saturated fatty alcohols with a 16-carbon chain were also produced as by-products when the reductase acted directly on the saturated substrate hexadecenyl-CoA.

[0220] When the fatty alcohol mixture recovered from the fermentation broth is chemically oxidized to aldehydes or acetylated to acetate, the corresponding aldehyde and acetate mixture is produced. In an exemplary sample preparation, the composition was as follows: 65% Z11-16:Ald, 4% Z9-16:Ald, 10% 16:Ald. Fermented Z11-16:Ald can be used to control the cotton bollworm (Helicoverpa armigera) by mating disruption. The pheromone glands of H. armigera contain Z11-16:Ald, Z9-16:Ald, and 16:Ald. The ratio between Z11-16:Ald and Z9-16:Ald varies from 99:1 to 90:10 (http: / / www.pherobase.com / database / species / species-Helicoverpa-armigera.php). Z9-16:Ald is a minor component of the H. armigera pheromone mixture, and it has been reported that when added to Z11-16:Ald, it enhances the activity of artificial pheromone formulations (Kehat et al., 1990). n-Hexadecanal 16:Ald is also present in the H. armigera pheromone gland at 4–20%, but it is neutral with respect to behavioral responses.

[0221] Fermented Z11-16:Ald can be used to control the rice stem borer (Chilo suppressalis) by mating disruption. The pheromone gland of C. suppressalis also contains Z11-16:Ald, Z9-16:Ald, and 16:Ald. The ratio between Z11-16:Ald and Z9-16:Ald is 10:1 (http: / / www.pherobase.com / database / species / species-Chilo-suppressalis.php). Z9-16:Ald is synergistic with the two major pheromone components, Z11-16:Ald and Z13-18:Ald (Tatsuki et al., 2013). n-Hexadecanal 16:Ald is a neutral compound and does not elicit a behavioral response.

[0222] Fermented Z11-16:Ald can be used to control the stag beetle (Scirpophaga incertulas) by mating disruption. The pheromone glands of S. incertulas contain Z11-16:Ald, Z9-16:Ald, and 16:Ald. The ratio between Z11-16:Ald and Z9-16:Ald ranges from 4:1 to 2:1 (http: / / www.pherobase.com / database / species / species-Scirpophaga-incertulas.php). Thus, the two important by-products produced by the genetically modified yeast are present in amounts similar to the natural range of pheromone compositions in insects. Z9-16 compounds are generally biologically active and behaviorally useful.

[0223] Example 6 - Strain Construction Strains ST3705 (Example 2 and Table 4 of WO 2016 / 207339) and ST5290 (Example 4 of WO 2018 / 109167) are Saccharomyces cerevisiae (budding yeast) strains genetically modified to produce (Z)-hexadecen-1-ol and Z-tetradecenyl acetate, respectively. Strain ST3705 expresses a Δ11 desaturase from Amielois transitella (the walnut moth) and a fatty acyl-CoA reductase from Helicoverpa armigera (the cotton bollworm). Strain ST5290 expresses the Δ9 desaturase from Drosophila melanogaster (Drosophila melanogaster), the fatty acyl-CoA reductase from Helicoverpa armigera (Helicoverpa armigera), and the acetyltransferase ATF1 from Saccharomyces cerevisiae (budding yeast).

[0224] Strain ST4840 is a Yarrowia lipolytica wild-type strain. Y. lipolytica strain ST6629 is a Yarrowia lipolytica strain previously described in WO 2018 / 109167 (Example 9 of WO 2018 / 109167). In this strain, genes HFD4 (YALI0B01298g), HFD3 (YALI0A17875), HFD2 (YALI0E15400), and HFD1 (YALI0F23793g), as well as nucleotides -1130 to -100 upstream of the coding sequence of GPAT (YALI0C00209g), were deleted. Premature stop codons and frameshifts were introduced into PEX10 (YALI0C01023g) and FAO1 (YALI0B14014g) to generate nonfunctional genes.

[0225] Y. lipolytica strain ST9426 was genetically modified to improve mevalonate pathway flux and used as a terpenoid platform strain. In this strain, genes KU70 (YALI0C08701g) and PEX10 (YALI0C01023g) and nucleotides -529 to -50 upstream of the coding sequence of squalene synthase (SQS1, YALI0A10076g) were deleted. In addition, genes isopentenyl-diphosphate delta isomerase (IDI1, YALI0F04015g), farnesyl diphosphate synthase (ERG20, YALI0E05753g), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG1, YALI0E04807g), and geranylgeranyl pyrophosphate synthase (GGPPS, SEQ ID NO: 46) were also overexpressed.

[0226] Y. lipolytica strains ST7982, ST8327, ST9253, ST10229, ST10230, ST10231, ST9423, ST9424, and ST10151 were constructed as follows. All heterologous genes were synthesized by GeneArt (Life Technologies) in codon-optimized Y. lipolytica. All genes were amplified by PCR using Phusion U Hot Start DNA Polymerase (ThermoFisher) to obtain fragments for cloning into yeast expression vectors. Primers and resulting DNA fragments (BioBricks) are listed in Table 4. PCR products were separated on a 1% agarose gel containing RedSafe™ (iNtRON Biotechnology). PCR products of the correct size were excised from the gel and purified using Nucleospin Gel and a PCR Clean-up Kit (Macherey-Nagel).

[0227] The integrative vector carrying the USER cassette was linearized using FastDigest SfaAI (ThermoFisher) for 2 hours at 37°C and then nicked using Nb.Bsml (New England BioLabs) for 1 hour at 65°C. The resulting vector containing cohesive ends was separated by gel electrophoresis, excised from the gel, and gel-purified using a Nucleospin Gel and PCR Clean-up Kit (Macherey-Nagel). The DNA fragment was inserted into the resulting vector by USER cloning, as described (Holkenbrink et al., 2018). The reaction was transformed into competent E. coli DHalpha cells, and the cells were plated on Lysogeny Broth (LB) agar plates containing 100 mg / L ampicillin. The plates were incubated overnight at 37°C, and the resulting colonies were screened by colony PCR. Plasmids were purified from overnight E. coli liquid cultures and correct cloning was confirmed by sequencing. The constructed vectors are listed in Table 5.

[0228] Yeast strains were constructed by transformation of DNA vectors as described in Holkenbrink et al., 2018. Integrating vectors were linearized with FastDigest NotI prior to transformation. Where necessary, helper vectors to facilitate integration into specific genomic regions were co-transformed with the integrating plasmid or DNA repair fragment (Tables 4 and 5). Strains were selected on yeast peptone dextrose (YPD) agar with appropriate antibiotic selection. Correct genotypes were confirmed by colony PCR and, where necessary, by sequencing. The resulting strains are listed in Table 6. [Table 4] TIFF0007821726000005.tif229159TIFF0007821726000006.tif236159TIFF0007821726000007.tif233159TIFF0007821726000008.tif40159 [Table 5] TIFF0007821726000010.tif92159 [Table 6] TIFF0007821726000012.tif69159

[0229] Example 7 - Surfactants: Selection of Ethoxylate / Ethoxylated Nonionic Surfactants Selected nonionic ethoxylated surfactants were tested (Table 7), showing their trade name, manufacturer, chemical name, CAS number, cloud point (°C (supplier's data)), cloud concentration (experimentally determined, v / v % in aqueous systems at room temperature), and recommended dosage for foam control (% (supplier's data)).

[0230] Simple model mixture experiments were performed in 2 mL spin tubes using 1 mL of production medium (50 g / L glycerol, 5 g / L yeast extract, 4 g / L KH2PO4, 1.5 g / L MgSO4, 0.2 g / L NaCl, 0.265 g / L CaCl2·2H2O, 2 mL / L trace element solution: 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L N2O). The fermentation was performed using a 0.1 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, and 15 g / L EDTA. The tubes were vortexed for 5 seconds to mimic the mixing conditions during the fermentation process. Spin tests were performed in a tabletop centrifuge at 15,000 g for 5 minutes at room temperature.

[0231] Spin tests revealed that when surfactants were applied at the low dosage recommended for foam control in fermentation (0.1% v / v, below their cloud concentration), no phase separation was detected (only one homogeneous aqueous phase). However, when surfactants were used at concentrations higher than their cloud concentration, the hydrophobic oil phase and the hydrophilic aqueous phase could be easily separated by the centrifugal force applied during the spin test. Application of surfactants at dosages higher than their recommended dosages for foam control and above their cloud concentration allowed for enhanced production and secretion of the hydrophobic pheromone product of fermentation by in situ extraction. In addition, simple mechanical separation facilitates easy and economical recovery of the hydrophobic pheromone product of fermentation. [Table 7] [Table 8] TIFF0007821726000015.tif96159

[0232] Example 8 - In situ extraction and recovery of fatty alcohols produced by fermentation Here, we investigated the effect of different amounts of added antifoam on the recovery of fatty alcohols in the separated phase. Substances similar to Antifoam A (Bekchem), which is often used as an antifoam in microbial fermentation, are known as emulsifiers. The cloud concentration of Antifoam A was experimentally determined to be approximately 1 v / v% antifoam in aqueous solution. The dosage recommended by the manufacturer for foam control is 0.1 v / v%.

[0233] Experiments were carried out using Y. lipolytica strain ST8327, which was genetically modified according to the procedures set forth in Example 1. Antifoam A was added at concentrations of 0, 0.4, 2, or 5% v / v. The results are shown in Table 8.

[0234] When Antifoam A was added at 0.4% v / v, below its cloud concentration measured in aqueous solution, it acted as an emulsifier in the fermentation culture, similar to that observed in Example 2. In this case, secretion of the target hydrophobic compound was 20-25%, with the majority of the product remaining intracellular. Application of centrifugation at 16,000 g for 5 min at room temperature resulted in separation of the solid cell fraction from the liquid phase. However, centrifugation at 16,000 g for 5 min at room temperature did not result in successful emulsion breaking, suggesting the need for complex recovery of the hydrophobic target compound using organic solvents and cell disruption.

