Methods for the production and recovery of volatile molecules

Abstract

The present disclosure provides systems and methods for the production and recovery of volatile molecules, such as terpenes, and in particular, monoterpenes. By employing the systems and methods disclosed herein, volatile molecules may be recovered from the liquid contents of a bioreactor as well as the air space or headspace of the bioreactor, significantly improving overall recovery.

Description

[0001] METHODS FOR THE PRODUCTION AND RECOVERY OF VOLATILE MOLECULES

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under a Biosynthesis of High-Density Endothermic Fuels Challenge awarded by the Air Force Research Laboratory. The government has certain rights in this invention.

[0004] FIELD OF THE INVENTION

[0005] The present disclosure relates to methods to produce and recover volatile product molecules produced during the course of a fermentation or other method of cell culture.

[0006] BACKGROUND

[0007] In the field of synthetic biology, microorganisms are genetically modified to produce a target molecule of interest. Once produced, the molecule of interest may be recovered from the culture medium. Known methods can be employed to extract and purify the molecule of interest, whether it is retained within the host cell or secreted into the culture medium.

[0008] However, in the case of volatile products, such as monoterpenes (e.g., myrcene, limonene), product recovery may be greatly reduced due to a substantial portion of the product evaporating out of the culture medium into the fermenter headspace. This off gas is typically vented through an exhaust, resulting in loss of product. There is therefore a need in the art for systems and methods of capturing and recovering such volatile products.

[0009] SUMMARY OF THE INVENTION

[0010] Provided herein are apparatuses and methods for improved recovery of volatile products from a bioreactor, including a fermentation vessel. The apparatuses and methods described herein can be used, for example, in connection with a population of host cells genetically modified to produce a desired volatile target molecule, including by fermentation. Increased recovery of the volatile target molecule according to the present invention provides for more efficient and cost-effective production processes.

[0011] In one aspect, the present invention provides a method of recovering a volatile product, involving culturing a population of cells in a culture medium in a bioreactor at a first temperature, wherein the cells produce the volatile product, introducing gas into the bioreactor at a first rate, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels contains a water-immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and recovering the volatile product from the water-immiscible solvent.

[0012] In an embodiment, the volatile product is a terpene. In another embodiment, the terpene is a monoterpene or a hemiterpene. In yet another embodiment, the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene, camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

[0013] In a further embodiment, the terpene is myrcene. In another embodiment, the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water- immiscible solvent.

[0014] In an additional embodiment, the terpene is limonene. In yet another embodiment, the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0015] In one embodiment, the water-immiscible solvent is chilled. In yet another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -20°C. In yet another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -15°C. In still another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -10°C.

[0016] In an embodiment, the water-immiscible solvent is a polyalphaolefin. In a further embodiment, the polyalphaolefin is Durasyn 164.

[0017] In another embodiment, the one or more collection vessels further contains an antifoam or an antioxidant. In another embodiment, the antifoam is L-81 . In yet another embodiment, the antioxidant is butylated hydroxytoluene (BHT).

[0018] In one embodiment, the first temperature is between 25-40°C. In another embodiment, the first temperature is 30°C. In a further embodiment, the first temperature is 37°C.

[0019] In another embodiment, the first rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0020] In a further embodiment, the method further involves passing the gas carrying the volatile product through a condenser.

[0021] In an additional embodiment, the method further involves adjusting the first temperature to a second temperature. In another embodiment, the second temperature is suboptimal for production of the volatile product by the population of host cells. In another embodiment, the second temperature is higher than the first temperature. In yet another embodiment, the second temperature is between 30- 50°C.

[0022] In one embodiment, the method further involves adjusting the first rate to a second rate. In a further embodiment, the second rate is suboptimal for production of the volatile product by the population of host cells. In a further embodiment, the second rate is higher than the first rate. In yet another embodiment, the second rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0023] In some embodiments, culturing the population of cells occurs in the presence of an overlay. In one embodiment, the overlay is Drakeol-10, Durasyn 164, ESTEREX A 51 , JARCOL 1-16, Drakeol 19, soybean oil, castor oil, or sunflower oil. In another embodiment, the overlay is Drakeol-10. In another embodiment, the overlay is Durasyn 164.

[0024] In one embodiment, the bioreactor is a fermentation vessel. In an additional aspect, the present invention provides a method for recovering a volatile product from a bioreactor, involving introducing gas into the bioreactor at a rate that is suboptimal for production of the volatile product by a population of host cells, setting a temperature within the bioreactor that is suboptimal for production of the volatile product by a population of host cells, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels contains a water-immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and recovering the volatile product from the water-immiscible solvent.

[0025] In one embodiment, the volatile product is a terpene. In another embodiment, the terpene is a monoterpene or a hemiterpene. In a further embodiment, the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene, camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

[0026] In another embodiment, the terpene is myrcene. In an additional embodiment, the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0027] In a further embodiment, the terpene is limonene. In yet a further embodiment, the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0028] In some embodiments, the water-immiscible solvent is chilled. In one embodiment, the water- immiscible solvent is chilled at a temperature between 1 -20°C. In another embodiment, the water- immiscible solvent is chilled at a temperature between 1 -15°C. In yet another embodiment, the water- immiscible solvent is chilled at a temperature between 1 -10°C.

[0029] In one embodiment, the water-immiscible solvent is a polyalphaolefin. In an additional embodiment, the polyalphaolefin is Durasyn 164.

[0030] In a further embodiment, the one or more collection vessels further contains an antifoam or an antioxidant. In another embodiment, the antifoam is L-81 . In yet another embodiment, the antioxidant is butylated hydroxytoluene (BHT).

[0031] In an embodiment, the temperature is between 30-50°C. In another embodiment, the temperature is 35°C. In further embodiment, the temperature is 45°C.

[0032] In one embodiment, the rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0033] In another embodiment, the method further involves passing the gas carrying the volatile product through a condenser.

[0034] In a further embodiment, the bioreactor is a fermentation vessel.

[0035] BRIEF DESCRIPTION OF THE FIGURES FIG. 1 provides an exemplary schematic representing bench scale fermentation and recovery of a volatile fermentation product.

[0036] FIG. 2 provides an exemplary pilot scale (300 L) fermentation and off gas capture setup. Arrows denote the direction of gas flow in the system.

[0037] FIG. 3 provides exemplary dimensions for a primary myrcene trap column, such as the exemplary primary myrcene trap column depicted in FIG. 3.

[0038] FIG. 4 depicts myrcene captured in an off gas trap over the fermentation at a low air flow rate (open diamonds) and high air flow rate (closed circles). The two lines denote the best linear regression of each trap’s data, with the slope ‘b’ denoting the myrcene stripping rate in units of g myrcene / hour. R2 of the regression is shown for reference.

