Carotenoid production by pressure fermentation

Abstract

A fermentative process for producing lycopene and / or beta-carotene as a product or intermediate is described herein. The process generally comprises: (a) providing a cell population of a eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene and / or beta-carotene from phytoene; (b) culturing the cell population under production conditions enabling the synthesis of phytoene; and (c) applying a degree of pressure to the fermenter sufficient to shift carbon flux from phytoene towards lycopene / beta-carotene production, thereby reducing phytoene accumulation in the eukaryotic microorganisms as compared to in the absence of the applied pressure. The production conditions may further comprise using a lipid feedstock as a primary carbon source. Also described herein are microorganisms engineered for the production of lycopene and / or beta-carotene, as a product or intermediate.

Description

[0001] CAROTENOID PRODUCTION BY PRESSURE FERMENTATION

[0002] The present technology relates to a fermentative process for the production of carotenoids such as lycopene and beta-carotene, as a product or intermediate, and microorganisms engineered for same. More particularly, described herein are fermentation conditions that advantageously shift carbon flux from phytoene towards lycopene / beta-carotene production.

[0003] The present description refers to a number of documents, the contents of which are herein incorporated by reference in their entirety.

[0004] BACKGROUND

[0005] Carotenoids refer to a group of isoprenoid-based compounds synthesized by plants, algae, and other microorganisms, that are responsible for many of the yellow, orange, and red pigments found in nature. To meet growing demand, commercially-relevant carotenoids are presently chemically synthesized or extracted from natural sources. While the former is generally less costly, demand for the latter is rapidly growing due to global environmental concerns and changing consumer preferences. Consequently, research and development efforts have focused on making non-synthetic production of carotenoids, and specifically microbial production of carotenoids, more cost-effective.

[0006] Microorganisms engineered for the production of carotenoids have been previously described and the influence of various fermentative conditions on carotenoid yields have been explored (e.g., Nishizaki et al., 2007; Reyes et al., 2014; Olson, 2014; Gomez et al., 2014; Trikka et al., 2015; Bobadilla Romero et al., 2020; and Paul et al., 2023). Despite this, there remains a need for improved processes and engineered microorganisms enabling the production of commercially-relevant carotenoids at industrial scales.

[0007] SUMMARY

[0008] In a first aspect, described herein is a process for producing lycopene as a product or intermediate. The process generally comprises: (a) providing a fermenter comprising a cell population of a eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene; (b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene; and (c) applying a degree of pressure to the fermenter sufficient to shift carbon flux from phytoene towards lycopene production, thereby reducing phytoene accumulation in the eukaryotic microorganisms as compared to in the absence of said pressure. In some embodiments, the pressure applied to the fermenter may be back-pressure. In a further aspect, described herein is a process for producing beta-carotene as a product or intermediate, wherein the process is as defined above and wherein the eukaryotic microorganism has a metabolic pathway for the synthesis of phytoene to lycopene to beta-carotene.

[0009] In a further aspect, described herein is an engineered eukaryotic microorganism as described herein suitable for the production of lycopene and / or beta-carotene, as a product or intermediate.

[0010] In a further aspect, described herein is a method for identifying and / or validating a eukaryotic microorganism that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure. The method generally comprises: (a) providing a fermenter comprising a cell population of the eukaryotic microorganism, the eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene; (b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene; (c) applying at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg of pressure to the fermenter during at least a portion of (b); and (d) measuring the amount of lycopene or downstream product thereof produced by the eukaryotic microorganism, wherein the eukaryotic microorganism is identified and / or validated as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, when the amount of lycopene or downstream product thereof produced is greater than that produced by a corresponding method lacking step (c).

[0011] General Definitions

[0012] Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., are presented merely for ease of reading the specification and claims. The use of headings or other identifiers in the specification or claims does not necessarily require the steps or elements be performed in alphabetical or numerical order or the order in which they are presented, and they do not necessarily imply that each heading or identifier is independent or is independently performed from one another.

[0013] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more”, “at least one”, and “one or more than one”.

[0014] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0015] The term “about” is used to indicate that a value includes the standard deviation of error for the device or method being employed in order to determine the value. In general, the terminology “about” is meant to designate a possible variation of up to 10%. Therefore, a variation of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term “about”. Unless indicated otherwise, use of the term “about” before a range applies to both ends of the range.

[0016] Other objects, advantages and features of the present description will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0017] SEQUENCE LISTING

[0018] This application contains a Sequence Listing in computer-readable form created October 13,

[0019] 2025. The computer-readable form is incorporated herein by reference.

[0020] DETAILED DESCRIPTION

[0021] Described herein are improved processes for producing carotenoids of commercial interest, such as lycopene and beta-carotene, as well as downstream products derived from these compounds, such as astaxanthin and vitamin A.

[0022] In a first aspect, described herein is a process for producing a phytoene-derived carotenoid by fermenting a suitable eukaryotic microorganism (e.g., engineered to have a metabolic pathway for the synthesis of lycopene from phytoene) under conditions of elevated pressure, which is demonstrated herein to shift carbon flux from phytoene towards lycopene production, thereby reducing intra-microorganism phytoene accumulation and improving the carotenoid yield.