[0235] When Antifoam A was added at 2 and 5% v / v, exceeding its cloud concentration measured in aqueous solution, it constituted a separate, immiscible light phase (as also observed in Figures 1C and 1D). This separate, immiscible oily phase apparently acted as an in situ extractant, resulting in the secretion of 73–74% and 67% of the target hydrophobic compounds. Centrifugation at 16,000 g for 5 min at room temperature successfully separated the three existing phases, resulting in the isolation of the hydrophobic target compounds in the oily phase without the need for expensive cell disruption techniques and organic solvent extraction for product recovery. [Table 9]

[0236] Example 9 - Enhanced production, secretion, and recovery of fatty alcohols by the yeast Saccharomyces cerevisiae (Budding yeast) Here, enhanced production, secretion, and recovery of fatty alcohols by the yeast Saccharomyces cerevisiae (Budding yeast) was demonstrated. Strain ST3705 was cultured on YPD agar plates (10 g / L yeast extract, 10 g / L peptone, 20 g / L glucose, 15 g / L agar) to an initial OD of 0.1–0.2. 600The cells were inoculated into 2.5 mL of YPD medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glucose) in a 24-well plate. The plate was incubated at 28°C and 300 rpm for 22 hours. The well plates were centrifuged at 3,500 g for 5 min at 4°C, the medium was removed, and the cells were resuspended in 1.25 mL of production medium (50 g / L glucose, 20 mg / L uracil, 5 g / L yeast extract, 4 g / L KH2PO4, 1.5 g / L MgSO4, 0.2 g / L NaCl, 0.265 g / L CaCl2·2H2O, 2 mL / L trace element solution: 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L N The solution was resuspended in 0.3 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, and 15 g / L EDTA). At the same time, the following surfactants were added: Control 1, 0% v / v surfactant; Control 2, 0.1% v / v surfactant (approximately the amount recommended by the manufacturer for foam control) and 3% v / v surfactant (amount exceeding the cloud concentration of surfactant): Antifoam A (Bekchem; ethoxylated and propoxylated C16-18 alcohol, CAS number 68002-96-0); Agnique BP420 (BASF; ethoxylated and propoxylated C16-18 alcohol, CAS number 68002-96-0); Plurafac® LF1300 (BASF; ethoxylated and propoxylated C16-18 alcohol, CAS number 68002-96-0); Dehipon® 2574 (BASF; fatty alcohol, ethoxylated and propoxylated, CAS number: 68154-97-2). Plates were incubated for 28 hours at 300 rpm at 28° C. Each experiment was performed in biological triplicate.

[0237] The concentration of extracellular fatty alcohols within cells was assessed as follows: 1000 μL of appropriately diluted culture broth was transferred to a 4 mL airtight glass extraction vial. The sample was centrifuged at 3,500 g for 5 minutes at room temperature. The supernatant was transferred to a new glass vial containing 1000 μL of hexane and 10 μL of internal standard (IS) solution (20 mg / L methyl nonadecanoate in ethyl acetate). The vial was vortexed for 10 seconds and centrifuged as described above. 250 μL of the upper hexane phase was transferred to a GC vial for GC-MS analysis of extracellular fatty alcohol concentration. The pellet remaining after removal of the supernatant from the centrifuged culture broth was resuspended in 1000 μL of a solvent mixture (EtOAc and EtOH) and 10 μL of the above-mentioned IS solution. The sample was incubated for 1 hour with periodic mixing. 300 μL of water was added, and the vial was centrifuged at 3,500 g for 5 minutes at room temperature. 250 μL of the upper organic phase was transferred to a GC vial for GC-MS analysis of intracellular fatty alcohols.

[0238] GC-MS analysis was performed on an Agilent 7820A GC coupled to an Agilent 5977B mass-selective detector. The GC was equipped with a DB Fatwax column (30 m x 0.25 mm x 0.25 μm) and helium was used as the carrier gas. The MS was operated in electron impact mode (70 eV), scanning between m / z 30 and 400, with the injector configured in split mode 20:1 at 220 °C. The oven temperature was set to 80 °C for 1 min, then increased to 210 °C at a rate of 20 °C / min, held at 210 °C for 7 min, and then increased to 230 °C at a rate of 20 °C / min. Compounds were identified by comparison of retention time and mass spectrum with those of authentic compounds. Compounds were quantified by the ion m / z of 55.1. Data were analyzed using Agilent Masshunter software. The concentrations of fatty alcohols were calculated based on a standard calibration curve generated with reference standards.

[0239] The results are shown in Table 9. Significant amounts of extracellular fatty alcohols were obtained when surfactant was added at high concentrations, i.e., above its cloud concentration. In addition, total fatty alcohol production also increased when surfactant was added at high concentrations (3 v / v%). When surfactant was added above its cloud concentration, it constituted a separate immiscible hydrophobic phase with the in situ extracted fatty alcohols. Therefore, phase separation and product recovery can be facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 10]

[0240] Example 10 - Enhanced production, secretion, and recovery of fatty alcohol acetate esters by the yeast Saccharomyces cerevisiae (Budding yeast) Here, enhanced production, secretion, and recovery of fatty alcohol acetate esters by the genetically engineered yeast Saccharomyces cerevisiae (Budding yeast) was demonstrated.

[0241] The experiment was carried out following the procedure using strain ST5290 as in Example 9, with the modification that the production medium was supplemented with an additional 76 mg / L histidine and 0.5 g / L myristate methyl ester. GC-MS analysis of acetate esters of fatty alcohols was carried out following the same procedure as in Example 9. Apart from acetate esters of fatty alcohols, untargeted screening of the data for other esters did not reveal significant production of other ester compounds.

[0242] The results are shown in Table 10. Significant production and secretion of fatty alcohol acetate esters was observed when the culture was supplemented with surfactant above its cloud concentration (3 v / v%). When surfactant was added above its cloud concentration, it constituted a separate immiscible hydrophobic phase along with the in situ extracted fatty alcohols. Therefore, phase separation and product recovery can be facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 11]

[0243] Example 11 - Enhanced production, secretion, and recovery of fatty alcohols by the yeast Yarrowia lipolytica (alkane-utilizing yeast) Here, enhanced production, secretion, and recovery of fatty alcohols by the genetically engineered yeast Yarrowia lipolytica (alkane-utilizing yeast) was demonstrated. Experiments with strains ST8327 and ST9253 were performed using YPG medium (10 g / L yeast extract, 10 g / L peptone, 40 g / L glycerol) with modifications to the production medium (50 g / L glycerol, 5 g / L yeast extract, 4 g / L KH2PO4, 1.5 g / L MgSO4, 0.2 g / L NaCl, 0.265 g / L CaCl2·2H2O, 2 mL / L trace element solution: 4.5 g / L CaCl2·2H2O, 4.5 g / L ZnSO4·7H2O, 3 g / L FeSO4·7H2O, 1 g / L H3BO3, 1 g / L MnCl2·4H2O, 0.4 g / L N The same procedure as in Example 9 was followed using 0.1 g / L Na2MoO4·2H2O, 0.3 g / L CoCl2·6H2O, 0.1 g / L CuSO4·5H2O, 0.1 g / L KI, and 15 g / L EDTA.

[0244] For strain ST8327, GC-MS analysis was performed as in Example 9. For strain ST9253, the oven temperature was set at 80°C for 1 min, then increased to 150°C at a rate of 20°C / min, then increased to 200°C at a rate of 1°C / min, and then increased to 230°C at a rate of 20°C / min.

[0245] The results are shown in Tables 11 and 12. A significant increase in total titer and secretion was observed when surfactants were supplemented above their cloud concentration (3% v / v). When surfactant was added above its cloud concentration, it constituted a separate immiscible hydrophobic phase with the in situ extracted fatty alcohols. Thus, phase separation and product recovery can be facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 12] [Table 13]

[0246] Example 12 - Enhanced production, secretion, and recovery of fatty alcohols by the yeast Yarrowia lipolytica (alkane-utilizing yeast) Here, enhanced production, secretion, and recovery of fatty alcohols by the genetically engineered yeast Yarrowia lipolytica (alkane-utilizing yeast) was demonstrated. Experiments were performed using strains ST10229, ST10230, and ST10231 according to the same procedure as in Example 11, with the modification that both the YPG and production medium were supplemented with 150 mg / L nourseothricin. When the cells were resuspended in production medium, Control 1 (0% v / v Antifoam A), Control 2 (0.1% v / v Antifoam A (approximately the amount recommended by the manufacturer for foam control)), and 3% v / v (amount exceeding the surfactant cloud concentration) Antifoam A (Bekchem; ethoxylated and propoxylated C16-C18 alcohol, CAS number 68002-96-0) were added. GC-MS analysis was performed as in Example 9.

[0247] The results are shown in Table 13. Significant production and secretion of fatty alcohols was observed when the culture was supplemented with surfactant above its cloud concentration (3 v / v%). When surfactant was added above its cloud concentration, it constituted a separate immiscible hydrophobic phase along with the in situ extracted fatty alcohols. Therefore, phase separation and product recovery can be facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 14]

[0248] Example 13 - Enhanced production, secretion, and recovery of fatty aldehydes by the yeast Yarrowia lipolytica (alkane-utilizing yeast) Here, enhanced secretion and recovery of fatty aldehydes by the genetically engineered yeast Yarrowia lipolytica (alkane-utilizing yeast) was demonstrated. Using strain ST8327, experiments were carried out according to the same procedure as in Example 10. GC-MS analysis was carried out as in Example 9.

[0249] The results are shown in Table 14. Significant secretion was observed when surfactants were supplemented above their cloud concentration (3 v / v%). When surfactant was added above its cloud concentration, it constituted a separate immiscible hydrophobic phase together with the in situ extracted fatty alcohols. Thus, phase separation and product recovery were facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 15]

[0250] Example 14 - Recovery of fatty alcohols and surfactants. Purification of fatty alcohols from the mixture. The purification of fatty alcohols from hydrophobic mixtures of fatty alcohols and surfactants was demonstrated by distillation. Model mixtures of various commercially available ethoxylated surfactants (Bekchem's Antifoam A, Plurafac® LF 1300, Dehipon® 2574) and technical-grade fatty alcohol mixtures were prepared. The model mixtures were subjected to reduced pressure distillation in a laboratory-scale distillation apparatus equipped with a Vigreux column. The experiments were carried out applying a 5 mbar vacuum, a final pot temperature of 200-210°C, and a final receiver temperature of 170-180°C. The light phase was collected and analyzed for mass and composition.

[0251] An appropriate amount of sample was transferred to a 50 mL volumetric flask and weighed on an analytical balance. The sample was dissolved in ethyl acetate in the volumetric flask and mixed thoroughly. 1 mL of diluted aliquots were transferred to GC vials, and 10 μL of internal standard (IS) solution (20 mg / L methyl nonadecanoate in ethyl acetate) was added to each GC vial. The vials were vortexed for 10 seconds before analysis. GC and data analysis were performed as described in Example 11.

[0252] The results are shown in Table 15. The composition of the light phase collected from the model mixture distillation experiments showed the enrichment of the target fatty alcohol compounds in the fractions. Distillation is a cost-effective solution for the purification of target compounds from the recovered hydrophobic surfactant-fatty alcohol, fatty alcohol ester, and fatty aldehyde mixtures presented in Examples 9-11 and 13. [Table 16]

[0253] Example 15 - Enhanced production, secretion, and recovery of isoprenoids by the yeast Yarrowia lipolytica (Yeast globulin) Here, increased secretion and recovery of isoprenoids by the genetically engineered yeast Yarrowia lipolytica (alkane-utilizing yeast) was demonstrated. The genetically modified Yarrowia lipolytica (alkane-utilizing yeast) ST10151 is capable of producing β-farnesene. This strain expresses β-farnesene synthase from Artemisia annua (artemis carrot) (SEQ ID NO: 47). This strain has additional modifications to increase mevalonate (MVA) pathway flux.