[0039] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0040] As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

[0041] As used herein, the term “bioreactor” refers to a device or system that supports biologically active organisms in a controlled environment. A bioreactor can be, for example, a fermentation vessel.

[0042] As used herein, the term “medium” refers to a culture medium and / or fermentation medium.

[0043] As used herein, the term “volatile” refers to the tendency of a substance to exist as a gas or vapor rather than as a liquid or solid.

[0044] As used herein, the term “water-immiscible solvent” refers to a solvent which does not form a homogenous mixture when added to water. A water-immiscible solvent may be an overlay. A water- immiscible solvent may be an alcohol (e.g., a C10-C20 alcohol). In some embodiments, the water- immiscible solvent is Drakeol 10. In other embodiments, the water-immiscible solvent is Durasyn 164. In other embodiments, the water-immiscible solvent is Jarcol-16. In some embodiments, the water- immiscible solvent is an oil. The water-immiscible solvent may also be, for example, corn oil, sunflower oil, soybean oil, mineral oil, polyalphaolefin, dodecane, hexadecane, oleyl alcohol, butyl oleate, dibutyl phthalate, dodecanol, dioctyl phthalate, farnesene, or isopropyl myristate.

[0045] Production and Recovery of Volatile Molecules

[0046] In one aspect, the present invention provides a method of recovering a volatile product, comprising culturing a population of cells in a culture medium in a bioreactor at a first temperature, wherein the cells produce the volatile product, introducing gas into the bioreactor at a first rate, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels comprises a water-immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and recovering the volatile product from the water-immiscible solvent.

[0047] In an embodiment, the volatile product is a terpene. In another embodiment, the terpene is a monoterpene or a hemiterpene. In yet another embodiment, the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene, camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

[0048] In a further embodiment, the terpene is myrcene. In another embodiment, the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water- immiscible solvent.

[0049] In an additional embodiment, the terpene is limonene. In yet another embodiment, the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0050] In one embodiment, the water-immiscible solvent is chilled. In yet another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -20°C. In yet another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -15°C. In still another embodiment, the water-immiscible solvent is chilled at a temperature between 1 -10°C.

[0051] In an embodiment, the water-immiscible solvent is a polyalphaolefin. In a further embodiment, the polyalphaolefin is Durasyn 164.

[0052] In another embodiment, the one or more collection vessels further comprises an antifoam or an antioxidant. In another embodiment, the antifoam is L-81 . In yet another embodiment, the antioxidant is butylated hydroxytoluene (BHT).

[0053] In one embodiment, the first temperature is between 25-40°C. In another embodiment, the first temperature is 30°C. In a further embodiment, the first temperature is 37°C.

[0054] In another embodiment, the first rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0055] In a further embodiment, the method further comprises passing the gas carrying the volatile product through a condenser.

[0056] In an additional embodiment, the method further comprises adjusting the first temperature to a second temperature. In another embodiment, the second temperature is suboptimal for production of the volatile product by the population of host cells. In another embodiment, the second temperature is higher than the first temperature. In yet another embodiment, the second temperature is between 30-50°C.

[0057] In one embodiment, the method further comprises adjusting the first rate to a second rate. In a further embodiment, the second rate is suboptimal for production of the volatile product by the population of host cells. In a further embodiment, the second rate is higher than the first rate. In yet another embodiment, the second rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0058] In some embodiments, culturing the population of cells occurs in the presence of an overlay. In one embodiment, the overlay is Drakeol-10, Durasyn 164, ESTEREX A 51 , JARCOL 1-16, Drakeol 19, soybean oil, castor oil, or sunflower oil. In another embodiment, the overlay is Drakeol-10. In another embodiment, the overlay is Durasyn 164.

[0059] In one embodiment, the bioreactor is a fermentation vessel. In an additional aspect, the present invention provides a method for recovering a volatile product from a bioreactor, comprising introducing gas into the bioreactor at a rate that is suboptimal for production of the volatile product by a population of host cells, setting a temperature within the bioreactor that is suboptimal for production of the volatile product by a population of host cells, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels comprises a water-immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and recovering the volatile product from the water-immiscible solvent.

[0060] In one embodiment, the volatile product is a terpene. In another embodiment, the terpene is a monoterpene or a hemiterpene. In a further embodiment, the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene, camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

[0061] In another embodiment, the terpene is myrcene. In an additional embodiment, the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0062] In a further embodiment, the terpene is limonene. In yet a further embodiment, the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

[0063] In some embodiments, the water-immiscible solvent is chilled. In one embodiment, the water- immiscible solvent is chilled at a temperature between 1 -20°C. In another embodiment, the water- immiscible solvent is chilled at a temperature between 1 -15°C. In yet another embodiment, the water- immiscible solvent is chilled at a temperature between 1 -10°C.

[0064] In one embodiment, the water-immiscible solvent is a polyalphaolefin. In an additional embodiment, the polyalphaolefin is Durasyn 164.

[0065] In a further embodiment, the one or more collection vessels further comprises an antifoam or an antioxidant. In another embodiment, the antifoam is L-81 . In yet another embodiment, the antioxidant is butylated hydroxytoluene (BHT).

[0066] In an embodiment, the temperature is between 30-50°C. In another embodiment, the temperature is 35°C. In further embodiment, the temperature is 45°C.

[0067] In one embodiment, the rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

[0068] In another embodiment, the method further comprises passing the gas carrying the volatile product through a condenser.

[0069] In a further embodiment, the bioreactor is a fermentation vessel.

[0070] Host Cells Host cells used for the production of volatile molecule products can be any cells deemed useful by those of skill in the art, including archae, prokaryotic, or eukaryotic cells.

[0071] Suitable prokaryotic hosts include, but are not limited, to any of a variety of gram-positive, gram-negative, or gram-variable bacteria. Examples include, but are not limited to, cells belonging to the genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus, Streptomyces, Synnecoccus, and Zymomonas. Examples of prokaryotic strains include, but are not limited to: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Staphylococcus aureus. In a particular embodiment, the host cell is an Escherichia coli cell.

[0072] Suitable archae hosts include, but are not limited to, cells belonging to the genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma. Examples of archae strains include, but are not limited to: Archaeoglobus fulgidus, Halobacterium sp., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Thermoplasma acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii, Pyrococcus abyss!, and Aeropyrum pernix.