[0023] Subjecting host microorganisms to fermentation conditions involving increased stress are known to have potentially negative impacts on their health and productivity and therefore be unfavorable to industrial fermentation. For example, Bobadilla Romero et al., 2020 reported that increased aeration improved beta-carotene production in engineered .S', cerevisiae, but that increased stress triggered by changes in solute-water concentration (osmotic pressure) or nutrient availability (carbon to nitrogen ratio) did not. Furthermore, increased stress has been shown to effect the metabolic activity and longevity of yeast, which was reported to ultimately hinder fermentation performance (Olton et al., 2014; Higgins et al., 2003). With regards to pressure, although fermentation under pressurized conditions has been suggested for achieving uncommonly high cell densities while neglecting product yields (Knoll et al., 2007), several groups have subsequently reported that fermenting under vacuum (i.e., reduced pressure) has generally resulted in an increase in the number of cells in suspension and a reduction in process time when compared to atmospheric pressure conditions, likely due to a reduction in dissolved carbon dioxide levels during fermentation, which is a known inhibitor of yeast metabolism (Guadalupe -Daqui et al., 2023). Based on this reasoning, fermentation under elevated pressure would be expected to have a negative impact on fermentation performance at least because of a predicted increase in dissolved carbon dioxide in the fermentation broth, compounded by the fact that the solubility in aqueous solution of carbon dioxide gas at 30 °C is 25 times higher than that of oxygen gas (Gevantman, 2017). Accordingly, it was an unexpected finding on the part of the preset inventors that carotenoid production could be improved by fermentation under conditions of elevated pressure, such as by applying a back-pressure to the fermenter sufficient to shift carbon flux from phytoene towards lycopene production, thereby reducing intra-microorganism phytoene accumulation and improving the carotenoid yield.

[0024] In a further aspect, described herein is a process for producing lycopene as a product or as an intermediate leading to the production of a downstream carotenoid. In some embodiments, the process generally comprises providing a fermenter comprising a cell population of a eukaryotic microorganism, wherein the eukaryotic microorganism natively has, or is engineered have, a metabolic pathway for the synthesis of lycopene from phytoene. In some embodiments, in a production phase, the cell population is cultured in the fermenter under production conditions enabling the synthesis of phytoene, and a degree of pressure is applied to the fermenter sufficient to shift carbon flux from phytoene towards lycopene production, thereby reducing phytoene accumulation in the eukaryotic microorganisms as compared to in the absence of said pressure. In some embodiments, the pressure applied to the fermenter is backpressure, such as controlled by a back-pressure regulator operably linked to the fermenter.

[0025] In some embodiments, the pressure or back-pressure applied may be sufficient to reduce phytoene accumulation by at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold, as compared to in the absence of the pressure. In some embodiments, the pressure or back-pressure applied may be sufficient to produce a final yield of lycopene that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 -fold higher than that of phytoene. In some embodiments, the pressure or back-pressure applied may be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg (bar gauge); and / or up to 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5 barg. In some embodiments, the maximum pressure is governed by the maximum pressure rating of the fermenter and / or the tolerance of the eukaryotic microorganism to elevated pressure.

[0026] In some embodiments, the production conditions described herein may comprise culturing the cell population in the presence of a lipid feedstock / carbon source as a carbon source. In some embodiments, the production conditions described herein may comprise culturing the cell population in the presence of a lipid feedstock / carbon source as the primary carbon source. As used herein, the expression “primary carbon source” refers to the type(s) of compound(s) in the culture that is / are most involved in the supply of carbon and energy for the host microorganism during a particular culture phase (e.g., a production phase or production conditions described herein). In conventional fermentations, sugars / carbohydrates are typically employed as primary carbon sources. In contrast, in some embodiments, the present inventors observed that the cells metabolizing lipid feedstocks developed lipid bodies in which the produced carotenoids are believed to advantageously accumulate. In some embodiments, the lipid feedstock / carbon source employed in the production conditions described herein may be or comprise: an edible oil (e.g., canola oil, vegetable oil, soybean oil, coconut oil, or any combination thereof); a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other nontoxic liquid hydrocarbon mixture); a fatty acid (e.g., palmitic acid, oleic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, arachidic acid, or any combination thereof), paraffin oil, mineral oil, oleic acid, a non-edible oil (e.g., castor oil, camelina oil, mahua oil, or any combination thereof); or any combination or mixture thereof.

[0027] In some embodiments, production conditions described herein may comprise culturing the cell population in the presence of glucose as a primary carbon source. In other embodiments, production conditions described herein may comprise culturing the cell population in the presence of glucose as a secondary (or non-primary) carbon source. As used herein, the expression “secondary carbon source” of “non-primary carbon source” refers to the type(s) of compound(s) in the culture that is / are not the most involved in the supply of carbon and energy for the host microorganism during a particular culture phase (e.g., production phase or production conditions described herein). In some embodiments, production conditions described herein may comprise culturing the cell population under nutrient-replete or nutrientexcess conditions. In some embodiments, production conditions described herein may comprise culturing the cell population under oxygen-replete or oxygen-excess conditions, such as wherein the dissolved oxygen (D.O.) is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%.

[0028] In some embodiments, culturing the cell population under the production conditions and pressure described herein may result in an increase in cell density of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold, as compared the cell population provided prior to the culturing. In some embodiments, culturing the cell population under the production conditions and pressure described herein may result in the cell population attaining a cell density corresponding to an optical density at 600 nm (OD600) of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400.

[0029] In some embodiments, the eukaryotic microorganism described herein may be a yeast, fungi, algae, or other suitable eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene. In some embodiments, the eukaryotic microorganism described herein may be an oleaginous microorganism. As used herein, the expression “oleaginous microorganism” refers to a microorganism capable of accumulating at least 20% of lipids with respect to its dry weight. In some embodiments, the eukaryotic microorganism described herein may be from Blastobotrys, Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowia. In some embodiments, the eukaryotic microorganism described herein may be from Blastobotrys adeninivorans, Yarrowia lipolytica, or Saccharomyces cerevisiae. In some embodiments, the eukaryotic microorganism employed in a process described herein may have been identified and / or validated according to a method described herein, as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure.

[0030] In some embodiments, the eukaryotic microorganism described herein may express or overexpress a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate from famesyl diphosphate and isopentenyl diphosphate. In some embodiments, the geranylgeranyl diphosphate synthase may comprise an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 11; or a fragment thereof having geranylgeranyl diphosphate synthase activity. As used herein, the expression “overexpress,” “overexpresses,” or “overexpression” refers to the production of a protein product in a genetically engineered microorganism that exceeds levels of production / expression in a corresponding normal (e.g., wild-type) or non-genetically engineered microorganism.

