Methods for the stable production of acetyl-coa derived compounds

Abstract

The present disclosure relates to the use of switches for the production of heterologous non-catabolic compounds in microbial host cells. In one aspect, provided herein are genetically modified microorganisms that more stably produce non-catabolic compounds when cultured continuously under aerobic conditions followed by microaerobic conditions, and methods of producing non-catabolic compounds by culturing the genetically modified microorganisms under such culture conditions. In another aspect, provided herein are genetically modified microorganisms that more stably produce non-catabolic compounds when cultured continuously in the presence of maltose followed by reduced or no maltose, and methods of producing non-catabolic compounds by culturing the genetically modified microorganisms under such culture conditions.

Description

1. TECHNICAL FIELD

[0001] The present disclosure relates to the use of oxygen-responsive promoters as gene switches for modulating the production of heterologous non-catabolic compounds by genetically modified host cells. 2. BACKGROUND

[0002] The advent of synthetic biology has brought the promise of fermentative microbial production of biofuels, chemicals, and biomaterials from renewable resources at industrial scale and quality. For example, functional non-natural biological pathways have been successfully constructed in microbial hosts for the production of precursors of the anti-malarial drug artemisinin (see, e.g., Martin et al., Nat Biotechnol 21 :796-802 (2003); fatty acid derived fuels and chemicals (e.g., fatty acid esters, fatty alcohols, and waxes; see, e.g., Steen et al., Nature 463:559-562 (2010); polyketide synthases that make cholesterol lowering drugs (see, e.g., Ma et al., Science 326:589-592 (2009); and polyketides (see, e.g., Kodumal, Proc Natl Acad Sci USA 101 :15573-15578 (2004). However, commercial success of synthetic biology will depend to a large extent on whether the production costs of renewable products can be made competitive or superior to those of their respective non-renewable counterparts.

[0003] Strain stability can be a major driver of industrial fermentation costs, as it affects the length of time that a continuous fermentation can be run efficiently. Strain stability generally refers to the ability of a microorganism to maintain favorable production characteristics of a non-catabolic fermentation product (i.e., high yield (grams (compound) / grams (substrate)) and productivity (grams / liter (broth) / hour)) over extended periods of cultivation. In particular, genetic stability is the propensity of a microbial population to maintain an almost unchanged expected allele frequency of the genes associated with production of a product over time, which plays a major role in the sustained output of the product.

[0004] The instability of non-catabolic fermentation of products other than biomass (which, by definition, consumes metabolic energy and carbon that would otherwise be used to produce more cells) is twofold: evolutionary mutation and selection. First, mutations that lose production arise spontaneously and randomly. Second, the growth rate or "fitness" of cells with reduced production is advantageous, leading to a takeover of the final population by low producers, reducing the overall performance of the culture. This phenomenon can be called "strain degeneration."

[0005] For a long time, Brazilian fuel ethanol fermentation achieved very high yields of ethanol from sugar, i.e., about 90% of the maximum theoretical yield. This was in part because ethanol production is catabolic: it generates 2 ATP per molecule of sugar produced and it is a redox balance without oxygen involvement. Cells that have mutated to not produce ethanol are less fit under the low oxygen conditions of the fermentor and also do not take over the population. This allowed industrial ethanol fermentation to recover most of the yeast biomass over time, leaving minimal sugar to be converted into yeast cell biomass and directing almost all sugar to ethanol production. This extended propagation and biomass recycling increased the efficiency of ethanol production: operating expenses were reduced because less sugar was converted into biomass per cycle (i.e., yield went up); and capital expenses were reduced because less and smaller fermentors were needed to build up the biomass for inoculation.

[0006] In contrast, the production of many acetyl-CoA-derived hydrocarbons (e.g., isoprenoids, fatty acids, and polyketides) is in fact usually non-catabolic; they usually require a net input of ATP, NADPH, and carbon, often with a supply of large amounts of oxygen to help balance the redox of the system. Such an environment allows evolution towards lower product, higher biomass of more fit genotypes, and leads to a higher rate of strain degeneration.

[0007] One way to reduce the negative selection pressure for producing non-catabolic products is to shut off the formation of the product during cycles when it is not needed, e.g., during the fermentation phase, where biomass must be produced in order to maximize the productivity of the fermentor. Thus, there is a need in the art for a switch that can control the time of production of acetyl-CoA-derived compounds during fermentation. 3. SUMMARY

[0008] Provided herein are fermentation methods for producing a heterologous non- catabolic compound from a genetically modified host cell. In some embodiments, the methods comprise two phases: a construction phase during which production of the non- catabolic compound is substantially reduced ("off phase") while cell biomass accumulates; and a production phase during which production of the non-catabolic compound is turned on. Thus, the negative selection pressure associated with non-catabolic compound production is alleviated during the fermentation phase where production is not required. During the construction phase, the reduction or elimination of non-catabolic compound production allows for (i) an increased growth rate of the cells during the construction phase; and (ii) an increased strain production stability during the production phase. This will result in a longer sustained production of the non-catabolic compound, thereby increasing the overall yield and / or productivity of the strain. Advantageously, the "off and "on" of non-catabolic compound production state in the fermentation methods provided herein is controlled by conditions that are readily available, cost effective, and industrially relevant.