[0073] Suitable eukaryotic hosts include, but are not limited to, fungal cells, algal cells, insect cells, and plant cells. In some embodiments, yeasts useful in the present methods include yeasts that have been deposited with microorganism depositories (e.g. IFO, ATCC, etc.) and belong to the genera Aciculoconidium, Ambrosiozyma, Arthroascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphopichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia, Nakazawaea, Nematospora, Ogataea, Oosporidium, Pachysolen, Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis, and Zygozyma, among others. In some embodiments, the host cell is a microbe that is suitable for industrial fermentation. In particular embodiments, the microbe is conditioned to subsist under high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due to sugar and salts, acidity, sulfite, and bacterial contamination, or combinations thereof, which are recognized stress conditions of the industrial fermentation environment.

[0074] Culture Media and Conditions

[0075] Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see, for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration must be given to appropriate culture medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the host cell, the fermentation, and the process.

[0076] The methods of producing volatile molecules provided herein may be performed in a suitable culture medium {e.g., with or without pantothenate supplementation) in a suitable container, including but not limited to a cell culture plate, a flask, or a fermentor. Further, the methods can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof. In particular embodiments utilizing Saccharomyces cerevisiae as the host cell, strains can be grown in a fermentor as described in detail by Kosaric, et al, in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co. KDaA, Weinheim, Germany.

[0077] In some embodiments, the culture medium is any culture medium in which a microbial cell can subsist, i.e., maintain growth and viability. In some embodiments, the culture medium includes an overlay. In some embodiments, the culture medium is an aqueous medium comprising assimilable carbon, nitrogen and phosphate sources. Such a medium can also include appropriate salts, minerals, metals and other nutrients. In some embodiments, the carbon source and each of the essential cell nutrients, are added incrementally or continuously to the fermentation media, and each required nutrient is maintained at essentially the minimum level needed for efficient assimilation by growing cells, for example, in accordance with a predetermined cell growth curve based on the metabolic or respiratory function of the cells which convert the carbon source to a biomass.

[0078] Suitable conditions and suitable media for culturing microorganisms are well known in the art. In some embodiments, the suitable medium is supplemented with one or more additional agents, such as, for example, an inducer {e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressor {e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressible promoter), or a selection agent {e.g., an antibiotic to select for microorganisms comprising the genetic modifications).

[0079] In some embodiments, the carbon source is an alcohol. In some embodiments, the carbon source is ethanol. In some embodiments, the carbon source is glycerol. In some embodiments, the carbon source is acetate. In some embodiments, the carbon source is a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or one or more combinations thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, xylose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol.

[0080] The concentration of a carbon source, such as glucose, in the culture medium should promote cell growth, but not be so high as to repress growth of the microorganism used. Typically, cultures are run with a carbon source, such as glucose, being added at levels to achieve the desired level of growth and biomass, but at undetectable levels (with detection limits being about <0.1 g / l). In other embodiments, the concentration of a carbon source, such as glucose, in the culture medium is greater than about 1 g / L, preferably greater than about 2 g / L, and more preferably greater than about 5 g / L. In addition, the concentration of a carbon source, such as glucose, in the culture medium is typically less than about 100 g / L, preferably less than about 50 g / L, and more preferably less than about 20 g / L. It should be noted that references to culture component concentrations can refer to both initial and / or ongoing component concentrations. In some cases, it may be desirable to allow the culture medium to become depleted of a carbon source during culture.

[0081] Sources of assimilable nitrogen that can be used in a suitable culture medium include, but are not limited to, simple nitrogen sources, organic nitrogen sources and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium hydroxide, ammonium salts and substances of animal, vegetable and / or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptone, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen sources in the culture medium is greater than about 0.1 g / L, preferably greater than about 0.25 g / L, and more preferably greater than about 1 .0 g / L. Beyond certain concentrations, however, the addition of a nitrogen source to the culture medium is not advantageous for the growth of the microorganisms. As a result, the concentration of the nitrogen sources, in the culture medium is less than about 20 g / L, preferably less than about 10 g / L and more preferably less than about 5 g / L. Further, in some instances it may be desirable to allow the culture medium to become depleted of the nitrogen sources during culture.

[0082] The effective culture medium can contain other compounds such as inorganic salts, vitamins, trace metals or growth promoters. Such other compounds can also be present in carbon, nitrogen or mineral sources in the effective medium or can be added specifically to the medium.

[0083] The culture medium can also contain a suitable phosphate source. Such phosphate sources include both inorganic and organic phosphate sources. Preferred phosphate sources include, but are not limited to, phosphate salts such as mono or dibasic sodium and potassium phosphates, ammonium phosphate and mixtures thereof. Typically, the concentration of phosphate in the culture medium is greater than about 1 .0 g / L, preferably greater than about 2.0 g / L and more preferably greater than about 5.0 g / L. Beyond certain concentrations, however, the addition of phosphate to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of phosphate in the culture medium is typically less than about 20 g / L, preferably less than about 15 g / L and more preferably less than about 10 g / L.

[0084] A suitable culture medium can also include a source of magnesium, preferably in the form of a physiologically acceptable salt, such as magnesium sulfate heptahydrate, although other magnesium sources in concentrations that contribute similar amounts of magnesium can be used. Typically, the concentration of magnesium in the culture medium is greater than about 0.5 g / L, preferably greater than about 1 .0 g / L, and more preferably greater than about 2.0 g / L. Beyond certain concentrations, however, the addition of magnesium to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of magnesium in the culture medium is typically less than about 10 g / L, preferably less than about 5 g / L, and more preferably less than about 3 g / L. Further, in some instances it may be desirable to allow the culture medium to become depleted of a magnesium source during culture.

[0085] In some embodiments, the culture medium can also include a biologically acceptable chelating agent, such as the dihydrate of trisodium citrate. In such instance, the concentration of a chelating agent in the culture medium is greater than about 0.2 g / L, preferably greater than about 0.5 g / L, and more preferably greater than about 1 g / L. Beyond certain concentrations, however, the addition of a chelating agent to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the concentration of a chelating agent in the culture medium is typically less than about 10 g / L, preferably less than about 5 g / L, and more preferably less than about 2 g / L.

[0086] The culture medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the culture medium. Biologically acceptable acids include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and mixtures thereof. Biologically acceptable bases include, but are not limited to, ammonium hydroxide, sodium hydroxide, potassium hydroxide and mixtures thereof. In some embodiments, the base used is ammonium hydroxide.

[0087] The culture medium can also include a biologically acceptable calcium source, including, but not limited to, calcium chloride. Typically, the concentration of the calcium source, such as calcium chloride, dihydrate, in the culture medium is within the range of from about 5 mg / L to about 2000 mg / L, preferably within the range of from about 20 mg / L to about 1000 mg / L, and more preferably in the range of from about 50 mg / L to about 500 mg / L.