[0031] In some embodiments, the eukaryotic microorganism described herein may express or overexpress a phytoene synthase (EC 2.5.1.32) that catalyzes the synthesis of phytoene from geranylgeranyl diphosphate. In some embodiments, the phytoene synthase may comprise an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 12; or a fragment thereof having phytoene synthase activity. In some embodiments, the eukaryotic microorganism described herein may express or overexpress a phytoene desaturase (EC 1.3.99.31) that catalyzes the synthesis of lycopene from phytoene. In some embodiments, the phytoene desaturase may comprise an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 13; or a fragment thereof having phytoene desaturase activity.

[0032] In some embodiments, the eukaryotic microorganism described herein may express or overexpress a bifunctional phytoene synthase (EC 2.5.1.32) / lycopene cyclase (EC 5.5.1.19) (CRTBY) that catalyzes the synthesis of phytoene from geranylgeranyl diphosphate. In some embodiments, the bifunctional phytoene synthase / lycopene cyclase may comprise an amino acid sequence at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 14; or a fragment thereof having bifunctional phytoene synthase / lycopene cyclase activity.

[0033] In some embodiments, the eukaryotic microorganism described herein further express or overexpress an acetyl-CoA C-acetyltransferase (EC 2.3. 1.9) that catalyzes the synthesis of acetoacetyl- CoA. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10) that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a hydroxymethylglutaryl-CoA reductase (EC 1.1.1.34) that catalyzes the conversion of hydroxymethylglutaryl-CoA to mevalonate. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a mevalonate kinase (EC 2.7.1.36) that catalyzes the conversion of mevalonate to a phosphomevalonate. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a phosphomevalonate kinase (EC 2.7.4.2) that catalyzes the conversion of phosphomevalonate to diphosphomevalonate. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a diphosphomevalonate decarboxylase (EC 4.1.1.33) that catalyzes the conversion of diphosphomevalonate to isopentenyl diphosphate. In some embodiments, the eukaryotic microorganism described herein further express or overexpress an isopentenyl-diphosphate Delta-isomerase (EC 5.3.3.2) that catalyzes the conversion of isopentenyl diphosphate to dimethylallyl diphosphate. In some embodiments, the eukaryotic microorganism described herein further express or overexpress a famesyl diphosphate synthase (EC 2.5. 1.1; EC 2.5.1.10) that catalyzes the conversion of isopentenyl diphosphate and dimethylallyl diphosphate to geranyl diphosphate, and / or catalyzes the conversion of geranyl diphosphate and isopentenyl diphosphate to famesyl diphosphate.

[0034] In some embodiments, one or more of the expressed or overexpressed enzymes described herein may be heterologous or exogenous with respect to the eukaryotic microorganism. In some embodiments, one or more of the expressed or overexpressed enzymes described herein may be encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the eukaryotic microorganism in one or more copies). In some embodiments, one or more of the expressed or overexpressed enzymes described herein may be encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid). In some embodiments, one or more of the expressed or overexpressed enzymes described herein may be encoded by polynucleotide(s) operably linked to a heterologous promoter.

[0035] In a further aspect, described herein is a process for producing beta-carotene as a product or an intermediate. In some embodiments, the process is as described herein for the production of lycopene, wherein the eukaryotic microorganism further expresses or overexpresses a lycopene cyclase described herein.

[0036] In some embodiments, one or more of the expressed or overexpressed enzymes described herein is or may be placed under the control of an inducible promoter. In some embodiments, the inducible promoter may be a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4-dienoyl CoA reductase), ICL1 (Isocitrate lyase), or FOX3 (3-ketoacyl CoA thiolase).

[0037] In some embodiments, the final yield of lycopene and / or beta-carotene in accordance with a process described herein may be at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10- fold higher than that of phytoene. In some embodiments, the processes described herein may further comprise purifying and / or isolating the lycopene and / or beta-carotene from the cell population.

[0038] In a further aspect, described herein is an engineered eukaryotic microorganism as described herein. In some embodiments, the engineered eukaryotic microorganism as described herein is for use in the production of lycopene and / or beta-carotene, as a product or intermediate. In some embodiments, the engineered eukaryotic microorganism as described herein is for use in the production of astaxanthin or vitamin A, which is produced from the intermediate lycopene and / or beta-carotene. In some embodiments, the engineered eukaryotic microorganism may be identified and / or validated according to a method described herein, as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure.

[0039] In a further aspect, described herein is a method for identifying and / or validating a eukaryotic microorganism that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure. In general, the method comprises providing a fermenter comprising a cell population of the eukaryotic microorganism, wherein the eukaryotic microorganism has a metabolic pathway for the synthesis of lycopene from phytoene (e.g., as described herein). In some embodiments, the method comprises a production phase comprising culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene, and applying a degree of pressure (e.g., at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg of pressure) to the fermenter during at least a portion of the production phase. In some embodiments, the method comprises measuring the amount of lycopene or downstream product thereof produced by the eukaryotic microorganism, and the eukaryotic microorganism is identified and / or validated as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, when the amount of lycopene or downstream product thereof produced is greater than that produced by a corresponding method lacking the application of pressure during the production phase. In some embodiments, the method further comprises employing the eukaryotic microorganism, if or when identified and / or validated, in an industrial-scale fermentative process for the production of lycopene as a product or intermediate.

[0040] In some embodiments of the method described herein, the pressure applied to the fermenter is as described herein, the production conditions are as described herein, the culturing of the cell population results in a cell density is as described herein, the eukaryotic microorganism is as described herein, the amount of lycopene or downstream product thereof is as described herein, or any combination thereof.