[0254] The experiment was carried out using strain ST10151 according to the same procedure as in Example 11. When the cells were resuspended in production medium, 0%, 0.1%, or 3% v / v of surfactant: Antifoam A (Bekchem) or A-204 (Sigma) was added. The plates were incubated at 28°C and 300 rpm for 28 hours. Each experiment was performed in biological triplicate.

[0255] Extracellular and intracellular samples for GC-MS analysis were analyzed according to the same procedure as in Example 9, except that patchouli alcohol (2 g / L patchouli alcohol in ethyl acetate) was used as the internal standard. GC-FID analysis for β-farnesene was performed using an Agilent GC7890B equipped with a flame ionization detector (FID) and a fused silica capillary column (BP5, 30 m x 0.32 mm ID, 0.25 μm, Agilent Technologies). Hydrogen was used as the carrier gas at a constant flow rate of 2.0 mL / min. The GC oven temperature was started at 50 °C for 1.5 min, then increased to 170 °C at 30 °C / min and held for 1.5 min. The temperature was then increased from 170 to 300 °C at 15 °C / min and held for 4.5 min. Both the injector and detector inlet / outlet were held at 300 °C, and the injector was operated in 20:1 split mode. For quantification of β-farnesene, calibration standards containing β-farnesene in the concentration range of 0.01 mg / ml to 1 mg / ml were prepared. 10 μl of 2 g / l patchouli alcohol in ethyl acetate was added to 990 μl of the standard to obtain a calibration curve. Data analysis was performed with Masshunter Quantitative Analysis version 10.1.

[0256] The results are shown in Table 16. Significant production and secretion of isoprenoids was observed when surfactants (Antifoam A and A-204) were supplied above their cloud concentration (3 v / v%). When surfactant was added above their cloud concentration, it constituted a separate immiscible hydrophobic phase along with the in situ extracted isoprenoids. Thus, phase separation and product recovery were facilitated by simple mechanical phase separation without the use of organic solvents or expensive separation methods. [Table 17]

[0257] References Anelli PL,Biffi C,Montanar F,and Quici S,J.Org.Chem.1987,52,12,2559-2562 Borodina,I.Understanding metabolite transport gives an upper hand in strain development.Microb Biotechnol.2019 Jan;12(1):69-70. Corey EJ;CUKim(1972).Journal of the American Chemical Society.94(21):7586-7587.doi:10.1021 / ja00776a056 Fritz,JSet al.Acid-catalysed acetylation of organic hydroxyl groups.Anal.Chem.1959,31,11,1808-1812 Holkenbrink C, Dam MI, Kildegaard KR, et al.EasyCloneYALI:CRISPR / Cas9-Based Synthetic Toolbox for Engineering of the Yeast Yarrowia lipolytica.Biotechnol J.2018;13(9):e1700543.doi:10.1002 / biot.201700543 Hoover J.M.et al.Highly Practical Copper(I) / TEMPO Catalyst System for Chemoselective Aerobic Oxidation of Primary Alcohols.J.Am.Chem.Soc.2011,133,42,16901-16910 Kehat et al.(1990),“Behavioral responses of male Heliothis armigera(Lepidoptera:Noctuidae)moths in a flight tunnel to combinations of components identified from female sex pheromone glands”.Journal of Insect Bhavior,3(1):75-83 Ley,Steven V.;Norman,Joanne;Griffith,William P.;Marsden,Stephen P.(1994).Synthesis.1994(7):639-666.doi:10.1055 / s-1994-25538 Marella ER,Dahlin J,Dam MI,ter Horst J,Christensen HB,Sudarsan S,Wang G,Holkenbrink C,Borodina I.A single-host fermentation process for the production of flavor lactones from non-hydroxylated fatty acids.Metabolic Engineering 2020.doi.org / 10.1016 / j.ymben.2019.08.009 Mattson,F.H.et al.Esterification of hydroxy compounds by fatty acid anhydrides.J Lipid Res.1964 Jul;5(3):374-7. Omura, K.; Swern, D. (1978). Tetrahedron. 34(11): 1651 - 1660. doi:10.1016 / 0040 - 4020(78)80197 - 5 Ratcliffe R and Rodehorst R(1970). J. Org. Chem. 35(11): 4000 - 4001. doi:10.1021 / jo00836a108 Steves, J. E. et al. Copper(I) / ABNO - catalyzed aerobic alcohol oxidation: alleviating steric and electronic constraints of Cu / TEMPO catalyst systems. J Am Chem Soc. 2013 Oct 23;135(42): 15742 - 5 Tatsuki, S., Kurihara, M., Usui, K., Ohguchi, Y., Uchiumi, K., and Fukami, J. 1983. Sex pheromone of the rice stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae): the third component, Z - 9 - hexadecenal. Appl. Entomol. Zool. 18: 443 - 446 WO 2016 / 207339 WO 2018 / 109163 WO 2018 / 109167 WO 2020 / 169389

[0258]