[0088] The culture medium can also include sodium chloride. Typically, the concentration of sodium chloride in the culture medium is within the range of from about 0.1 g / L to about 5 g / L, preferably within the range of from about 1 g / L to about 4 g / L, and more preferably in the range of from about 2 g / L to about 4 g / L.

[0089] In some embodiments, the culture medium can also include trace metals. Such trace metals can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Typically, the amount of such a trace metals solution added to the culture medium is greater than about 1 ml / L, preferably greater than about 5 mL / L, and more preferably greater than about 10 mL / L. Beyond certain concentrations, however, the addition of a trace metals to the culture medium is not advantageous for the growth of the microorganisms. Accordingly, the amount of such a trace metals solution added to the culture medium is typically less than about 100 mL / L, preferably less than about 50 mL / L, and more preferably less than about 30 mL / L. It should be noted that, in addition to adding trace metals in a stock solution, the individual components can be added separately, each within ranges corresponding independently to the amounts of the components dictated by the above ranges of the trace metals solution.

[0090] In some embodiments, the culture medium may include a surfactant. In some embodiments, the surfactant may be an anionic surfactant; for example, the surfactant may be alkyl-naphthalene sulfonate, alkyl benzene sulfonate, or the like. In some embodiments, the surfactant is a nonionic surfactant. Suitable surfactants include biocompatible nonionic surfactants such as Brij (e.g., polyoxyethylene (4) lauryl ether, also known as Brij-30; polyoxyethylene (2) oleyl ether; polyoxyethylene (2) stearyl ether; etc.); micelles; and the like. In some embodiments, the surfactant is a secondary ether polyol. In some embodiments, the surfactant is TERGITOL L-62 (Dow Chemical Company). In some embodiments, the surfactant is TERGITOL L-81 (Dow Chemical Company). In some embodiments, the surfactant is a Jarcol™ alcohol. In some embodiments, the surfactant is TERGAZYME (Alconox), which may be used in an amount of between 0% (w / v) and about 1% (w / v).

[0091] The culture media can include other vitamins, such as pantothenate, biotin, calcium, pantothenate, inositol, pyridoxine-HCI, and thiamine-HCl. Such vitamins can be added to the culture medium as a stock solution that, for convenience, can be prepared separately from the rest of the culture medium. Beyond certain concentrations, however, the addition of vitamins to the culture medium is not advantageous for the growth of the microorganisms.

[0092] The fermentation methods described herein can be performed in conventional culture modes, which include, but are not limited to, batch, fed-batch, cell recycle, continuous and semi-continuous. In some embodiments, the fermentation is carried out in fed-batch mode. In such a case, some of the components of the medium are depleted during culture, including pantothenate during the production stage of the fermentation. In some embodiments, the culture may be supplemented with relatively high concentrations of such components at the outset, for example, of the production stage, so that growth and / or molecule production is supported for a period of time before additions are required. The preferred ranges of these components are maintained throughout the culture by making additions as levels are depleted by culture. Levels of components in the culture medium can be monitored by, for example, sampling the culture medium periodically and assaying for concentrations. Alternatively, once a standard culture procedure is developed, additions can be made at timed intervals corresponding to known levels at particular times throughout the culture. As will be recognized by those in the art, the rate of consumption of nutrient increases during culture as the cell density of the medium increases. Moreover, to avoid introduction of foreign microorganisms into the culture medium, addition is performed using aseptic addition methods, as are known in the art. In addition, an anti-foaming agent may be added during the culture.

[0093] The temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and / or production of a target molecule of interest. For example, in some embodiments, prior to inoculation of the culture medium with an inoculum, the culture medium can be brought to and maintained at a temperature in the range of from about 20°C to about 45°C, preferably to a temperature in the range of from about 25°C to about 40°C, and more preferably in the range of from about 28°C to about 32°C. In other embodiments, the temperature can be maintained in a range from about 20°C to about 45°C, preferably to a temperature in the range of from about 25°C to about 40°C, and more preferably in the range of from about 35°C to about 40°C.

[0094] The pH of the culture medium can be controlled by the addition of acid or base to the culture medium. In such cases when ammonia is used to control pH, it also conveniently serves as a nitrogen source in the culture medium. Preferably, the pH is maintained from about 3.0 to about 9.0, more preferably from about 3.5 to about 8.0, and most preferably from about 4.0 to about 7.0.

[0095] In some embodiments, the carbon source concentration, such as the glucose concentration, of the culture medium is monitored during culture. Glucose concentration of the culture medium can be monitored using known techniques, such as, for example, use of the glucose oxidase enzyme test or high pressure liquid chromatography, which can be used to monitor glucose concentration in the supernatant, e.g., a cell-free component of the culture medium. As stated previously, the carbon source concentration should be kept below the level at which cell growth inhibition occurs. Although such concentration may vary from organism to organism, for glucose as a carbon source, cell growth inhibition occurs at glucose concentrations greater than at about 60 g / L, and can be determined readily by trial. Accordingly, when glucose is used as a carbon source the glucose is preferably fed to the fermentor and maintained below detection limits. Alternatively, the glucose concentration in the culture medium is maintained in the range of from about 1 g / L to about 100 g / L, more preferably in the range of from about 2 g / L to about 50 g / L, and yet more preferably in the range of from about 5 g / L to about 20 g / L. Although the carbon source concentration can be maintained within desired levels by addition of, for example, a substantially pure glucose solution, it is acceptable, and may be preferred, to maintain the carbon source concentration of the culture medium by addition of aliquots of the original culture medium. The use of aliquots of the original culture medium may be desirable because the concentrations of other nutrients in the medium {e.g. the nitrogen and phosphate sources) can be maintained simultaneously. Likewise, the trace metals concentrations can be maintained in the culture medium by addition of aliquots of the trace metals solution.

[0096] Product Recovery

[0097] Once produced, a volatile molecule may be recovered or isolated according to methods described in the present disclosure, including with the overlay traps describe herein. In addition, despite its volatility, some portion of the volatile molecule may remain in the culture medium and / or bioreactor, and may be recovered or isolated using any other suitable separation and purification methods known in the art. In some embodiments, an aqueous phase comprising the product molecule is separated from the bioreactor by centrifugation. In other embodiments, an aqueous phase comprising the molecule is separated from the culture medium by adding a deemulsifier and / or a nucleating agent into the bioreactor. Illustrative examples of deemulsifiers include flocculants and coagulants. Illustrative examples of nucleating agents include organic solvents such as chloroform, ethyl acetate, acetone, hexane, ethanol, heptane, dodecane, isopropyl myristrate, and methyl oleate.