[0041] As used herein, the expression “downstream product of lycopene” refers to a metabolite whose synthesis pathway involves lycopene as an intermediate. In some embodiments, the downstream product of lycopene may comprise or consist of: beta-carotene, astaxanthin, retinal, retinol (Vitamin A), alphacarotene, gamma-carotene, zeaxanthin, lutein, violaxanthin, neoxanthin, , canthaxanthin, abscisic acid (ABA), strigolactone, or any combination thereof.

[0042] ITEMS

[0043] In various embodiments, described herein are one or more of the following items:

[0044] Item 1. A process for producing lycopene as a product or intermediate, the process comprising: (a) providing a fermenter comprising a cell population of a eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene; (b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene; and (c) applying a degree of pressure to the fermenter sufficient to shift carbon flux from phytoene towards lycopene production, thereby reducing phytoene accumulation in the eukaryotic microorganisms as compared to in the absence of said pressure.

[0045] Item 2. The process of item 1, wherein the pressure applied to the fermenter is back-pressure.

[0046] Item 3. The process of item 1 or 2, wherein the pressure or back-pressure applied is: (i) sufficient to reduce phytoene accumulation by at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold, as compared to in the absence of said pressure or back-pressure; (ii) sufficient to produce a final yield of lycopene that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15-fold higher than that of phytoene; (iii) at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg; or (iv) any combination of (i) to (iii).

[0047] Item 4. The process of any one of items 1 to 3, wherein said production conditions comprise culturing the cell population in the presence of a lipid carbon source.

[0048] Item 5. The process of item 4, wherein the lipid carbon source is or comprises: an edible oil (e.g., canola oil, vegetable oil, soybean oil, coconut oil, or any combination thereof); a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other liquid hydrocarbon mixture); a fatty acid (e.g., palmitic acid, oleic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, arachidic acid, or any combination thereof), paraffin oil, mineral oil, oleic acid, a non-edible oil (e.g., castor oil, camelina oil, mahua oil, or any combination thereof); or any combination thereof.

[0049] Item 6. The process of any one of items 1 to 5, wherein said production conditions comprise: (i) culturing the cell population in the presence of glucose as a primary or secondary carbon source; (ii) culturing the cell population under nutrient-replete or nutrient-excess conditions; (iii) culturing the cell population under oxygen-replete or oxygen-excess conditions (e.g., wherein the dissolved oxygen (D.O.) is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%); or (iv) both (i) and (iii).

[0050] Item 7. The process of any one of items 1 to 6, wherein the culturing of the cell population under said pressure results in: (i) an increase in cell density of the cell population of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold as compared the cell population provided in item 1(a); (ii) the cell population attaining a cell density corresponding to an OD600 of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400; or (iii) both (i) and (ii).

[0051] Item 8. The process of any one of items 1 to 7, wherein the eukaryotic microorganism is: (i) a yeast, fungi, algae, or other eukaryotic microorganism; (ii) an oleaginous microorganism; (iii) from Blastobotrys, Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowicr, or (iv) any combination of (i) to (in).

[0052] Item 9. The process of any one of items 1 to 8, wherein the eukaryotic microorganism is from Blastobotrys adeninivorans, Yarrowia lipolytica, or Saccharomyces cerevisiae.

[0053] Item 10. The process of any one of items 1 to 9, wherein the eukaryotic microorganism expresses or overexpresses: (i) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate from famesyl diphosphate and isopentenyl diphosphate; (ii) a phytoene synthase (EC 2.5.1.32) that catalyzes the synthesis of phytoene from geranylgeranyl diphosphate; (iii) a phytoene desaturase (EC 1.3.99.31) that catalyzes the synthesis of lycopene from phytoene; or (iv) any combination of (i) to (iii). Item 11. A process for producing beta-carotene as a product or intermediate, wherein the process is as defined in any one of items 1 to 10, wherein the eukaryotic microorganism expresses or overexpresses a lycopene cyclase (EC 5.5.1.19) that catalyzes the synthesis lycopene to beta-carotene.

[0054] Item 12. The process of item 10 or 11, wherein the expressed or overexpressed enzyme is: (i) heterologous or exogenous with respect to the eukaryotic microorganism; (ii) encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the eukaryotic microorganism in one or more copies); (iii) encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid); (iv) encoded by polynucleotide(s) operably linked to a heterologous promoter; or (v) any combination of (i) to (iv).

[0055] Item 13. The process of any one of items 10 to 12, wherein one or more of the expressed or overexpressed enzymes is / are under the control of an inducible promoter.

[0056] Item 14. The process of item 13, wherein the inducible promoter is a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4-dienoyl CoA reductase), ICL1 (Isocitrate lyase), or FOX3 (3-ketoacyl CoA thiolase).

[0057] Item 15. The process of any one of items 1 to 14, wherein the final yield of lycopene and / or betacarotene is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10-fold higher than that of phytoene.

[0058] Item 16. The process of any one of items 1 to 15, further comprising purifying or isolating the lycopene and / or beta-carotene from the cell population.

[0059] Item 17. An engineered eukaryotic microorganism as defined in any one of items 10 to 14.

[0060] Item 18. The engineered eukaryotic microorganism of item 17, for use in the production of lycopene and / or beta-carotene, as a product or intermediate.

[0061] Item 19. The engineered eukaryotic microorganism of item 18, for use in the production of astaxanthin or vitamin A, which is produced from the intermediate lycopene and / or beta-carotene.

[0062] Item 20. A method for identifying and / or validating a eukaryotic microorganism that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, the method comprising: (a) providing a fermenter comprising a cell population of the eukaryotic microorganism, the eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene; (b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene; (c) applying at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg of pressure to the fermenter during at least a portion of (b); and (d) measuring the amount of lycopene or downstream product thereof produced by the eukaryotic microorganism, wherein the eukaryotic microorganism is identified and / or validated as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, when the amount of lycopene or downstream product thereof produced is greater than that produced by a corresponding method lacking step (c).