Table 18

[0259] Item 1. A method for producing hydrophobic compounds such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and / or terpenes such as terpenoids in fermentation, comprising the steps of providing a microorganism, preferably a yeast cell, capable of producing said hydrophobic compound, and culturing said microorganism in a medium under conditions allowing the production of said hydrophobic compound, wherein the medium comprises an extractant in an amount equal to or greater than its cloud concentration in an aqueous solution, the extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The polyethoxylated surfactant is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, and optionally the method further comprises recovering the hydrophobic compound. 2. A method for increasing the titer of hydrophobic compounds such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and / or terpenes such as terpenoids in fermentation, comprising culturing a microorganism, preferably yeast cells, capable of producing said hydrophobic compounds in a medium under conditions allowing the production of said hydrophobic compounds, said medium comprising an extractant in an amount equal to or greater than its cloud concentration in an aqueous solution, said extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18The polyethoxylated surfactant is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, whereby the titer of the hydrophobic compound is increased compared to fermentation carried out under similar conditions in the absence of the extractant or in the presence of the extractant in an amount less than its cloud concentration in aqueous solution. 3. A method for enhancing secretion of hydrophobic compounds, such as fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes and / or terpenes, such as terpenoids, from a microorganism, preferably a yeast cell, capable of producing said hydrophobic compounds by fermentation, comprising culturing said microorganism in a medium under conditions allowing production of said hydrophobic compounds, said medium comprising an extractant in an amount equal to or greater than its cloud concentration in an aqueous solution, said extractant being a non-ionic surfactant such as an antifoaming agent, preferably polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 The method is a polyethoxylated surfactant selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof, whereby secretion of hydrophobic compounds from the microorganism is enhanced compared to fermentation carried out under similar conditions in the absence of the extractant or in the presence of the extractant in an amount less than its cloud concentration in aqueous solution. 4. The method according to any one of items 1 to 3, wherein the extractant is a nonionic ethoxylated surfactant. 5. The extractant is a fatty alcohol alkoxylate, or preferably a mixture of polyethylene polypropylene glycol, polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 185. The method according to any one of items 1 to 4, wherein the ethoxylated surfactant is selected from antifoaming agents including alcohol-based antifoaming agents and combinations thereof. 6. The method according to any one of items 1 to 5, wherein the fatty alcohol is a saturated fatty alcohol, an unsaturated fatty alcohol, or a mixture thereof. 7. The method according to any one of items 1 to 6, wherein the fatty acyl acetate is a saturated fatty acyl acetate, an unsaturated fatty acyl acetate, or a mixture thereof. 8. The method according to any one of items 1 to 7, wherein the fatty aldehyde is a saturated fatty aldehyde, an unsaturated fatty aldehyde, or a mixture thereof. 9. The method according to any one of items 1 to 8, wherein the fatty alcohol ester is a saturated fatty alcohol ester, an unsaturated fatty alcohol ester, or a mixture thereof. 10. The method according to any one of items 1 to 9, wherein the fatty alcohol, fatty alcohol ester, fatty acyl acetate and / or fatty aldehyde has a carbon chain length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22. 11. The method according to any one of items 1 to 10, wherein the terpene is a hemiterpene, monoterpene, sesquiterpene, diesterterpene, triterpene, sesquaterpene, tetraterpene, or polyterpene. 12. The method according to any one of items 1 to 11, wherein the terpene is a terpenoid, such as a hemiterpenoid, monoterpenoid, sesquiterpenoid, di-sesterterpenoid, triterpenoid, sesquaterpenoid, tetraterpenoid or polyterpenoid. 13. The extractant is ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 13. The method according to any one of items 1 to 12, wherein the antifoaming agent is alcohol-based, preferably having a cloud concentration of 1% vol / vol in aqueous solution. 14. The extractant is C16 -C 18 14. The method according to any one of items 1 to 13, wherein the surfactant is selected from surfactants including alkyl alcohol ethoxylate propoxylate (CAS number 68002-96-0), Agnique BP420 (CAS number 68002-96-0), polyethylene polypropylene glycol, Antifoam 204, polyethylene glycol monostearate, and fatty alcohol alkoxylates such as Kolliphor® P407 (CAS number 9003-11-6), simethicone, Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2), or Imbentin SG / 251 (CAS number 68002-96-0). 15. The method according to any one of items 1 to 14, wherein the culture medium comprises at least 1% vol / vol of extractant, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol of extractant. 16. The method according to any one of items 1 to 15, wherein the nonionic surfactant is an antifoaming agent. 17. Nonionic surfactants include ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers, e.g. C 16 -C 18Alkyl alcohol ethoxylate propoxylate (CAS no. 68002-96-0), and the medium contains at least 1% vol / vol C 16 -C 18 Alkyl alcohol ethoxylates propoxylates, for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol of C 16 -C 18 17. The method according to any one of items 1 to 16, comprising an alkyl alcohol ethoxylate propoxylate. 18. The method according to any one of items 1 to 17, wherein the non-ionic surfactant is polyethylene polypropylene glycol, such as Kolliphor® P407 (CAS number 9003-11-6), and the culture medium comprises at least 10% vol / vol of polyethylene polypropylene glycol such as Kolliphor® P407, such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol such as Kolliphor® P407. 19. The method according to any one of items 1 to 18, wherein the non-ionic surfactant is Agnique BP420 (CAS No. 68002-96-0) and the culture medium comprises at least 10% vol / vol of Agnique BP420 (CAS No. 68002-96-0), such as at least 11% vol / vol, for example at least 12% vol / vol, such as at least 13% vol / vol, for example at least 14% vol / vol, such as at least 15% vol / vol, for example at least 16% vol / vol, such as at least 17% vol / vol, for example at least 18% vol / vol, such as at least 19% vol / vol, for example at least 20% vol / vol, such as at least 25% vol / vol, for example at least 30% vol / vol, such as at least 35% vol / vol or more of polyethylene polypropylene glycol of Agnique BP420 (CAS No. 68002-96-0). 20. The method according to any one of items 1 to 19, wherein the non-ionic surfactant is a mixture of polyether dispersions, such as Antifoam 204, and the culture medium comprises at least 1% vol / vol of the mixture of polyether dispersions such as Antifoam 204, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more of the mixture of polyether dispersions such as Antifoam 204. 21. The method according to any one of items 1 to 20, wherein the non-ionic surfactant is simethicone and the medium comprises at least 1% vol / vol simethicone, such as at least 1.5%, for example at least 2%, such as at least 2.5%, for example at least 3%, such as at least 3.5%, for example at least 4%, such as at least 5%, for example at least 6%, such as at least 7%, for example at least 8%, such as at least 9%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 17.5%, for example at least 20%, such as at least 22.5%, for example at least 25%, such as at least 27.5%, for example at least 30% vol / vol or more simethicone. 22. The nonionic surfactant is Plurafac® LF300 (CAS number 196823-11-7), Plurafac® LF1300 (CAS number 68002-96-0), Plurafac® SLF180 (CAS number 196823-11-7), Dehipon® 2574 (CAS number 68154-97-2) or Imbentin SG / 251 (CAS No. 68002-96-0), preferably Plurafac® LF300 or Dehipon® 2574, and the medium contains at least 1% vol / vol of Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (CAS No. 68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), Imbentin SG / 251 (CAS No. 68002-96-0), for example at least 1.5%, such as at least 2%, for example at least 2.5%, such as at least 3%, for example at least 3.5%, such as at least 4%, for example at least 5%, such as at least 6%, for example at least 7%, such as at least 8%, for example at least 9%, such as at least 10%, for example at least 12.5%, such as at least 15%, for example at least 17.5%, for example at least 20%, e.g. 22.5%, such as at least 25%, for example at least 27.5%, for example at least 30% vol / vol of or more Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (CAS No. 68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), Imbentin SG / 251 (CAS No. 68002-96-0). 23. The method according to any one of items 1 to 22, wherein the medium comprises the extractant in an amount at least 50%, such as at least 100%, for example at least 150%, such as at least 200%, for example at least 250%, such as at least 300%, for example at least 350%, such as at least 400%, for example at least 500%, for example at least 750%, such as at least 1000% more than its cloud concentration. 24. The method according to any one of items 1 to 23, wherein the culture medium comprises the extractant in an amount of at least 2 times the cloud concentration, such as at least 3 times the cloud concentration, for example at least 4 times the cloud concentration, such as at least 5 times the cloud concentration, for example at least 6 times the cloud concentration, such as at least 7 times the cloud concentration, for example at least 8 times the cloud concentration, such as at least 9 times the cloud concentration, for example at least 10 times the cloud concentration, such as at least 12.5 times the cloud concentration, for example at least 15 times the cloud concentration, such as at least 17.5 times the cloud concentration, for example at least 20 times the cloud concentration, such as at least 25 times the cloud concentration, for example at least 30 times the cloud concentration. 25. The method according to any one of items 1 to 24, wherein the hydrophobic compounds produced by the microorganisms are present in an emulsion in the fermentation broth, and further comprising the step of breaking the emulsion, thereby obtaining a composition comprising a product phase comprising the extractant and the hydrophobic compounds. 26. The method according to item 25, wherein the step of breaking the emulsion comprises or consists of a phase separation step, such as a centrifugation step, of the fermentation broth, thereby obtaining a composition consisting of three phases: an aqueous phase, a phase containing cells and necrotic debris, and a product phase containing the extractant and hydrophobic compounds. 27. The method according to item 26, wherein the product phase comprises at least 50%, such as at least 55%, for example at least 60%, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 85%, for example at least 90%, such as at least 95% or more of the hydrophobic compounds initially present in the fermentation broth. 28. The method according to item 26 or 27, further comprising recovering a product phase comprising the extractant and the hydrophobic compound from the composition, and optionally further separating the hydrophobic compound from the extractant, wherein the separation is preferably by distillation, such as distillation under reduced pressure, or column purification. 29. The hydrophobic compound is one or more fatty alcohols; - recovering said one or more fatty alcohols by a distillation step, such as distillation, preferably under reduced pressure, or by column purification, thereby obtaining a mixture of fatty alcohols, - chemically converting at least a portion of the fatty alcohols of said mixture into the corresponding fatty acyl acetates and / or into the corresponding fatty aldehydes, 29. The method according to any one of items 1 to 28, further comprising: 30. The method according to item 29, wherein at least a portion of the fatty alcohols are converted to the corresponding fatty acyl acetates by acetylation. 31. The method according to item 29 or 30, wherein at least a portion of the fatty alcohol is converted to the corresponding fatty aldehyde by oxidation. 32. The method according to any one of items 29 to 31, further comprising recovering the corresponding fatty acyl acetate and / or the corresponding fatty aldehyde. 33. The method of any one of items 1 to 32, wherein the extractant is recovered from the fermentation broth and optionally recycled to the fermentation broth. 34. The potency of the hydrophobic compound is increased by at least 5%, such as at least 10%, for example at least 15%, for example at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, for example at least 40%, for example at least 45%, for example at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, for example at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the potency obtained in a fermentation carried out under similar or identical conditions but either in the absence of an extractant or in the presence of an extractant in an amount lower than its cloud concentration in aqueous solution, more preferably the nonionic surfactant is Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers or ethoxylated and propoxylated C 16 -C 18 34. The method according to any one of items 1 to 33, wherein the antifoaming agent is selected from the group consisting of alcohol-based agents and combinations thereof. 