[0098] The product molecule may be present in the culture medium and / or associated with the host cells. In embodiments where the molecule is associated with the host cell, the recovery of the molecule may comprise a method of permeabilizing or lysing the cells. Alternatively or simultaneously, the molecule in the culture medium can be recovered using a recovery process including, but not limited to, chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization and crystallization.

[0099] In some embodiments, the product molecule is separated from other products that may be present. In some embodiments, the product molecule is separated from other products that may be present in the aqueous phase. In some embodiments, the product molecule is separated from other products that may be present in the organic phase. In some embodiments, separation is achieved using adsorption, distillation, gas-liquid extraction (stripping), liquid-liquid extraction (solvent extraction), ultrafiltration, and standard chromatographic techniques.

[0100] Methods of Making Genetically Modified Host Cells

[0101] Also described herein are methods for producing a host cell that is genetically engineered to comprise one or more of modifications, e.g., one or more heterologous nucleic acids encoding biosynthetic pathway enzymes, e.g., for production of a target molecule. Expression of a heterologous enzyme in a host cell can be accomplished by introducing into the host cells a nucleic acid comprising a nucleotide sequence encoding the enzyme under the control of regulatory elements that permit expression in the host cell. In some embodiments, the nucleic acid is an extrachromosomal plasmid. In other embodiments, the nucleic acid is a chromosomal integration vector that can integrate the nucleotide sequence into the chromosome of the host cell.

[0102] Nucleic acids encoding these proteins can be introduced into the host cell by any method known to one of skill in the art without limitation (see, for example, Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75:1292-3; Cregg et al. (1985) Mol. Cell. Biol. 5:3376-3385; Goeddel et al. eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc. , CA; Krieger, 1990, Gene Transfer and Expression - A Laboratory Manual, Stockton Press, NY; Sambrook et al. , 1989, Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al. , eds. , Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY). Exemplary techniques include, but are not limited to, spheroplasting, electroporation, PEG 1000 mediated transformation, and lithium acetate or lithium chloride mediated transformation.

[0103] The copy number of an enzyme in a host cell may be altered by modifying the transcription of the gene that encodes the enzyme. This can be achieved for example by modifying the copy number of the nucleotide sequence encoding the enzyme {e.g., by using a higher or lower copy number expression vector comprising the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the genome of the host cell or by deleting or disrupting the nucleotide sequence in the genome of the host cell), by changing the order of coding sequences on a polycistronic mRNA of an operon or breaking up an operon into individual genes each with its own control elements, or by increasing the strength of the promoter or operator to which the nucleotide sequence is operably linked. Alternatively or in addition, the copy number of an enzyme in a host cell may be altered by modifying the level of translation of an mRNA that encodes the enzyme. This can be achieved for example by modifying the stability of the mRNA, modifying the sequence of the ribosome binding site, modifying the distance or sequence between the ribosome binding site and the start codon of the enzyme coding sequence, modifying the entire intercistronic region located “upstream of” or adjacent to the 5’ side of the start codon of the enzyme coding region, stabilizing the 3’-end of the mRNA transcript using hairpins and specialized sequences, modifying the codon usage of enzyme, altering expression of rare codon tRNAs used in the biosynthesis of the enzyme, and / or increasing the stability of the enzyme, as, for example, via mutation of its coding sequence.

[0104] The activity of an enzyme in a host cell can be altered in a number of ways, including, but not limited to, expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme that lacks a domain through which the activity of the enzyme is inhibited, expressing a modified form of the enzyme that has a higher or lower Kcat or a lower or higher Km for the substrate, or expressing an altered form of the enzyme that is more or less affected by feed-back or feed-forward regulation by another molecule in the pathway.

[0105] In some embodiments, a nucleic acid used to genetically modify a host cell comprises one or more selectable markers useful for the selection of transformed host cells and for placing selective pressure on the host cell to maintain the foreign DNA.

[0106] In some embodiments, the selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to, the BLA, NAT1, PAT, AUR1- C, PDR4, SMR1, CAT, mouse dhfr, HPH, DSDA, KANR, and SH BLE gene products. The BLA gene product from E. coli confers resistance to beta-lactam antibiotics {e.g. , narrow-spectrum cephalosporins, cephamycins, and carbapenems (ertapenem), cefamandole, and cefoperazone) and to all the anti-gram-negative-bacterium penicillins except temocillin; the NAT1 gene product from S. noursei confers resistance to nourseothricin; the PAT gene product from S. viridochromogenes TuQA- confers resistance to bialophos; the AURTCgene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the PDR4 gene product confers resistance to cerulenin; the SMR1 gene product confers resistance to sulfometuron methyl; the CAT gene product from Tn9 transposon confers resistance to chloramphenicol; the mouse dhfr gene product confers resistance to methotrexate; the HPH gene product of Klebsiella pneumonia confers resistance to Hygromycin B; the DSDA gene product of E. coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KANRgene of the Tn903 transposon confers resistance to G418; and the SH BLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin). In some embodiments, the antibiotic resistance marker is deleted after the genetically modified host cell disclosed herein is isolated.

[0107] In some embodiments, the selectable marker rescues an auxotrophy {e.g., a nutritional auxotrophy) in the genetically modified microorganism. In such embodiments, a parent microorganism comprises a functional disruption in one or more gene products that function in an amino acid or nucleotide biosynthetic pathway and that when non-functional renders a parent cell incapable of growing in media without supplementation with one or more nutrients. Such gene products include, but are not limited to, the HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast. The auxotrophic phenotype can then be rescued by transforming the parent cell with an expression vector or chromosomal integration construct encoding a functional copy of the disrupted gene product, and the genetically modified host cell generated can be selected for based on the loss of the auxotrophic phenotype of the parent cell. Utilization of the URA3, TRP1, and LYS2 genes as selectable markers has a marked advantage because both positive and negative selections are possible. Positive selection is carried out by auxotrophic complementation of the URA3, TRP1, and LYS2 mutations, whereas negative selection is based on specific inhibitors, i.e., 5- fluoro-orotic acid (FOA), 5-fluoroanthranilic acid, and aminoadipic acid (aAA), respectively, that prevent growth of the prototrophic strains but allows growth of the URA3, TRP1, and LYS2 mutants, respectively. In other embodiments, the selectable marker rescues other non-lethal deficiencies or phenotypes that can be identified by a known selection method.

[0108] Described herein are specific genes and proteins useful in the methods, compositions and organisms of the disclosure; however it will be recognized that absolute identity to such genes is not necessary. For example, changes in a particular gene or polynucleotide comprising a sequence encoding a polypeptide or enzyme can be performed and screened for activity. Typically such changes comprise conservative mutations and silent mutations. Such modified or mutated polynucleotides and polypeptides can be screened for expression of a functional enzyme using methods known in the art.