[0063] Item 21. The method of item 20, further comprising (e) employing the eukaryotic microorganism, if identified and / or validated, in an industrial-scale fermentative process for the production of lycopene as a product or intermediate.

[0064] Item 22. The method of item 20 or 21, wherein: (i) the pressure applied to the fermenter is as defined in item 2 or 3; (ii) the production conditions are as defined in item 4 to 6; (iii) the culturing of the cell population under said pressure results in a cell density as defined in item 7; (iv) the eukaryotic microorganism is as defined in any one of items 8 to 14; (v) the amount of lycopene or downstream product thereof is as defined in item 15; or (vi) any combination of (i) to (v).

[0065] Item 23. The method of any one of items 20 to 22, wherein the downstream product of lycopene comprises or consists of: beta-carotene, astaxanthin, retinal, retinol (Vitamin A), alpha-carotene, gammacarotene, zeaxanthin, lutein, violaxanthin, neoxanthin, , canthaxanthin, abscisic acid (ABA), strigolactone, or any combination thereof.

[0066] Item 24. The process of any one of items 1 to 16, wherein the eukaryotic microorganism is one identified and / or validated by the method of any one of items 20 to 23.

[0067] EXAMPLES

[0068] Example 1: Transformation Procedure

[0069] 5 mL YPD starter cultures were inoculated with a colony of Blastobotrys adeninivorans strain ATCC 76597 (LCYBS1) and incubated overnight at 30 °C with shaking at about 200 rpm. The following day, fresh 25 mL YPD cultures were inoculated to an initial OD600 nm of 0.4 and the culture incubated at 30 °C with shaking at about 200 rpm until an OD600 nm of 1.0-2.0 was reached. Cells were pelleted by centrifugation at 1,000 x g for 10 minutes at 4 °C. Cells were washed by resuspending in 10 mL sterile water, pelleted, resuspended in 1 mL sterile water, and transferred to a 1.5 mL microcentrifuge tube. The cells were then washed in 1 mL sterile TE / LiOAC solution (100 mM Tris-EDTA (TE), 100 mM LiOAC, pH 7.5), pelleted, resuspended in 0.25 mL TE / LiOAC solution and incubated with shaking at 30 °C for 30 minutes. The cell solution was divided into 50 pL aliquots in 1.5 mL tubes to which was added 1-3 pg of both donor DNA, guide RNA, and 5 pL of carrier DNA (boiled and cooled salmon sperm DNA, 10 mg / mL). 300 pL of sterile PEG solution (40% polyethylene glycol (PEG) 3350, 100 mM TE, 100 mM LiOAC) was added, mixed thoroughly, and incubated at 30 °C for 60 minutes with gentle mixing every 15 minutes. 40 pL of dimethyl sulfoxide (DMSO) was added, mixed thoroughly and the cell solution was incubated at 42 °C for 15 minutes. Cells were then pelleted by centrifugation at 1,000 x g for 30 seconds, resuspended in 500 pL of YPD media and incubated at 30 °C with shaking at about 200 rpm for 2 hours. Cells were then pelleted by centrifugation and resuspended in 1 mb 100 mM TE, cells were pelleted again, resuspended in 0.2 m 100 mM TE and plated on selective media. Plates were incubated at 30 °C for growth of transformants.

[0070] Example 2: Construction of Strain LCYBSA1 (SFK1::CRTE)

[0071] An SFK1 deletion cassette was constructed by assembling three DNA fragments using overlap polymerase chain reaction (PCR) to produce a strain with the endogenous SFK1 gene (SEQ ID NO: 1) interrupted with the geranylgeranyl diphosphate synthase (EC 2.5.1.29) (CRTE) gene (SEQ ID NO: 2). The SFK1 upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The SFK1 downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized CRTE gene under the control of an HDE1 promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), which was created using overlapping PCR. All three fragments were assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBAl, which contains an SFK1 deletion cassette that overexpresses the CRTE gene. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBAl and suitable primers. The SFK1 deletion cassette was then purified and used to replace the SFK1 gene in strain LCYBS1 using a CRISPR / Cas9-based system, generating strain LCYBSA1.

[0072] Example 3: Construction of Strain LCYBSA2 (YER134C::CRTI_CRTBY)

[0073] A YER134C deletion cassette was constructed by assembling three DNA fragments using overlap PCR to produce a strain with the endogenous YER134C gene (SEQ ID NO.: 5) interrupted with the phytoene desaturase (EC 1.3.99.31) (CRTI) (SEQ ID NO: 6) and phytoene synthase (EC 2.5.1.32) / lycopene cyclase (EC 5.5.1.19) (CRTBY genes (SEQ ID NO. 7). The YER134C upstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The YER134C downstream fragment was amplified from LCYBS1 genomic DNA using suitable primers. The third fragment contained a synthesized and codon-optimized Crtl gene under the control of aaHDEl promoter (SEQ ID NO: 3) aaA HDEl terminator (SEQ ID NO: 4), and codon-optimized CrtBY gene under the control of a Pox promoter (SEQ ID NO: 8) and Pox terminator (SEQ ID NO: 9), which was created using overlapping PCR. All three fragments were then assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBA2, which contains a YER134C deletion cassette that overexpresses both Crtl and CrtBY genes. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBA2 and suitable primers. The YER134C deletion cassette was then purified and used to replace the YER134C gene in strain LCYBSA1 using a CRISPR / Cas9-based system, generating strain LCYBSA2.