35. The secretion of hydrophobic compounds is increased by at least 5%, for example at least 7.5%, for example at least 10%, for example at least 12.5%, for example at least 15%, for example at least 20%, for example at least 25%, for example at least 30%, for example at least 35%, for example at least 36%, for example at least 37%, for example at least 38%, for example at least 39%, for example at least 40%, for example at least more than that, compared to fermentation carried out under similar or identical conditions but either in the absence of the extractant or in the presence of an amount of the extractant lower than its cloud concentration measured in aqueous solution, more preferably the nonionic surfactant is Agnique BP420 (CAS number 68002-96-0), polyethylene polypropylene glycol, a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone and ethoxylated and propoxylated C 16 -C 18 Alcohol-based defoamers or ethoxylated and propoxylated C 16 -C 18 35. The method according to any one of items 1 to 34, wherein the antifoaming agent is selected from the group consisting of alcohol-based agents and combinations thereof. 36. The hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, and / or a fatty aldehyde, and the extractant is an ethoxylated and propoxylated C 16 -C 18 Alcohol-based agents or antifoam agents, preferably containing more than 1% vol / vol of ethoxylated and propoxylated C 16 -C 18 36. The method according to any one of items 1 to 35, comprising an alcohol-based agent or an antifoaming agent. 37. Fatty acyl acetate titer and / or fatty aldehyde titer are under similar or identical conditions, but are not ethoxylated or propoxylated. 16 -C 18 Ethoxylated and propoxylated C in the absence of alcohol-based agents or antifoaming agents or at amounts below its cloud concentration in aqueous solution 16 -C 1837. The method according to item 36, wherein the titer is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 46%, for example at least 47%, such as at least 48%, for example at least 49%, such as at least 50%, for example at least 51%, such as at least 52%, for example at least 53%, such as at least 54%, for example at least 55% or more compared to the titer obtained in a fermentation carried out either in the presence of an alcohol-based agent or an antifoam agent. 38. The secretion of fatty alcohols, fatty acyl acetates and / or fatty aldehydes is under similar or identical conditions, but with ethoxylated and propoxylated C 16 -C 18 Ethoxylated and propoxylated C in the absence of alcohol-based agents or antifoaming agents or at amounts below its cloud concentration in aqueous solution 16 -C 18 38. The method according to item 36 or 37, wherein the yield of the fermentation product is increased by at least 5%, such as at least 7.5%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 36%, for example at least 37%, such as at least 38%, for example at least 39%, such as at least 40%, for example at least more than that, compared to fermentation carried out either in the presence of an alcohol-based agent or an antifoaming agent. 39. The method according to any one of items 1 to 38, wherein the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate and / or a fatty aldehyde, the extractant is polyethylene polypropylene glycol, and preferably the culture medium contains more than 1% vol / vol polyethylene polypropylene glycol. 40. The method according to item 39, wherein the fatty alcohol titer, fatty acyl acetate titer and / or fatty aldehyde titer is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, such as at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the titer obtained in a fermentation carried out under similar or identical conditions but either in the absence of polyethylene polypropylene glycol or in the presence of polyethylene polypropylene glycol in an amount lower than its cloud concentration in aqueous solution. 41. The method according to item 39 or 40, wherein the secretion of fatty alcohols, fatty acyl acetates and / or fatty aldehydes is increased by at least 5%, such as at least 7.5%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 36%, for example at least 37%, for example at least 38%, for example at least 39%, such as at least 40%, for example at least more than that, compared to fermentation carried out under similar or identical conditions but either in the absence of polyethylene polypropylene glycol or in the presence of polyethylene polypropylene glycol in an amount lower than its cloud concentration in aqueous solution. 42. The method according to any one of items 1 to 41, wherein the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate and / or a fatty aldehyde, the extractant is a mixture of polyether dispersions, preferably the medium comprises more than 1% vol / vol of the mixture of polyether dispersions. 43. The method according to item 42, wherein the fatty alcohol titer, fatty acyl acetate titer and / or fatty aldehyde titer is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, such as at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, for example at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the titer obtained in a fermentation carried out under similar or identical conditions but either in the absence of the mixture of polyether dispersions or in the presence of the mixture of polyether dispersions in an amount lower than its cloud concentration in aqueous solution. 44. The method according to item 42 or 43, wherein the secretion of fatty alcohols, fatty acyl acetates and / or fatty aldehydes is increased by at least 5%, such as at least 7.5%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 36%, for example at least 37%, for example at least 38%, for example at least 39%, for example at least 40%, such as at least more than that, compared to fermentation carried out under similar or identical conditions but either in the absence of the mixture of polyether dispersions or in the presence of the mixture of polyether dispersions in an amount lower than its cloud concentration in aqueous solution. 45. The method according to any one of items 1 to 44, wherein the hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate and / or a fatty aldehyde, the extractant is simethicone, and preferably the medium contains more than 1% vol / vol simethicone. 46. ​​The method according to item 45, wherein the fatty alcohol titer, fatty acyl acetate titer and / or fatty aldehyde titer is increased by at least 5%, such as at least 10%, for example at least 15%, for example at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, for example at least 40%, for example at least 45%, such as at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, for example at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the titer obtained in a fermentation carried out under similar or identical conditions but either in the absence of simethicone or in the presence of simethicone in an amount lower than its cloud concentration in aqueous solution. 47. The method according to item 45 or 46, wherein the secretion of fatty alcohols, fatty acyl acetates and / or fatty aldehydes is increased by at least 5%, such as at least 7.5%, for example at least 10%, such as at least 12.5%, for example at least 15%, such as at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, such as at least 36%, for example at least 37%, for example at least 38%, for example at least 39%, for example at least 40%, such as at least more than that, compared to fermentation carried out under similar or identical conditions but either in the absence of simethicone or in the presence of simethicone in an amount lower than its cloud concentration in aqueous solution. 48. The hydrophobic compound is a fatty alcohol, a fatty alcohol ester, a fatty acyl acetate, and / or a fatty aldehyde, and the extractant is C 16 -C 18surfactants including alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0), Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, Antifoam 204; polyethylene glycol monostearate, and Kolliphor® P407 (CAS No. 9003-11-6), simethicone, Plurafac® LF300 (CAS No. 196823-11-7), Plurafac® LF1300 (68002-96-0), Plurafac® SLF180 (CAS No. 196823-11-7), Dehipon® 2574 (CAS No. 68154-97-2), or Imbentin 48. The method according to any one of items 1 to 47, wherein the extractant is a fatty alcohol alkoxylate, such as SG / 251 (CAS number 68002-96-0), and preferably the medium comprises more than 1% vol / vol of the extractant. 49. The method according to item 48, wherein the fatty alcohol titer, fatty acyl acetate titer and / or fatty aldehyde titer is increased by at least 5%, such as at least 10%, for example at least 15%, such as at least 20%, for example at least 25%, for example at least 30%, for example at least 35%, such as at least 40%, for example at least 45%, for example at least 46%, for example at least 47%, for example at least 48%, for example at least 49%, for example at least 50%, for example at least 51%, for example at least 52%, for example at least 53%, for example at least 54%, for example at least 55% or more compared to the titer obtained in a fermentation carried out under similar or identical conditions but either in the absence of extractant or in the presence of extractant in an amount lower than its cloud concentration in aqueous solution. 50. The method according to item 48 or 49, wherein the secretion of fatty alcohols, fatty acyl acetates and / or fatty aldehydes is increased by at least 5%, such as at least 7.5%, for example at least 10%, for example at least 12.5%, such as at least 15%, for example at least 20%, for example at least 25%, such as at least 30%, for example at least 35%, for example at least 36%, for example at least 37%, for example at least 38%, for example at least 39%, for example at least 40%, such as at least more than that, compared to fermentation carried out under similar or identical conditions but either in the absence of the extractant or in the presence of an amount of the extractant lower than its cloud concentration in aqueous solution. 51. The method according to any one of items 1 to 50, wherein the microorganism is a yeast, such as a yeast of the genus Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces, preferably a yeast of the genus Saccharomyces or Yarrowia. 52. The method according to any one of items 1 to 51, wherein the microorganism is a yeast of a species selected from Saccharomyces cerevisiae (budding yeast), Pichia pastoris (methanol-utilizing yeast), Kroiveromyces marxianus (thermotolerant yeast), Cryptococcus albidus, Lipomyces lipofera, Lipomyces starchii (oleaginous yeast), Rhodosporidium toruloides (oleaginous yeast), Rhodotorula glutinis (red yeast), Trichosporon pullulans (psychrophilic β-galactosidase-producing bacterium), and Yarrowia lipolytica (alkane-utilizing yeast), preferably wherein the yeast cell is a Saccharomyces cerevisiae (budding yeast) or Yarrowia lipolytica (alkane-utilizing yeast) cell. 53. The hydrophobic compound is (Z)-11-hexadecen-1-ol, and the microorganism is a yeast cell capable of producing (Z)-11-hexadecen-1-ol at a titer of 0.2 mg / L, preferably the yeast cell is a Saccharomyces cerevisiae (budding yeast) cell, and the yeast cell is Amielois transitella (walnut moth) Δ11-desaturase (Atr_Δ11; SEQ ID NO: 1), Spodoptera littoralis (African armyworm) Δ11-desaturase (Sl_Δ11; SEQ ID NO: 2), Agrotis segetum (turnip moth) Δ11-desaturase (As_Δ11; SEQ ID NO: 3), Lobesia botrana (grass leaf moth) desaturase (Lbo_PPTQ; SEQ ID NO: 43), Drosophila grimshaui (Hawaiian fruit fly) desaturase (Dgd9; SEQ ID NO: 44), Drosophila virilis (Drosophila virilis) desaturase (Dvd9; SEQ ID NO: 45), and Trichoprasia ni (nettle looper moth) Δ11-desaturase (Tni_Δ11; SEQ ID NO:4), or a functional variant thereof having at least 65% homology to Atr_Δ11 (SEQ ID NO:1), Sl_Δ11 (SEQ ID NO:2), As_Δ11 (SEQ ID NO:3), Lbop_PPTQ (SEQ ID NO:43), Dgd9 (SEQ ID NO:44), Dvd9 (SEQ ID NO:45) or Tni_Δ11 (SEQ ID NO:4), such as at least 70% homology, for example at least 71% homology, such as at least 72%, for example at least 73%, such as at least 74%, for example at least 75%, such as at least 80%, for example at least 85%, such as at least 90%, for example at least 95%, such as 100% homology; and an alcohol-forming fatty acyl-CoA reductase (FAR) selected from the group consisting of Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), and Has_FAR (SEQ ID NO: 7), or a variant thereof having at least 80% homology with Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), or Has_FAR (SEQ ID NO: 7), such as at least 85%, for example at least 90%, such as at least 95%, for example 100% homology; thereby expressing a Δ11-desaturase capable of converting at least a portion of the hexadecanoyl-CoA to (Z)11-hexadecenoyl-CoA; and -FAR can convert at least a portion of the (Z)11-hexadecenol into (Z)11-hexadecenol; 53. The method according to any one of items 1 to 52. 54. The method of item 53, wherein the yeast cell further expresses a fatty acyl synthetase (FAA) such as Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9), or a variant thereof having at least 75% homology to Sc_FAA1 (SEQ ID NO: 8) or Yl_FAA (SEQ ID NO: 9), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology. 55. The method further comprising converting at least a portion of the (Z)-11-hexadecen-1-ol to (Z)-11-hexadecen-1-yl acetate by chemical conversion, or by expression from the yeast cell of an acetyltransferase, such as a heterologous acetyltransferase (AcT), or by overexpression of a native acetyltransferase from the yeast cell, wherein the acetyltransferase is capable of converting at least a portion of the (Z)-11-hexadecen-1-ol to (Z)11-hexadecen-1-yl acetate, preferably the acetyltransferase is selected from the group consisting of S 55. The method according to item 53 or 54, wherein the variant has at least 75% homology to c_Atf1 (SEQ ID NO: 10), such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology to Sc_Atf1 (SEQ ID NO: 10). 56. The hydrophobic compound is an unsaturated fatty alcohol, and the microorganism is an oleaginous yeast cell, such as a Yarrowia cell, e.g., a Yarrowia lipolytica (alkane-utilizing Yeast) cell, capable of producing the unsaturated fatty alcohol, wherein the oleaginous yeast cell: - expressing at least one heterologous desaturase capable of introducing at least one double bond into fatty acyl-CoA; and - expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol; and - has a mutation resulting in a reduced activity of Fao1 (SEQ ID NO: 11) and a mutation resulting in a reduced activity of at least one of Hfd1 (SEQ ID NO: 12), Hfd4 (SEQ ID NO: 13), Pex10 (SEQ ID NO: 14) and GPAT (SEQ ID NO: 15), or has a mutation resulting in a reduced activity of at least one protein having at least 90% homology with Fao1 (SEQ ID NO: 11) and at least one of Hfd1 (SEQ ID NO: 12), Hfd4 (SEQ ID NO: 13), Pex10 (SEQ ID NO: 14) and GPAT (SEQ ID NO: 15), such as at least 91% homology, for example at least 92% homology, such as at least 93% homology, for example at least 94% homology, such as at least 95% homology, for example at least 96% homology, such as at least 97% homology, for example at least 98% homology, for example at least 99% homology, 56. The method according to any one of items 1 to 55. 