[0109] Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantially the same or functionally equivalent polypeptides can also be used to clone and express the polynucleotides encoding such enzymes.

[0110] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, in a process sometimes called “codon optimization” or “controlling for species codon bias.”

[0111] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (Murray et al., 1989, Nucl Acids Res. 17: 477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non-optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coll commonly use UAA as the stop codon (Dalphin et al., 1996, Nucl Acids Res. 24: 216-8).

[0112] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of DNA molecules differing in their nucleotide sequences can be used to encode a given enzyme of the disclosure. The native DNA sequence encoding the biosynthetic enzymes described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes DNA molecules of any sequence that encode the amino acid sequences of the polypeptides and proteins of the enzymes utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with different amino acid sequences than the specific proteins described herein so long as the modified or variant polypeptides have the enzymatic anabolic or catabolic activity of the reference polypeptide. Furthermore, the amino acid sequences encoded by the DNA sequences shown herein merely illustrate embodiments of the disclosure.

[0113] In addition, homologs of enzymes useful for the compositions and methods provided herein are encompassed by the disclosure. In some embodiments, two proteins (or a region of the proteins) are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes {e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In one embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0114] When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties {e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art {See, e.g., Pearson W. R., 1994, Methods in Mol 8 / 0 / 25: 365-89).

[0115] The following six groups each contain amino acids that are conservative substitutions for one another: 1 ) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0116] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. A typical algorithm used to compare a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST. When searching a database containing sequences from a large number of different organisms, it is typical to compare amino acid sequences.

[0117] Furthermore, any of the genes encoding the foregoing enzymes (or any others mentioned herein (or any of the regulatory elements that control or modulate expression thereof)) may be optimized by genetic / protein engineering techniques, such as directed evolution or rational mutagenesis, which are known to those of ordinary skill in the art. Such action allows those of ordinary skill in the art to optimize the enzymes for expression and activity in yeast.

[0118] In addition, genes encoding these enzymes can be identified from other fungal and bacterial species and can be expressed for the modulation of this pathway. A variety of organisms could serve as sources for these enzymes, including, but not limited to, Saccharomyces spp., including S. cerevisiae and S. uvarum, Kluyveromyces spp., including K. thermotolerans, K. lactis, and K. marxianus, Pichia spp., Hansenula spp., including H. polymorpha, Candida spp., Trichosporon spp., Yamadazyma spp., including Y. spp. stipitis, Torulaspora pretoriensis, Issatchenkia orientalis, Schizosaccharomyces spp., including S. pombe, Cryptococcus spp., Aspergillus spp., Neurospora spp., or Ustilago spp. Sources of genes from anaerobic fungi include, but are not limited to, Piromyces spp., Orpinomyces spp., or Neocallimastix spp. Sources of prokaryotic enzymes that are useful include, but are not limited to, Escherichia, coll, Zymomonas mobilis, Staphylococcus aureus, Bacillus spp., Clostridium spp., Corynebacterium spp., Pseudomonas spp., Lactococcus spp., Enterobacter spp., Salmonella spp., or X. dendrorhous.

[0119] Techniques known to those skilled in the art may be suitable to identify additional homologous genes and homologous enzymes. Generally, analogous genes and / or analogous enzymes can be identified by functional analysis and will have functional similarities. Techniques known to those skilled in the art may be suitable to identify analogous genes and analogous enzymes. For example, to identify homologous or analogous PK, PTA, RHR2, HOR2, or carotenogic genes, proteins, or enzymes, techniques may include, but are not limited to, cloning a gene by PCR using primers based on a published sequence of a gene / enzyme of interest, or by degenerate PCR using degenerate primers designed to amplify a conserved region among a gene of interest. Further, one skilled in the art can use techniques to identify homologous or analogous genes, proteins, or enzymes with functional homology or similarity. Techniques include examining a cell or cell culture for the catalytic activity of an enzyme through in vitro enzyme assays for said activity (e.g. as described herein or in Kiritani, K., Branched-Chain Amino Acids Methods Enzymology, 1970), then isolating the enzyme with said activity through purification, determining the protein sequence of the enzyme through techniques such as Edman degradation, design of PCR primers to the likely nucleic acid sequence, amplification of said DNA sequence through PCR, and cloning of said nucleic acid sequence. To identify homologous or similar genes and / or homologous or similar enzymes, analogous genes and / or analogous enzymes or proteins, techniques also include comparison of data concerning a candidate gene or enzyme with databases such as BRENDA, KEGG, or MetaCYC. The candidate gene or enzyme may be identified within the above mentioned databases in accordance with the teachings herein.

[0120] EXAMPLES

[0121] Example 1 : Yeast Sample Preparation Conditions for Analysis of Monoterpene Titers

[0122] To quantify the amount of monoterpene produced, 50 pL whole cell broth or overlay was diluted into 950 pL of methanol in a 2ml Eppendorf tube and vortexed for 5 minutes. The sample was then centrifuged for 3 minutes at 10,000 RPM to pellet solids and 100 pL of the organic layer was transferred to a GC vial containing 900 pL ethyl acetate (1 Ox dilution). A subsequent 10x dilution step into ethyl acetate was utilized for high titer samples. Samples were then submitted for analysis by gas chromatography-mass spectrometry (GC-MS).

[0123] Example 2: Analytical Methods

[0124] Samples derived from yeast producing monoterpenes were routinely analyzed using GC-MS. GC-MS samples were loaded onto Gerstel MSP2 (Gerstel, Inc, Linthicum, MD, USA) autosampler and analyses were performed using a GC-MSD system (5975C Agilent Technologies Inc., Santa Rosa, CA, USA) equipped with DB-1 MS (Agilent Technologies Inc., Santa Rosa, CA, USA) capillary column of 20 m x 0.10 mm with a phase thickness of 0.10mm. The injection volume of each sample was 1 pL using split mode with split ratio of 20:1 . Helium (99.999%) was used as the carrier gas at a flowrate of 0.45 mL / min. The temperature of the injection port was 275° C, and the column temperature program was as follows: 60° C for 0 min, followed by an increase to 320° C at a rate of 30° C / min. The MS conditions included an El ion source temperature of 230° C, quad temperature of 150°C, an ionization energy of ~ 1100 to 1250 eV, and a mass scan range of 40-600 Amu. Quantification of monoterpenes in crude extracts was calculated by comparing peak area of target molecules to calibration curves of authentic standards. If an authentic chemical standard was not available for calibration for a molecule, a calibration curve for trans-p-farnesene was used as a surrogate instead.