[0074] Example 4: Construction of Strain LCYBSA7 (CRTBY::CRTB)

[0075] A deletion cassette for the codon-optimized CrtBY (SEQ ID NO: 7) insertion in LCYBSA2 was next constructed by assembling three DNA fragments using overlap PCR to produce a variant of the LCYBSA2 strain, with the inserted CrtBY gene (SEQ ID NO: 7) interrupted with the phytoene synthase (EC 2.5.1.32) CRTB gene (SEQ ID NO: 10). The CrtBY upstream fragment was amplified from the bifunctional phytoene synthase (EC 2.5.1.32) / lycopene cyclase (EC 5.5.1.19) (CRTBY) cDNA using suitable primers. The CrtBY downstream fragment was amplified from the bifunctional phytoene synthase (EC 2.5.1.32) / lycopene cyclase (EC 5.5.1.19) (CRTBY) cDNA using suitable primers. The third fragment contained a synthesized and codon-optimized CRTB gene under the control of an HDE1 promoter (SEQ ID NO: 3) and HDE1 terminator (SEQ ID NO: 4), which was created using overlapping PCR. All three fragments were then assembled using Gibson cloning (Gibson et al., 2009) to generate the plasmid pLCYBA7, which contains the CrtBY deletion cassette and overexpresses the CrtB gene. The deletion cassette was then generated via a PCR reaction containing plasmid pLCYBA7 and suitable primers. The CrtBY deletion cassette was then purified and used to replace the CrtBY gene in strain LCYBSA2 using a CRISPR / Cas9-based system, generating strain LCYBSA7.

[0076] Example 5: Batch-fed fermentation of LCYBSA2 strain in the absence of back-pressure

[0077] Strain LCYBSA2, engineered as described in Example 3, expresses the enzymes necessary for the conversion of geranylgeranyl diphosphate to phytoene (phytoene synthase; CrtB), phytoene to lycopene (phytoene desaturase; Crtl), and lycopene to beta-carotene (lycopene cyclase; CrtBY), all under control of a fatty acid inducible promoter (HDE1 promoter).

[0078] LCYBSA2 was grown using a batch-fed process conducted in a bench-scale fermenter. The fermentation was a two-phase process comprising an initial growth phase (characterized by rapid cellular growth over a period of 18-24 hours) and a subsequent production phase (characterized by slower cellular growth and carotenoid production). The initial growth phase medium contained 60 g / L glucose as the primary carbon source, with agitation and airflow rates being set to provide oxygen-replete conditions without added back-pressure. After the initial batch glucose had been consumed, an observed rapid rise in the fermenter dissolved oxygen level indicated the end of the initial growth phase and the OD600 nm of the cells at the end of the growth phase was approximately 75. Next, during the production phase, the LCYBSA2 strain was fed with 100% canola oil at a feed rate set at 4 g / L / hour with the agitation and airflow rates being kept the same as in the initial growth phase. During the production phase, the cells metabolizing the lipid feedstock (canola oil) were observed to develop lipid bodies in which the produced carotenoids are believed to accumulate.

[0079] Cell samples collected at 48 h after the start of the production phase, with cells reaching an OD600 nm of approximately 200, were lysed and carotenoid levels were measured by high-performance liquid chromatography (HPLC). The results revealed that 70% of total carotenoids produced was phytoene, and 25% was beta-carotene.

[0080] Example 6: Batch-fed fermentation of LCYBSA2 strain with back-pressure

[0081] The LCYBSA2 strain was grown using a batch-fed process conducted in a bench-scale fermenter as described in Example 5, except that 0.5 to 1 barg of back-pressure was applied throughout the production phase to shift carbon flux from phytoene to beta-carotene, with higher back-pressures being required for higher cell densities. Cell samples collected at 48 h after the start of the production phase were lysed and carotenoid levels were measured by HPLC. The results revealed that 7% of total carotenoids produced was phytoene, and 89% was beta-carotene, with the total yield of the carotenoids being substantially the same as in Example 5.

[0082] Example 7: Batch-fed fermentation of LCYBSA7 strain in the absence of back-pressure

[0083] Strain LCYBSA7, engineered as described in Example 4, expresses the enzymes necessary for the conversion of geranylgeranyl diphosphate to phytoene (phytoene synthase; CrtB) and phytoene to lycopene (phytoene desaturase; Crtl), under the control of fatty acid inducible promoters (Pox promoter and HDE1 promoter, respectively). Unlike LCYBSA2, the LCYBSA7 strain lacks a lycopene cyclase (CrtY) enzyme required to convert lycopene to beta-carotene.

[0084] The LCYBSA7 strain was grown using a batch-fed process conducted in a bench-scale fermenter as described in Example 5, in the absence of any back-pressure in the production phase. Cell samples collected at 48 h after the start of the production phase were lysed and carotenoid levels were measured by HPLC. The results revealed that 65% of total carotenoids produced was phytoene, and 30% was lycopene, with the total yield of the carotenoids being comparable to those in Examples 5 and 6.

[0085] Example 8: Batch-fed fermentation of LCYBSA7 strain with back-pressure

[0086] The LCYBSA7 strain was grown using a batch-fed process conducted in a bench-scale fermenter as described in Example 7, except that 0.5 to 1 barg of back-pressure was applied throughout the production phase. Cell samples collected at 48 h after the start of the production phase were lysed and carotenoid levels were measured by HPLC. The results revealed that 5% of total carotenoids produced was phytoene, and 90% was lycopene, with the total yield of the carotenoids being substantially the same as in Example 7.

[0087] Example 9: Batch-fed fermentation of LCYBSA7 strain with delayed back-pressure

[0088] The LCYBSA7 strain was grown using a batch-fed process conducted in a bench-scale fermenter as described in Example 7, except that no back-pressure was added for the first 24 h of the production phase and 0.5 to 1 barg of back-pressure was applied, depending on the stage of fermentation, for the second 24 h of the production phase. Cell samples collected at 24 h and 48 h after the start of the production phase were lysed and carotenoid levels were measured by HPLC. The results revealed that, at 24 h after the start of the production phase, 65% of total carotenoids produced was phytoene, and 30% was lycopene. However, at 48 h after the start of the production phase, 5% of total carotenoids produced was phytoene, and 95% was lycopene. The total yield of the carotenoids at the end of fermentation was substantially the same as in Examples 7 and 8. These results suggest that 95% of the phytoene that was produced during the first 24 h of the production phase under no back-pressure was converted in the next 24 h when cultured under back-pressure.