57. The at least one heterologous desaturase is selected from the group consisting of Δ3 desaturase, Δ5 desaturase, Δ6 desaturase, Δ7 desaturase, Δ8 desaturase, Δ9 desaturase, Δ10 desaturase, Δ11 desaturase, Δ12 desaturase, Δ13 desaturase and Δ14 desaturase, preferably the desaturase is from an insect, such as from the order Lepidoptera, preferably the desaturase is a Δ11 desaturase having at least 60% homology to the Δ11 desaturase from Amielois transitella (walnut moth) set forth in SEQ ID NO:1, or the Δ11 desaturase from Drosophila spp. set forth in SEQ ID NO:16. 57. The method of Item 56, wherein the desaturase is a Δ9 desaturase having at least 60% identity to the Δ9 desaturase derived from Drosophila melanogaster (Drosophila melanogaster), a desaturase having at least 60% identity to the desaturase derived from Lobesia botulana (the grain leaf moth) as set forth in SEQ ID NO:43, a desaturase having at least 60% identity to the desaturase derived from Drosophila grimshauii (Hawaiian fruit fly) as set forth in SEQ ID NO:44, or a desaturase having at least 60% identity to the desaturase derived from Drosophila virilis (Drosophila virilis) as set forth in SEQ ID NO:45. 58. Fatty acyl reductase (FAR) is: i) a FAR having at least 80% homology to the FAR from Helicoverpa armigera (Helicoverpa armigera) set forth in SEQ ID NO: 5; ii) a FAR having at least 80% homology with FAR from Helicoverpa assulta (tobacco budworm) as set forth in SEQ ID NO: 7; iii) a FAR having at least 80% homology to the FAR from Heliothis subflexa (the nightshade moth) set forth in SEQ ID NO: 6; and iv) FAR having at least 80% homology with FAR from Bicyclus annina (Satyridae) as set forth in SEQ ID NO: 17; 58. The method according to item 56 or 57, wherein the FAR is selected from the group consisting of: FAR from Helicoverpa armigera (helicobacterium armigera) and FAR from Heliothis subflexa (ground cherry moth), and preferably FAR has at least 80% homology with FAR from Helicoverpa armigera (helicobacterium armigera) or FAR from Heliothis subflexa (ground cherry moth). 59. The method of claim 1, further comprising converting at least a portion of the unsaturated fatty alcohols to fatty acyl acetates by chemical conversion, or by expression from the oleaginous yeast cell of an acetyltransferase, such as a heterologous acetyltransferase (AcT), or by overexpression of a native acetyltransferase from the oleaginous yeast cell, wherein the acetyltransferase is capable of converting at least a portion of the unsaturated fatty alcohols to fatty acyl acetates, preferably the acetyltransferase is selected from the group consisting of Sc_Atf1 (SEQ ID NO: 10), Sc_Atf1 (SEQ ID NO: 11), Sc_Atf1 (SEQ ID NO: 12), Sc_Atf1 (SEQ ID NO: 13), Sc_Atf1 (SEQ ID NO: 14), Sc_Atf1 (SEQ ID NO: 15), Sc_Atf1 (SEQ ID NO: 16), Sc_Atf1 (SEQ ID NO: 17), Sc_Atf1 (SEQ ID NO: 18), Sc_Atf1 (SEQ ID NO: 19), Sc_Atf1 (SEQ ID NO: 20), Sc_Atf1 (SEQ ID NO: 21), Sc_Atf1 (SEQ ID NO: 22), Sc_Atf1 (SEQ ID NO: 23), Sc_Atf1 (SEQ ID NO: 24), Sc_Atf1 (SEQ ID NO: 25), Sc_Atf1 (SEQ ID NO: 26), Sc_Atf1 (SEQ ID NO: 27), Sc_Atf1 (SEQ ID NO: 28), Sc_Atf1 (SEQ ID NO: 29), Sc_Atf1 (SEQ ID NO: 30), Sc_Atf1 (SEQ ID NO: 31), Sc_Atf1 (SEQ ID NO: 32), 59. The method according to any one of items 56 to 58, wherein the polypeptide is a variant thereof having at least 75% homology, such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology to the polypeptide of item 10). 60. The hydrophobic compound is an unsaturated fatty alcohol, and the microorganism is a yeast cell, such as a Yarrowia cell, e.g., a Yarrowia lipolytica (alkane-utilizing Yeast) cell, capable of producing the unsaturated fatty alcohol, wherein the yeast cell: at least one heterologous desaturase capable of introducing at least one double bond into a fatty acyl-CoA having a carbon chain length of −14; and at least one heterologous fatty acyl-CoA reductase (FAR) capable of converting at least a portion of said unsaturated fatty acyl-CoAs into unsaturated fatty alcohols, 53. The method according to any one of items 1 to 52, wherein the gene expresses 61. The at least one heterologous desaturase is derived from an organism selected from Pelargonium holtorum (geranium), Ricinus communis (castor bean), Drosophila melanogaster (Drosophila melanogaster), Spodoptera litura (spodoptera litura) and Tribolium castanium (red flour beetle), preferably the desaturase is derived from Drosophila melanogaster (Drosophila melanogaster), preferably the at least one heterologous desaturase is derived from: i) a Δ9 desaturase having at least 60% homology to the Δ9 desaturase from Drosophila melanogaster (Drosophila melanogaster) set forth in SEQ ID NO: 16; ii) a Δ9 desaturase having at least 60% homology to the Δ9 desaturase from Spodoptera litura (common cutworm) set forth in SEQ ID NO: 18; iii) a desaturase having at least 60% homology to the desaturase from Lobesia botlana (grass leaf moth) set forth in SEQ ID NO: 43; iv) a desaturase having at least 60% identity to the desaturase from Drosophila grimshawii (Hawaiian fruit fly) set forth in SEQ ID NO: 44; v) a desaturase having at least 60% homology to the desaturase from Drosophila virilis (fruit fly) set forth in SEQ ID NO: 45; vi) a Δ11 desaturase having at least 60% identity to the Δ11 desaturase from Choristoneura parallela (Lepidoptera: Tortricidae) set forth in SEQ ID NO: 42; vii) a Δ11 desaturase having at least 60% identity to the Δ11 desaturase from Choristoneura rosacea (lotus band moth) set forth in SEQ ID NO: 35; 61. The method of item 60, selected from the group consisting of: 62. Fatty acyl-CoA reductase (FAR) is: i) a FAR having at least 80% homology to the FAR from Helicoverpa armigera (Helicoverpa armigera) set forth in SEQ ID NO: 5; ii) a FAR having at least 80% homology with FAR from Helicoverpa assulta (tobacco budworm) as set forth in SEQ ID NO: 7; iii) a FAR having at least 80% homology to the FAR from Heliothis subflexa (the nightshade moth) set forth in SEQ ID NO: 6; and iv) FAR having at least 80% homology with FAR from Bicyclus annina (Satyridae) as set forth in SEQ ID NO: 17; The method of claim 60 or 61, wherein the FAR is selected from the group consisting of, preferably a FAR having at least 80% homology to FAR from Helicoverpa armigera (helicoverpa armigera) as shown in SEQ ID NO: 5. 63. The method of claim 1, further comprising converting at least a portion of the unsaturated fatty alcohols to fatty acyl acetates by chemical conversion, or by expression from the oleaginous yeast cell of an acetyltransferase, such as a heterologous acetyltransferase (AcT), or by overexpression from the oleaginous yeast cell of a native acetyltransferase, wherein the acetyltransferase is capable of converting at least a portion of the unsaturated fatty alcohols to fatty acyl acetates, preferably the acetyltransferase is selected from the group consisting of Sc_Atf1 (SEQ ID NO: 10), Sc_Atf1 (SEQ ID NO: 11), Sc_Atf1 (SEQ ID NO: 12), Sc_Atf1 (SEQ ID NO: 13), Sc_Atf1 (SEQ ID NO: 14), Sc_Atf1 (SEQ ID NO: 15), Sc_Atf1 (SEQ ID NO: 16), Sc_Atf1 (SEQ ID NO: 17), Sc_Atf1 (SEQ ID NO: 18), Sc_Atf1 (SEQ ID NO: 19), Sc_Atf1 (SEQ ID NO: 20), Sc_Atf1 (SEQ ID NO: 21), Sc_Atf1 (SEQ ID NO: 22), Sc_Atf1 (SEQ ID NO: 23), Sc_Atf1 (SEQ ID NO: 24), Sc_Atf1 (SEQ ID NO: 25), Sc_Atf1 (SEQ ID NO: 26), Sc_Atf1 (SEQ ID NO: 27), Sc_Atf1 (SEQ ID NO: 28), Sc_Atf1 (SEQ ID NO: 29), Sc_Atf1 (SEQ ID NO: 30), Sc_Atf1 (SEQ ID NO: 31), Sc_Atf1 (SEQ ID NO: 32), 63. The method according to any one of items 60 to 62, wherein the polypeptide is a variant thereof having at least 75% homology, such as at least 80% homology, for example at least 85% homology, such as at least 90% homology, for example at least 91% homology, such as at least 92% homology, for example at least 93% homology, such as at least 94% homology, for example at least 95% homology, such as at least 96% homology, for example at least 97% homology, such as at least 98% homology, for example at least 99% homology, such as 100% homology to the polypeptide of item 10). 64. The hydrophobic compound is an unsaturated fatty alcohol, the microorganism is a yeast cell capable of producing the unsaturated fatty alcohol, and the yeast cell: -having one or more mutations that result in reduced activity of one or more native acyl-CoA oxidases; and expressing at least one first group of enzymes including at least one acyl-CoA oxidase capable of oxidizing a fatty acyl-CoA, wherein the first group of enzymes is capable of shortening a fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', where X'≦X-2; and - expressing at least one heterologous desaturase capable of introducing at least one double bond into said fatty acyl-CoA and / or into said truncated fatty acyl-CoA; and expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol; 53. The method according to any one of items 1 to 52. 65. The method according to item 64, wherein the native acyl-CoA oxidase and / or heterologous acyl-CoA oxidase is a peroxisomal acyl-CoA oxidase. 66. At least one acyl-CoA oxidase of the first group of enzymes is a native acyl-CoA oxidase or a heterologous acyl-CoA oxidase, which is optionally overexpressed compared to a reference yeast strain that does not express said at least one first group of enzymes, preferably at least one acyl-CoA oxidase of the first group of enzymes is a heterologous acyl-CoA oxidase, and optionally at least one first group of enzymes is selected from the group consisting of Yarrowia, Agrotis, Arabidopsis, Aspergillus, and Cucurbita. , Homo, Penalthrobacter and Rattus, and preferably at least one of the enzymes of the first group is derived from an organism of a genus selected from Yarrowia lipolytica (alkane-utilizing yeast), Agrotis segetum (turnip moth), Arabidopsis thaliana (Arabidopsis thaliana), Aspergillus nidulans (pseudo-infesting aspergillus), Cucurbita maxima (chestnut squash), Homo sapiens (human), Penalthrobacter ureafaciens (nylon-eating bacteria) or Rattus norvegicus (seed oil plant). The present invention relates to an acyl-CoA oxidase derived from a plant, and the acyl-CoA oxidase of the first group of enzymes is preferably selected from the group consisting of Yli_POX1 (SEQ ID NO: 19), Yli_POX2 (SEQ ID NO: 20), Yli_POX3 (SEQ ID NO: 21), Yli_POX4 (SEQ ID NO: 22), Yli_POX5 (SEQ ID NO: 23), Yli_POX6 (SEQ ID NO: 24), Ase_POX (SEQ ID NO: 25), Ath_POX1 (SEQ ID NO: 26), Ath_POX2 (SEQ ID NO: 27), Ani_POX (SEQ ID NO: 28), Cma_POX (SEQ ID NO: 29), Hsa_POX (SEQ ID NO: 30), Hsa_POX (SEQ ID NO: 31), Hsa_POX (SEQ ID NO: 32), Hsa_POX (SEQ ID NO: 33), Hsa_POX (SEQ ID NO: 34), Hsa_POX (SEQ ID NO: 35), Hsa_POX (SEQ ID NO: 36), Hsa_POX (SEQ ID NO: 37), Hsa_POX (SEQ ID NO: 38), Hsa_POX (SEQ ID NO: 39), Hsa_POX (SEQ ID NO: 40), Hsa_POX (SEQ ID NO: 41), Hsa_POX (SEQ ID NO: 42), Hsa_POX (SEQ ID NO: 43), Hsa_POX (SEQ ID NO: 44), Hsa_POX (SEQ ID NO: 45), Hsa_POX (SEQ ID NO: 46), Hsa_POX (SEQ ID NO: 47), Hsa_POX (SEQ ID NO: 48), Hsa_POX (S POX1-2 (SEQ ID NO: 30), Pur_POX (SEQ ID NO: 31), and Rno_POX2 (SEQ ID NO: 32), or at least 60% homology thereto, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%,66. The method according to item 64 or 65, wherein the acyl-CoA oxidase is selected from the group consisting of, for example, an acyl-CoA oxidase having at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% homology thereto, and functional variants thereof. 67. The at least one heterologous desaturase is selected from the group consisting of Δ3 desaturase, Δ5 desaturase, Δ6 desaturase, Δ7 desaturase, Δ8 desaturase, Δ9 desaturase, Δ10 desaturase, Δ11 desaturase, Δ12 desaturase, Δ13 desaturase and Δ14 desaturase, and / or the desaturase is from a yeast, such as Saccharomyces or Yarrowia, such as Saccharomyces cerevisiae (budding yeast) or Yarrowia lipolytica (alkane-utilizing yeast), or from an organism such as Drosophila melanogaster (Drosophila melanogaster), Amielois transitella (walnut moth), Choristoneura rosacea (lotus band moth), 67. The method according to any one of items 64 to 66, wherein the insect is from an insect such as from the order Diptera, Coleoptera, or Lepidoptera, such as from the genus Amielois, Chorystoneura, Drosophila, Ostrinia, Thaumetpoea, Dendrophilus, Grahorita, Scidia, Epiphias, or Spodoptera, such as Ostrinia nubilalis (European corn borer), Thaumetpoea piciocampa (pine moth), Dendrophilus punctatus, Grahorita molesta (pear fruit moth), Scidia pomonella (codling moth), Epiphias postificittana (apple leaf moth), Spodoptera littoralis (African armyworm) or Chorystoneura parallella (Lepidoptera: Tortricidae). 68. Desaturase is Δ Z9 -desaturase, such as Sce_OLE1 (SEQ ID NO: 33), Yli_OLE1 (SEQ ID NO: 34) or Dme_D9 (SEQ ID NO: 16), Δ Z11-desaturases, such as Atr_D11 (SEQ ID NO: 1), Cro_Z11 (SEQ ID NO: 35), Onu_11 (SEQ ID NO: 36), Tpi_D13 (SEQ ID NO: 37), Δ E9 -desaturases, such as Dpu_E9-14 (SEQ ID NO: 38), Δ Z / E10 a desaturase, such as Gmo_CPRQ (SEQ ID NO: 39), or a desaturase, such as Epo_E11 (SEQ ID NO: 40), Sls_ZE11 (SEQ ID NO: 41), Lbo_PPTQ (SEQ ID NO: 43), Dgd9 (SEQ ID NO: 44), Dvd9 (SEQ ID NO: 45) or Cpa_E11 (SEQ ID NO: 42), or at least 60% homology thereto, such as at least 65%, for example at least 70%, such as at least 75%, for example at least 80%, such as at least 81%, for example at least 82%, such as at least 83% 68. The method according to any one of items 64 to 67, wherein the nucleic acid sequence of the present invention is a functional variant thereof having at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% homology thereto. 69. The method according to any one of items 64 to 68, wherein the fatty acyl-CoA reductase is derived from an insect such as an insect of the order Lepidoptera, such as the genus Helicoverpa, Heliothis or Bicyclus, preferably the fatty acyl-CoA reductase is a fatty acyl-CoA reductase characteristic of Helicoverpa armigera (helicobacterium armigera), Helicoverpa assulta, Heliothis su...