[0125] Example 3: Volatile Myrcene and Limonene Product Recovery at the Bench Scale

[0126] Myrcene and limonene producing yeast strains were cultured in 2-L Biostat B plus vessels (Sartorius, Germany) containing 800 mL growth medium and 40 g / L sucrose, 10 g / L maltose, and 3 g / L lysine. The fermentation temperature was controlled at 30°C. To control foaming 0.1 mL of L-81 was added to at inoculation. Additionally, 10% vol / vol of durasyn 164 mineral oil was added as an overlay for in situ detoxification of monoterpenes. pH 5 was maintained throughout the run using 30% w / w ammonium hydroxide. Stirring was controlled between 600 to 1 ,100 RPM and the fermentor was continuously sparged with between 0.5 L min-1 to 1 L min-1 of air (equivalent to a volume of air to volume of broth per minute (VVM) ratio range of 0.5 to 1 ). Fermentations were fed a sucrose- molasses blend with a total reducing sugar (TRS) concentration of 66.6% g / g. This feed was delivered at a rate to hold the dissolved oxygen constant at 30% or lower set point. Feed was periodically reduced to confirm substrate limitation and then restored to rate required to hold the dissolved oxygen set point. Tanks were sampled daily starting 24 hours after inoculation. Whole cell broth (WCB) was collected and stored at -20°C. At the end of the fermentation run, samples were thawed and extracted as described previously. Run lengths were approximately 7 days.

[0127] A significant proportion of the monoterpenes produced during the fermentation was stripped from the fermentation broth by the sparged air. This product fraction was recovered by flowing the off fermentation off gas air through two 1 -L external traps in series containing 200 mL of chilled durasyn 164 overlay at 4C (FIG. 1 ). The monoterpenes in the gas phase were absorbed from the gas phase into the overlay in the traps. To control foaming, 15 ml L-81 antifoam was added to each trap. Off gas traps were sampled at the end of the fermentation and submitted for titer analysis as described above.

[0128] Table 1. Representative bench scale final titer, total mass, myrcene mass, and % of total myrcene in the fermentor, the first external overlay trap and the second overlay trap.

[0129] Table 2. Representative bench scale final titer, total mass, limonene mass, and % of total limonene in the fermentor, the first external overlay trap and the second overlay trap

[0130] Example 4: Volatile Myrcene Product Recovery at the Pilot Scale Fermentation at the pilot scale was a fed-batch process that used cane syrup as the feedstock. The production process consisted of three phases in series: 1 . Initial Phase (IP), 2. Growth Phase (GP), and 3. Production Phase (PP). A feedback-controlled feed algorithm was used in GP and PP phases to avoid over feeding. Aeration and agitation rates were set to deliver an average oxygen transfer rate (OTR) of ~ 60mmol / L / h in the PP. The pH was controlled at 5 by addition of NH4OH (28% NH4OH) in all phases. The temperature was ramped up to 30°C. Antifoam Tergitol L-81 was added as needed to control foaming. The air sparge rate was maintained at 75L min-1 throughout the fermentation (0.43 VVM) to provide oxygen and remove CO2 and myrcene from the fermentation broth. The fermentation ended after 140.5h of feeding.

[0131] The myrcene-containing off gas was first passed through a condenser to remove water from the gas stream (FIG. 2). The dehydrated air then passed to the Primary Myrcene Trap (FIG. 3) containing 24.6kg Durasyn 164, 15ml antifoam, and 300g butylated hydroxytoluene (BHT) to prevent oxidation of the myrcene. This trap had an external cooling jacket attached to the lower part of the column (FIG. 3) through which cooling water was circulated to chill the Durasyn 164 and improve the absorption kinetics of myrcene. Approximately 93% of the myrcene in the gas phase was removed by this trap. However, a Secondary Myrcene Trap was employed to further remove myrcene from the air prior to venting. It was smaller and contained 6.6kg Durasyn 164, 4 ml antifoam, 80g BHT.

[0132] Table 3. Myrcene production and location at the end of the 140.5h fermentation process.

[0133] Example 5: Increased Myrcene Recovery by Additional Gas Stripping after Fermentation Completion.

[0134] After completion of the fermentation, the whole cell broth was allowed to settle for several hours and 125 kg of broth was drained from the bottom of the fermentor. This removed a biomass- enriched heavy phase, with the majority of the light organic phase containing 2.8 kg of myrcene retained in the fermentor (Table 4). The myrcene traps were drained and 30L of fresh durasyn 164 overlay was added to the Primary Myrcene Trap and 4L was added to the Secondary Trap, with the same concentrations of L-81 antifoam and BHT antioxidant as described above. To increase the rate of myrcene stripping from the fermentation broth, the temperature of the fermentor was increased to 35C from 30C, and the air sparge rate was increased to 100 L min-1 (0.63 VVM) from 75 L min-1 . After 4 days of sparging at these conditions, an additional 1 .25 kg of myrcene (44.6% of what was remaining) was transferred from the fermentor to the Primary Myrcene Trap, thereby doubling the overall amount of myrcene recovered from the off gas.

[0135] Table 4. Myrcene recovery by additional air stripping post fermentation.

[0136] Example 6: Increasing Air Flow Rate Increases the Rate of Myrcene Stripping from a Fermentation

[0137] Myrcene producing yeast strains were cultured in 0.5L fermentation vessels (Sartorius, Germany) containing 125 mL growth medium with 7 g / L maltose, and 5 g / L lysine and 50g of Drakeol 10. The fermentation temperature was controlled at 30°C. To control foaming 0.1 mL of L-81 was added to at inoculation. pH 5 was maintained throughout the run using 30% w / w ammonium hydroxide. Stirring was controlled between 400 and 900 RPM to maintain an oxygen transfer rate target of 110 mmol O2 / L / h. One fermentor was continuously sparged with a low air flow rate of 0.2 L min-1 of air (0.67 VVM) while the other fermentor was continuously sparged with a high air flow rate of 0.6 L min-1 of air (2 VVM). Fermentations were fed cane syrup with a total reducing sugar (TRS) concentration of 56.6% g / g. This feed was delivered at a rate to hold the dissolved oxygen near 0% while minimizing overfeeding. Feed was periodically reduced to confirm substrate limitation and then restored to rate required to hold the dissolved oxygen set point. Tanks were sampled daily starting 24 hours after inoculation. Whole cell broth (WCB) was collected and stored at -20°C. At the end of the fermentation run, samples were thawed and extracted as described previously. Run durations were approximately 170h.