[0089] Example 10: Batch-fed fermentation of LCYBSA2 and LCYBSA7 strains with different lipid feedstocks

[0090] The LCYBSA2 and LCYBSA7 strains were grown using a batch-fed process conducted in a bench-scale fermenter as described in Examples 5 to 8, except that canola oil was substituted with vegetable oil, soybean oil, coconut oil, paraffin oil, mineral oil, oleic acid, or non-edible oils (e.g., castor oil). The results were consistent with those observed with canola oil, with the addition of back-pressure during the production phase significantly shifting the proportion of total carotenoids towards lycopene (LCYBSA7) or beta-carotene (LCYBSA2).

[0091] Example 11: Batch-fed fermentation of LCYBSA2 and LCYBSA7 strains with lipid / glucose feedstocks

[0092] The LCYBSA2 and LCYBSA7 strains were grown using a batch-fed process conducted in a bench-scale fermenter as described in Examples 5 to 8, except that, in additional to canola oil, the cells were additionally fed with a limited amount of glucose during the production phase that was completely consumed by the cells (i.e., without being allowed to accumulate in the media). The results were comparable or better than those observed with canola oil alone, with the addition of back-pressure during the production phase significantly shifting the proportion of total carotenoids towards lycopene (LCYBSA7) or beta-carotene (LCYBSA2).

[0093] REFERENCES

[0094] Bobadilla Romero et al., “Evaluation of bioreactor operation modes for heterologous -carotene production in Saccharomyces cerevisiae '' Department of Chemical Engineering, Universidad de los Andes, Bogota, Colombia, 2020. Disponible en: http: / / hdl.handle.net / 1992 / 44816

[0095] Dixon et al., “The inhibition by CO2 of the growth and metabolism of micro-organisms.” J Appl Bacterial. 1989;67(2): 109-136. doi: 10.1111 / j. 1365-2672.1989.tb03387.x

[0096] Gibson et al., “Enzymatic assembly of DNA molecules up to several hundred kilobases”. Nat Methods . 2009;6(5):343-345. doi: 10.1038 / nmeth,1318

[0097] Gevantman, “Solubility of selected gases in water.” In: Haynes, W.M. (ed.) CRC Handbook of Chemistry and Physics 2016-2017, 97th edn., pp. 5-134 - 5-135. CRC Press, Boca Raton (2017)

[0098] Guadalupe-Daqui et al., “The effect of CO2 concentration on yeast fermentation: rates, metabolic products, and yeast stress indicators.” J Ind Microbiol Biotechnol. 2023;50(l):kuad001. doi: 10. 1093 / jimb / kuad001

[0099] Higgins et al., “Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation”. Appl Environ Microbiol. doi: 10.1128 / AEM.69.8.4777-4787.

[0100] Knoll et al., “High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors.” J Biotechnol. 2007; 132(2): 167-179. doi: 10.1016 / j.jbiotec.2007.06.010

[0101] Nishizaki et al., “Metabolic engineering of carotenoid biosynthesis in Escherichia coli by ordered gene assembly in Bacillus siibiilis." Appl Environ Microbiol . 2007;73(4): 1355-1361. doi: 10. 1128 / AEM.02268-06

[0102] Olson, “Metabolic engineering of .S', cerevisiae for carotenoid production optimization,” Master’s Thesis, Texas A&M University, Master of Science, 2014.

[0103] Paul et al., ‘Yeast Carotenoids: Cost-Effective Fermentation Strategies for Health Care Applications.” Fermentation. 2023; 9(2): 147. https: / / doi.org / 10.3390 / fermentation9020147

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[0105] Trikka et al. “Iterative carotenogenic screens identify combinations of yeast gene deletions that enhance sclareol production. ” Mier ob Cell Fact. 2015;14:60. Published 2015 Apr 24. doi: 10. 1186 / s 12934- 015-0246-0

Claims

CLAIMS1. A process for producing lycopene as a product or intermediate, the process comprising:(a) providing a fermenter comprising a cell population of a eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene;(b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene; and(c) applying a degree of pressure to the fermenter sufficient to shift carbon flux from phytoene towards lycopene production, thereby reducing phytoene accumulation in the eukaryotic microorganisms as compared to in the absence of said pressure.

2. The process of claim 1, wherein the pressure applied to the fermenter is back-pressure.

3. The process of claim 1 or 2, wherein the pressure or back-pressure applied is:(i) sufficient to reduce phytoene accumulation by at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold, as compared to in the absence of said pressure or backpressure;(ii) sufficient to produce a final yield of lycopene that is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 11, 12, 13, 14, or 15 -fold higher than that of phytoene;(iii) at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg; or(iv) any combination of (i) to (iii).

4. The process of any one of claims 1 to 3, wherein said production conditions comprise culturing the cell population in the presence of a lipid carbon source.

5. The process of claim 4, wherein the lipid carbon source is or comprises: an edible oil (e.g., canola oil, vegetable oil, soybean oil, coconut oil, or any combination thereof); a liquid alkane and / or alkene mixture (e.g., mineral oil, paraffin oil, or other liquid hydrocarbon mixture); a fatty acid (e.g., palmitic acid, oleic acid, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, stearic acid, arachidic acid, or any combination thereof), paraffin oil, mineral oil, oleic acid, a non-edible oil (e.g., castor oil, camelina oil, mahua oil, or any combination thereof); or any combination thereof.