Claims

1. 1. A method for producing a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes by fermentation, comprising the steps of providing yeast cells capable of producing the hydrophobic compound and culturing the yeast cells in a medium under conditions that allow production of the hydrophobic compound, wherein the culturing step is carried out at a culture temperature, the medium containing an extractant in an amount equal to or greater than its cloud concentration as measured in an aqueous solution, and the extractant is a non-ionic ethoxylated surfactant, and the method further comprises the step of recovering the hydrophobic compound.

2. 1. A method for increasing the titer of a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes in fermentation, the method comprising culturing yeast cells capable of producing the hydrophobic compound in a medium under conditions that allow the production of the hydrophobic compound, wherein the culturing step is carried out at a culture temperature, the medium containing an extractant in an amount equal to or greater than its cloud concentration measured in aqueous solution at the culture temperature, and the extractant is a non-ionic ethoxylated surfactant, whereby the titer of the hydrophobic compound is increased compared to fermentation carried out under the same conditions but in the absence of the extractant or in the presence of the extractant in an amount less than its cloud concentration measured in aqueous solution at the culture temperature.

3. 1. A method for enhancing secretion of a hydrophobic compound selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes from yeast cells capable of producing said hydrophobic compound during fermentation, the method comprising culturing said yeast cells in a medium under conditions that allow production of said hydrophobic compound, wherein said culturing step is carried out at a culture temperature, and said medium comprises an extractant in an amount equal to or greater than its cloud concentration measured in aqueous solution at said culture temperature, said extractant being a non-ionic ethoxylated surfactant, whereby secretion of said hydrophobic compound from said yeast cells is enhanced compared to fermentation carried out under the same conditions but in the absence of the extractant or in the presence of the extractant in an amount less than its cloud concentration measured in aqueous solution at said culture temperature.

4. The nonionic ethoxylated surfactant is a fatty alcohol alkoxylate or a polyethoxylated surfactant, or the nonionic ethoxylated surfactant is a polyethoxylated nonionic surfactant comprising or consisting essentially of a mixture of polyether dispersions, polyethylene glycol monostearate, simethicone, and ethoxylated and propoxylated C １６ -C １８ Alcohol-based agents or ethoxylated and propoxylated C １６ -C １８ The method of any one of claims 1 to 3, wherein the antifoaming agent is selected from the group consisting of alcohol-based antifoaming agents and combinations thereof.

5. 5. The method according to claim 1, wherein the fatty alcohol is a saturated fatty alcohol, an unsaturated fatty alcohol, or a mixture thereof, and / or the fatty acyl acetate is a saturated fatty acyl acetate, an unsaturated fatty acyl acetate, or a mixture thereof, and / or the fatty aldehyde is a saturated fatty aldehyde, an unsaturated fatty aldehyde, or a mixture thereof.

6. The method according to any one of claims 1 to 5, wherein the terpene is a sesquiterpene.

7. The method of any one of claims 1 to 6, wherein the fatty alcohol ester is a fatty alcohol acetate ester.

8. The nonionic ethoxylated surfactant is C １６ -C １８ 8. The method of any one of claims 1 to 7, wherein the surfactant is selected from alkyl alcohol ethoxylate propoxylate (CAS No. 68002-96-0), Agnique BP420 (CAS No. 68002-96-0), polyethylene polypropylene glycol, Antifoam 204, surfactants including polyethylene glycol monostearate, fatty alcohol alkoxylates, and Imbentin SG / 251 (CAS No. 68002-96-0).

9. 9. The method of any one of claims 1 to 8, wherein the medium comprises at least 1% vol / vol of the non-ionic ethoxylated surfactant, and / or the medium comprises the extractant in an amount at least 50% greater than its cloud concentration, and / or the medium comprises the extractant in an amount at least twice its cloud concentration.

10. 10. The method of any one of claims 1 to 9, wherein the hydrophobic compounds produced by the microorganism are present in an emulsion in the fermentation broth, and further comprising the step of breaking the emulsion, thereby obtaining a composition comprising the extractant and a product phase comprising the hydrophobic compounds.

11. 11. The method of claim 10, wherein the step of breaking the emulsion comprises or consists of a step of phase separation of the fermentation broth, thereby obtaining a composition consisting of three phases: an aqueous phase, a phase containing cells and necrotic cell debris, and a product phase containing extractant and hydrophobic compounds, and / or wherein the product phase comprises at least 50% of the hydrophobic compounds initially present in the fermentation broth.

12. 12. The method of claim 10 or 11, further comprising recovering a product phase comprising the extractant and the hydrophobic compound from the composition.

13. 13. The method of any one of claims 1 to 12, wherein the hydrophobic compounds are one or more fatty alcohols, and further comprising recovering the one or more fatty alcohols.

14. 14. The method of claim 13, further comprising chemically converting at least a portion of the fatty alcohols of the mixture of fatty alcohols to the corresponding fatty acyl acetates.

15. 15. The method of any one of claims 10 to 14, further comprising chemically converting at least a portion of the fatty alcohols of the mixture of fatty alcohols to the corresponding fatty aldehydes.

16. 16. The method of any one of claims 1 to 15, wherein the extractant is recovered from a fermentation broth.

17. 17. The method according to any one of claims 1 to 16, wherein the titer of the hydrophobic compounds is increased by at least 5% compared to the titer obtained in a fermentation carried out under the same conditions but either in the absence of extractant or in the presence of extractant in an amount lower than its cloud concentration measured in aqueous solution, and / or the secretion of the hydrophobic compounds is increased by at least 5% compared to a fermentation carried out under the same conditions but either in the absence of extractant or in the presence of extractant in an amount lower than its cloud concentration measured in aqueous solution.

18. 18. The method according to any one of claims 1 to 17, wherein the yeast is a yeast of the genus Saccharomyces, Pichia, Yarrowia, Kluyveromyces, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon or Lipomyces, and / or the yeast is a yeast of a species selected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces marxianus, Cryptococcus albidus, Lipomyces lipofera, Lipomyces starkii, Rhodosporidium toruloides, Rhodotorula glutinis, Trichosporon pullulans or Yarrowia lipolytica.

19. The method of any one of claims 1 to 18, wherein the yeast is Yarrowia lipolytica or Saccharomyces cerevisiae.

20. 20. The method of any one of claims 1 to 19, wherein the hydrophobic compound is selected from the group consisting of unsaturated fatty alcohols, acetate esters of saturated or unsaturated fatty alcohols, and fatty aldehydes.

21. the unsaturated fatty alcohol is (Z)-11-hexadecen-1-ol, (Z)9-hexadecen-1-ol, (Z)11-tetradecen-1-ol, (E)11-tetradecen-1-ol, (Z)9-tetradecen-1-ol, or E8,E10-dodecadien-1-ol, and / or b. the acetate ester of an unsaturated fatty alcohol is (Z) 9-tetradecen-1-yl acetate or E8, E10-dodecadienyl acetate, and / or c. the fatty aldehyde is an unsaturated fatty aldehyde, and / or d) The method of claim 20, wherein the fatty aldehyde is (Z)11-hexadecen-1-al or E8,E10-dodecadienal.

22. 22. The method of any one of claims 1 to 21, wherein the yeast cells are capable of producing the hydrophobic compound at a titer of at least 0.2 mg / L.

23. The yeast cell comprises: Amielois transitella (walnut moth) Δ11-desaturase (Atr_Δ11; SEQ ID NO: 1), Spodoptera littoralis (African armyworm) Δ11-desaturase (Sl_Δ11; SEQ ID NO: 2), Agrotis segetum (turnip cutworm moth) Δ11-desaturase (As_Δ11; SEQ ID NO: 3), Lobesia botlana (grass leaf moth) desaturase (Lbo_PPTQ; SEQ ID NO: 43), Drosophila grimshaui (Hawaiian fruit fly) desaturase (Dgd9; SEQ ID NO: 44), Drosophila bili a Δ11-desaturase selected from the group consisting of Drosophila virilis (Drosophila virilis) desaturase (Dvd9; SEQ ID NO:45) and Trichoplasia ni (stem looper) Δ11-desaturase (Tni_Δ11; SEQ ID NO:4), or a functional variant thereof having at least 90% identity to Atr_Δ11 (SEQ ID NO:1), Sl_Δ11 (SEQ ID NO:2), As_Δ11 (SEQ ID NO:3), Lbop_PPTQ (SEQ ID NO:43), Dgd9 (SEQ ID NO:44), Dvd9 (SEQ ID NO:45) or Tni_Δ11 (SEQ ID NO:4); and an alcohol-forming fatty acyl-CoA reductase (FAR) selected from the group consisting of Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), and Has_FAR (SEQ ID NO: 7), or a variant thereof having at least 90% identity to Har_FAR (SEQ ID NO: 5), Hs_FAR (SEQ ID NO: 6), or Has_FAR (SEQ ID NO: 7); thereby expressing - the Δ11-desaturase is capable of converting at least a portion of the hexadecanoyl-CoA to (Z)11-hexadecenoyl-CoA; and the FAR is capable of converting at least a portion of the (Z)11-hexadecenoyl-CoA to (Z)11-hexadecenol; The method according to any one of claims 1 to 22.