[0138] One 1 -L external off gas trap containing 200 mL of durasynl 64 overlay was used to capture myrcene from the off gas for each fermentor. The off gas traps were sampled every 24h over the fermentation and submitted for titer analysis as described above. The rate of myrcene stripping from each fermentor was estimated by fitting a line to the trap titer data from 48h to 160h (FIG. 4). A nearly 9-fold faster rate of removal was observed for the higher air flow rate of 0.6 L min-1 (2 VVM) relative to the lower air flow rate of 0.2 L min-1 (0.67 VVM) even though the total myrcene produced during the fermentation were nearly identical (Table 5). Table 5. Impact of air flow rate on myrcene stripping rate from fermentation broth.

[0139] Other Embodiments

[0140] While the invention has been described in connection with the specific embodiments herein, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the invention that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims. Other embodiments are within the claims.

[0141] All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Claims

CLAIMS1 . A method of recovering a volatile product, comprising:(a) culturing a population of cells in a culture medium in a bioreactor at a first temperature, wherein the cells produce the volatile product,(b) introducing gas into the bioreactor at a first rate, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels comprises a water- immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and(c) recovering the volatile product from the water-immiscible solvent.

2. The method of claim 1 , wherein the volatile product is a terpene.

3. The method of claim 2, wherein the terpene is a monoterpene or a hemiterpene.

4. The method of claim 2, wherein the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene, camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

5. The method of claim 4, wherein the terpene is myrcene.

6. The method of claim 5, wherein the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

7. The method of claim 4, wherein the terpene is limonene.

8. The method of claim 7, wherein the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

9. The method of any one of claims 1 -8, wherein the water-immiscible solvent is chilled.

10. The method of claim 9, wherein the water-immiscible solvent is chilled at a temperature between 1 -20°C.11 . The method of claim 10, wherein the water-immiscible solvent is chilled at a temperature between 1 -15°C.

12. The method of claim 11 , wherein the water-immiscible solvent is chilled at a temperature between 1 -10°C.

13. The method of any one of claims 1 -12, wherein the water-immiscible solvent is a polyalphaolefin.

14. The method of claim 13, wherein the polyalphaolefin is Durasyn 164.

15. The method of any one of claims 1 -14, wherein the one or more collection vessels further comprises an antifoam or an antioxidant.

16. The method of claim 15, wherein the antifoam is L-81 .

17. The method of claim 15, wherein the antioxidant is butylated hydroxytoluene (BHT).

18. The method of any one of claims 1 -17, wherein the first temperature is between 25-40°C.

19. The method of claim 18, wherein the first temperature is 30°C.

20. The method of claim 18, wherein the first temperature is 37°C.21 . The method of claim any one of claims 1 -20, wherein the first rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

22. The method of any one of claims 1 -21 , further comprising passing the gas carrying the volatile product through a condenser.

23. The method of claim any one of claims 1 -22, further comprising adjusting the first temperature to a second temperature.

24. The method of claim 23, wherein the second temperature is suboptimal for production of the volatile product by the population of host cells.

25. The method of claim 23, wherein the second temperature is higher than the first temperature.

26. The method of claim 23, wherein the second temperature is between 30-50°C.

27. The method of any one of claimsl -26, further comprising adjusting the first rate to a second rate.

28. The method of claim 27, wherein the second rate is suboptimal for production of the volatile product by the population of host cells.

29. The method of claim 27, wherein the second rate is higher than the first rate.

30. The method of claim 27, wherein the second rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).31 . The method of any one of claims 1 -30, wherein culturing the population of cells occurs in the presence of an overlay.

32. The method of claim 31 , wherein the overlay comprises Drakeol-10, Durasyn 164, ESTEREXA 51 , JARCOL 1-16, Drakeol 19, soybean oil, castor oil, or sunflower oil.

33. The method of claim 32, where in the overlay is Drakeol-10.

34. The method of claim 32, wherein the overlay is Durasyn 164.

35. The method of any one of claims 1 -34, wherein the bioreactor is a fermentation vessel.

36. A method for recovering a volatile product from a bioreactor, comprising:(a) introducing gas into the bioreactor at a rate that is suboptimal for production of the volatile product by a population of host cells,(b) setting a temperature within the bioreactor that is suboptimal for production of the volatile product by a population of host cells, wherein the gas carries the volatile product to one or more collection vessels, wherein the one or more collection vessels comprises a water- immiscible solvent, wherein the volatile product partitions into the water-immiscible solvent, and(c) recovering the volatile product from the water-immiscible solvent.

37. The method of claim 36, wherein the volatile product is a terpene.

38. The method of claim 37, wherein the terpene is a monoterpene or a hemiterpene.

39. The method of claim 37, wherein the terpene is myrcene, limonene, linalool, isoprene, isoprenol, menthol, a-copaene, p-elemene, a-barbatene, p-caryophyllene, thujopsene, a-humulene, p-farnesene, p-chamigrene, cuparene, bisabolene, bisabolol, sesquiphellandrene, a-pinene, p-pinene,camphene, citranellol, citronellal, geraniol, geronial, perillyl alcohol, thujone, hinokitiol, carvacrol, anethole, cuminaldehyde, eucalyptol, isopulegol, nerol, neral, or carvone.

40. The method of claim 39, wherein the terpene is myrcene.41 . The method of claim 40, wherein the total myrcene captured is between 1 -20 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

42. The method of claim 39, wherein the terpene is limonene.

43. The method of claim 42, wherein the total limonene captured is between 1 -10 g per kg of the combined mass of the culture medium and the water-immiscible solvent.

44. The method of any one of claims 36-43, wherein the water-immiscible solvent is chilled.

45. The method of claim 44, wherein the water-immiscible solvent is chilled at a temperature between 1 -20°C.

46. The method of claim 45, wherein the water-immiscible solvent is chilled at a temperature between 1 -15°C.

47. The method of claim 46, wherein the water-immiscible solvent is chilled at a temperature between 1 -10°C.

48. The method of any one of claims 36-47, wherein the water-immiscible solvent is a polyalphaolefin.

49. The method of claim 48, wherein the polyalphaolefin is Durasyn 164.

50. The method of any one of claims 36-49, wherein the one or more collection vessels further comprises an antifoam or an antioxidant.51 . The method of claim 50, wherein the antifoam is L-81 .

52. The method of claim 50, wherein the antioxidant is butylated hydroxytoluene (BHT).

53. The method of any one of claims 36-52, wherein the temperature is between 30-50°C.

54. The method of claim 53, wherein the temperature is 35°C.

55. The method of claim 53, wherein the temperature is 45°C.

56. The method of any one of claims 36-55, wherein the rate is between 0.1 and 5.0 VVM (volume of gas per volume of culture medium per minute).

57. The method of any one of claims 36-56, further comprising passing the gas carrying the volatile product through a condenser.

58. The method of any one of claims 36-57, wherein the bioreactor is a fermentation vessel.

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