6. The process of any one of claims 1 to 5, wherein said production conditions comprise:(i) culturing the cell population in the presence of glucose as a primary or secondary carbon source;(ii) culturing the cell population under nutrient-replete or nutrient-excess conditions;(iii) culturing the cell population under oxygen-replete or oxygen-excess conditions (e.g., wherein the dissolved oxygen (D.O.) is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95%); or(iv) both (i) and (iii).

7. The process of any one of claims 1 to 6, wherein the culturing of the cell population under said pressure results in:(i) an increase in cell density of the cell population of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10-fold as compared the cell population provided in claim 1(a);(ii) the cell population attaining a cell density corresponding to an OD600 of at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, or 400; or(iii) both (i) and (ii).

8. The process of any one of claims 1 to 7, wherein the eukaryotic microorganism is:(i) a yeast, fungi, algae, or other eukaryotic microorganism;(ii) an oleaginous microorganism;(iii) from Blastobotrys, Saccharomyces, Aspergillus, Pichia, Hansenula, Phycomyces, Mucor, Rhodotorula, Sporobolomyces, Xanthophyllomyces, Phaffia, Blakeslea, or Yarrowicr, or(iv) any combination of (i) to (iii).

9. The process of any one of claims 1 to 8, wherein the eukaryotic microorganism is from Blastobotrys adeninivorans, Yarrowia lipolytica, or Saccharomyces cerevisiae.

10. The process of any one of claims 1 to 9, wherein the eukaryotic microorganism expresses or overexpresses:(i) a geranylgeranyl diphosphate synthase (EC 2.5.1.29) that catalyzes the synthesis of geranylgeranyl diphosphate from famesyl diphosphate and isopentenyl diphosphate;(ii) a phytoene synthase (EC 2.

5. 1.32) that catalyzes the synthesis of phytoene from geranylgeranyl diphosphate;(iii) a phytoene desaturase (EC 1.3.99.31) that catalyzes the synthesis of lycopene from phytoene; or(iv) any combination of (i) to (iii).

11. A process for producing beta-carotene as a product or intermediate, wherein the process is as defined in any one of claims 1 to 10, wherein the eukaryotic microorganism expresses or overexpresses a lycopene cyclase (EC 5.5.1.19) that catalyzes the synthesis lycopene to beta-carotene.

12. The process of claim 10 or 11, wherein the expressed or overexpressed enzyme is:(i) heterologous or exogenous with respect to the eukaryotic microorganism;(ii) encoded by polynucleotide(s) comprised in a single polycistronic expression cassette (e.g., integrated into the genome of the eukaryotic microorganism in one or more copies);(iii) encoded by polynucleotide(s) comprised in an expression vector (e.g., plasmid);(iv) encoded by polynucleotide(s) operably linked to a heterologous promoter; or(v) any combination of (i) to (iv).

13. The process of any one of claims 10 to 12, wherein one or more of the expressed or overexpressed enzymes is / are under the control of an inducible promoter.

14. The process of claim 13, wherein the inducible promoter is a promoter of a fatty acid inducible gene, such as: HDE1, POX, PEX11 (peroxin), SPS19 (Peroxisomal 2-4-dienoyl CoA reductase), ICL1 (Isocitrate lyase), or FOX3 (3 -ketoacyl CoA thiolase).

15. The process of any one of claims 1 to 14, wherein the final yield of lycopene and / or beta-carotene is at least 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10-fold higher than that of phytoene.

16. The process of any one of claims 1 to 15, further comprising purifying or isolating the lycopene and / or beta-carotene from the cell population.

17. An engineered eukaryotic microorganism as defined in any one of claims 10 to 14.

18. The engineered eukaryotic microorganism of claim 17, for use in the production of lycopene and / or beta-carotene, as a product or intermediate.

19. The engineered eukaryotic microorganism of claim 18, for use in the production of astaxanthin or vitamin A, which is produced from the intermediate lycopene and / or beta-carotene.

20. A method for identifying and / or validating a eukaryotic microorganism that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, the method comprising:(a) providing a fermenter comprising a cell population of the eukaryotic microorganism, the eukaryotic microorganism having a metabolic pathway for the synthesis of lycopene from phytoene;(b) culturing the cell population in the fermenter under production conditions enabling the synthesis of phytoene;(c) applying at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, or 1.5 barg of pressure to the fermenter during at least a portion of (b); and(d) measuring the amount of lycopene or downstream product thereof produced by the eukaryotic microorganism, wherein the eukaryotic microorganism is identified and / or validated as one that shifts carbon flux from phytoene towards lycopene under fermentative conditions of elevated pressure, when the amount of lycopene or downstream product thereof produced is greater than that produced by a corresponding method lacking step (c).

21. The method of claim 20, further comprising (e) employing the eukaryotic microorganism, when identified and / or validated, in an industrial-scale fermentative process for the production of lycopene as a product or intermediate.

22. The method of claim 20 or 21, wherein:(i) the pressure applied to the fermenter is as defined in claim 2 or 3;(ii) the production conditions are as defined in claim 4 to 6;(iii) the culturing of the cell population under said pressure results in a cell density as defined in claim 7;(iv) the eukaryotic microorganism is as defined in any one of claims 8 to 14;(v) the amount of lycopene or downstream product thereof is as defined in claim 15; or(vi) any combination of (i) to (v).

23. The method of any one of claims 20 to 22, wherein the downstream product of lycopene comprises or consists of: beta-carotene, astaxanthin, retinal, retinol (Vitamin A), alpha-carotene, gamma-carotene, zeaxanthin, lutein, violaxanthin, neoxanthin, , canthaxanthin, abscisic acid (ABA), strigolactone, or any combination thereof.

24. The process of any one of claims 1 to 16, wherein the eukaryotic microorganism is one having been identified and / or validated by the method of any one of claims 20 to 23.

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