24. 24. The method of claim 23, wherein the yeast cell further expresses a fatty acyl synthetase.

25. 25. The method of claim 24, further comprising converting at least a portion of the (Z)-11-hexadecen-1-ol to (Z)-11-hexadecen-1-yl acetate by chemical conversion or by expression of an acetyltransferase, wherein the acetyltransferase is capable of converting at least a portion of the (Z)-11-hexadecen-1-ol to the (Z)-11-hexadecen-1-yl acetate.

26. The acetyltransferase a. Heterologous acetyltransferase (AcT); b. a native AcT that is native to the yeast cell and is overexpressed; c. Sc_Atf1 (SEQ ID NO: 10) or a functional variant thereof having at least 90% identity with Sc_Atf1 (SEQ ID NO: 10); 26. The method of claim 25, wherein the compound is selected from the group consisting of:

27. wherein the hydrophobic compound is an unsaturated fatty alcohol and the yeast is an oleaginous yeast cell capable of producing the unsaturated fatty alcohol, the oleaginous yeast cell comprising: - expressing at least one heterologous desaturase capable of introducing at least one double bond into fatty acyl-CoA; and expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol; and - has a mutation resulting in a reduced activity of Fao1 and a mutation resulting in a reduced activity of at least one of Hfd1, Hfd4, Pex10 and GPAT, or has a mutation resulting in a reduced activity of at least one protein having at least 90% identity with Fao1 as set forth in SEQ ID NO: 11, and a mutation resulting in a reduced activity of at least one of Hfd1 as set forth in SEQ ID NO: 12, Hfd4 as set forth in SEQ ID NO: 13, Pex10 as set forth in SEQ ID NO: 14 and GPAT as set forth in SEQ ID NO: 15, The method according to any one of claims 1 to 26.

28. a. the at least one heterologous desaturase is selected from the group consisting of a Δ3 desaturase, a Δ5 desaturase, a Δ6 desaturase, a Δ7 desaturase, a Δ8 desaturase, a Δ9 desaturase, a Δ10 desaturase, a Δ11 desaturase, a Δ12 desaturase, a Δ13 desaturase, and a Δ14 desaturase; b. the at least one heterologous desaturase is derived from an insect, and / or c. the at least one heterologous desaturase is a Δ11 desaturase having at least 90% identity to the Δ11 desaturase from Amielois transitella set forth in SEQ ID NO:1, a Δ9 desaturase having at least 90% identity to the Δ9 desaturase from Drosophila melanogaster set forth in SEQ ID NO:16, a desaturase having at least 90% identity to the desaturase from Lobesia botulana set forth in SEQ ID NO:43, a desaturase having at least 90% identity to the desaturase from Drosophila grimshauii set forth in SEQ ID NO:44, and a desaturase having at least 90% identity to the desaturase from Drosophila bililis set forth in SEQ ID NO:45; 28. The method of claim 27.

29. the hydrophobic compound is an unsaturated fatty alcohol and the yeast is capable of producing the unsaturated fatty alcohol, and the yeast cells: at least one heterologous desaturase capable of introducing at least one double bond into a fatty acyl-CoA having a carbon chain length of −14; and at least one heterologous fatty acyl-CoA reductase (FAR) capable of converting at least a portion of the unsaturated fatty acyl-CoAs into unsaturated fatty alcohols; The method according to any one of claims 1 to 28, wherein the vector expresses

30. a. the at least one heterologous desaturase is derived from an organism selected from Pelargonium holtorum, Ricinus communis, Drosophila melanogaster, Spodoptera litura, and Tribolium castanium, and / or b. At least one heterologous desaturase comprises: i) a Δ9 desaturase having at least 90% identity to the Δ9 desaturase from Drosophila melanogaster as set forth in SEQ ID NO:16; ii) a Δ9 desaturase having at least 90% identity to the Δ9 desaturase from Spodoptera litura set forth in SEQ ID NO:18; iii) a desaturase having at least 90% identity to the desaturase from Lobesia botolana set forth in SEQ ID NO: 43; iv) a desaturase having at least 90% identity to the desaturase from Drosophila grimshauii set forth in SEQ ID NO:44; and v) a desaturase having at least 90% identity to the desaturase derived from Drosophila bililis set forth in SEQ ID NO: 45; 30. The method of claim 29, selected from the group consisting of:

31. The hydrophobic compound is codlemone (E8,E10-dodecadien-1-ol) or one or more of its derivatives E8,E10-dodecadienyl acetate and / or E8,E10-dodecadienal, and the yeast cell expresses at least one heterologous desaturase capable of introducing one or more double bonds into fatty acyl-CoAs having a carbon chain length of 12, thereby converting the fatty acyl-CoAs into unsaturated fatty acyl-CoAs, and at least a portion of the unsaturated fatty acyl-CoAs are E8,E10- 31. The method of any one of claims 1 to 30, further expressing at least one heterologous fatty acyl-CoA reductase (EC 1.2.1.84) capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol, wherein the fatty acyl-CoA reductase is capable of converting at least a portion of the E8,E10-dodecadienyl coenzyme A (E8,E10-C12:CoA) into E8,E10-dodecadien-1-ol.

32. the at least one desaturase a. Cpo_CPRQ (SEQ ID NO: 48), or a functional variant thereof having at least 90% identity to SEQ ID NO: 48, or b. a variant of Cpo_CPRQ with a mutation at position 85, or c. at least two desaturases, wherein at least one of the two desaturases is Cpo_CPRQ (SEQ ID NO:48) or a functional variant thereof having at least 90% identity to SEQ ID NO:48, and another desaturase is: i. a variant of Cpo_CPRQ with a mutation at position 85; ii. a desaturase capable of introducing at least one double bond into a fatty acyl-CoA having a carbon chain length of 12; iii. Z9-12 desaturase, iv. Cpo_NPVE (SEQ ID NO: 49) or a functional variant thereof having at least 90% identity to SEQ ID NO: 49, and v. Cpo_SPTQ (SEQ ID NO: 50) or a functional variant thereof having at least 90% identity to SEQ ID NO: 50; 32. The method of claim 31 , wherein the compound is selected from the group consisting of:

33. the hydrophobic compound is an unsaturated fatty alcohol and the yeast cells are - have one or more mutations that result in reduced activity of one or more native acyl-CoA oxidases; and expressing at least one first group of enzymes comprising at least one acyl-CoA oxidase capable of oxidizing a fatty acyl-CoA, wherein the first group of enzymes is capable of shortening a fatty acyl-CoA of a first carbon chain length X to a shortened fatty acyl-CoA having a second carbon chain length X', where X'≦X-2; - expressing at least one heterologous desaturase capable of introducing at least one double bond into said fatty acyl-CoA and / or into said truncated fatty acyl-CoA; and expressing at least one heterologous fatty acyl-CoA reductase capable of converting at least a portion of the unsaturated fatty acyl-CoA into an unsaturated fatty alcohol; 33. The method according to any one of claims 1 to 32.

34. and / or wherein the at least one first group enzyme comprises an acyl-CoA oxidase selected from the group consisting of Yli_POX1 (SEQ ID NO: 19), Yli_POX2 (SEQ ID NO: 20), Yli_POX3 (SEQ ID NO: 21), Yli_POX4 (SEQ ID NO: 22), Yli_POX5 (SEQ ID NO: 23), Yli_POX6 (SEQ ID NO: 24), Ase_POX (SEQ ID NO: 25), Ath_POX1 (SEQ ID NO: 26), Ath_POX2 (SEQ ID NO: 27), Ani_POX (SEQ ID NO: 28), Cma_POX (SEQ ID NO: 29), Hsa_POX1-2 (SEQ ID NO: 30), Pur_POX (SEQ ID NO: 31), and Rno_POX2 (SEQ ID NO: 32), and a functional variant thereof having at least 90% identity thereto; and / or The at least one heterologous desaturase is selected from the group consisting of: Ｚ９ -desaturase, Δ Ｚ１１ -desaturase, Δ Ｅ９ -desaturase, Δ Ｚ／Ｅ１０ - desaturases selected from the group consisting of Epo_E11 (SEQ ID NO:40), Sls_ZE11 (SEQ ID NO:41), Lbo_PPTQ (SEQ ID NO:43), Dgd9 (SEQ ID NO:44), Dvd9 (SEQ ID NO:45) and Cpa_E11 (SEQ ID NO:42), or functional variants thereof having at least 90% identity thereto; and / or 34. The method of claim 33, wherein the fatty acyl-CoA reductase is selected from the group consisting of a fatty acyl-CoA reductase having at least 90% identity to Har_FAR (SEQ ID NO:5), Has_FAR (SEQ ID NO:7), Ban_FAR (SEQ ID NO:17), or Hs_FAR (SEQ ID NO:6).

35. 35. The method of any one of claims 26 to 34, further comprising converting at least a portion of the unsaturated fatty alcohol to a fatty acyl acetate by chemical conversion or by expression of an acetyltransferase from the yeast cell or by overexpression of a native acetyltransferase from the yeast cell, wherein the acetyltransferase is capable of converting at least a portion of the unsaturated fatty alcohol to an unsaturated fatty acyl acetate.

36. 36. The method of any one of claims 26 to 35, further comprising converting at least a portion of the unsaturated fatty alcohols to unsaturated fatty aldehydes by expression of at least one alcohol dehydrogenase and / or at least one fatty alcohol oxidase from the yeast cell.

37. 1. A method for monitoring the presence of or disrupting the mating of pests, comprising: i) producing a hydrophobic compound by a method according to any one of claims 1 to 36, wherein said hydrophobic compound is as defined in any one of claims 1 to 36, ii) formulating an unsaturated fatty alcohol, an unsaturated fatty acyl acetate, and / or an unsaturated fatty aldehyde as a pheromone composition; and iii) using said pheromone composition as an incorporated pest management composition; A method comprising:

38. 38. The method of claim 37, wherein the hydrophobic compound is selected from fatty alcohols, fatty alcohol esters, fatty acyl acetates, fatty aldehydes, and terpenes.

Images