[0009] In one aspect, the "off and "on" of non-catabolic compound production state in a fermentation culture is controlled by the level of oxygen in the fermentation, e.g., the amount of dissolved oxygen in the culture medium, in conjunction with the use of an oxygen sensitive promoter that drives the expression of genes for pathway enzymes that affect production of the heterologous non-catabolic compound. These methods take advantage of the fact that when cells that have been engineered to produce a heterologous non-catabolic compound are cultured, a limited amount of oxygen can be provided. These cells can be maintained in a microaerophilic condition for growth and viability, thereby saving costs associated with running a fully aerobic fermentation process. In some embodiments, the microaerophilic conditions can be achieved once the host cell population reaches a density sufficient to equal the rate of oxygen consumption and oxygen supply. Advantageously, by coupling the expression of pathway genes to an oxygen sensitive promoter, compound production is turned on only when the oxygen consumption of the host cell population is high enough to achieve microaerophilic conditions in the fermentor, which effectively occurs at the end of the construction phase, i.e., when the optimal cell density has been achieved for efficient compound production. Thus, the expression of pathway genes is tightly linked to achieving a population density that is ideal for the start of the production phase. Accordingly, the methods provided herein utilize oxygen levels and a genetic switch to achieve the "on and "off phases of an improved fermentation process for producing a heterologous non-catabolic compound.

[0010] Accordingly, provided herein are methods for producing a heterologous non- catabolic compound in a genetically modified host cell, the method comprising:

[0011] (a) culturing a population of genetically modified host cells under aerobic conditions in a culture medium comprising a carbon source, wherein the host cells comprise one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for making a heterologous non-catabolic compound, wherein expression of the one or more enzymes is positively regulated by promoter activity that is responsive to microaerobiosis, wherein the aerobic conditions limit the amount of the heterologous non-catabolic compound produced by the host cells; and

[0012] (b) culturing the population or subpopulation thereof under microaerobic conditions in a culture medium comprising a carbon source, wherein the microaerobic conditions increase the non-catabolic compound produced by the population or subpopulation thereof.

[0013] In some embodiments, the microaerobiosis-responsive promoter is a mutated DAN1 promoter. In some embodiments, the mutated DAN1 promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the mutated DAN1 promoter sequence comprises SEQ ID NO: 1. In some embodiments, the mutated DAN1 promoter sequence comprises SEQ ID NO: 2.

[0014] In some embodiments, the microaerobiosis-responsive promoter is operably linked to one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway, and the microaerobic conditions increase expression of the one or more enzymes of the enzyme pathway. In some embodiments, the microaerobiosis-responsive promoter is operably linked to a heterologous nucleic acid encoding a transcriptional regulator that positively regulates expression of one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway, and the microaerobic conditions increase expression of the transcriptional regulator. In some embodiments, the transcriptional regulator is Gal4p, and each of the one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway is operably linked to a Gal4p-responsive promoter selected from the group consisting of pGAL1, pGAL7, and pGAL10. In some embodiments, the host cell further comprises a functional disruption of Gal80p.

[0015] In some embodiments, the microaerobic conditions include a dissolved oxygen concentration in the culture medium of less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some embodiments, the microaerobic conditions include a dissolved oxygen concentration in the culture medium of about 0%. In some embodiments, the microaerobic conditions result in an oxygen consumption rate of the host cell of less than about 50 millimoles, less than about 40 millimoles, less than about 30 millimoles, less than about 20 millimoles, or less than about 10 millimoles per liter of culture medium. In some embodiments, the microaerobic conditions result in a specific oxygen consumption rate of the host cell of less than about 30 millimoles, less than about 25 millimoles, less than about 20 millimoles, less than about 15 millimoles, less than about 10 millimoles, or less than about 5 millimoles per gram of cell dry weight per hour.

[0016] In some embodiments, during the culturing of step (b), the production of the heterologous non- assimilated compound by the population of genetically modified host cells is improved compared to the production of the compound achieved in an aerobic fermentation process in which expression of the one or more enzymes of the enzyme pathway is not limited by the activity of the microaerobic-responsive promoter.

[0017] Also provided herein are methods for producing a heterologous isoprenoid in a genetically modified host cell, the method comprising:

[0018] (a) culturing a population of genetically modified host cells in a culture medium comprising a carbon source under aerobic conditions, wherein the host cells comprise:

[0019] (i) one or more heterologous nucleic acids encoding one or more enzymes of the mevalonate (MEV) pathway, each operably linked to a Gal4p-responsive promoter selected from the group consisting of pGALl, pGAL7, and pGALlO; and

[0020] (ii) a nucleic acid encoding Gal4p operably linked to a microaerobic-responsive promoter; wherein the aerobic conditions limit the amount of heterologous isoprenoid produced by the host cells; and

[0021] (b) culturing the population or a subpopulation thereof in a culture medium comprising a carbon source under microaerobic conditions, wherein the microaerobic conditions increase the production of the heterologous isoprenoid by the population or the subpopulation thereof.

[0022] In another aspect, the "on" and "off states of non-catabolic compound production in a fermentation culture are controlled by the amount of the sugar maltose in the culture medium in combination with a maltose-responsive promoter that regulates the expression of a gene that affects a pathway enzyme for the production of the heterologous non-metabolic compound. Advantageously, by coupling the expression of a pathway gene to a maltose-sensitive promoter, compound production can be turned on or off by controlling the amount of maltose in the feedstock. For example, a maltose-responsive promoter can be wired as an "on" switch to induce heterologous non-catabolic compound production in the presence of maltose. Alternatively, a maltose-responsive promoter can also be wired as an "off switch to induce the expression of a negative regulator of the enzyme pathway for compound production in the presence of maltose. Thus, the methods provided herein utilize maltose levels in the culture medium and gene switches to affect the "on" and "off stages of an improved fermentation process for the production of a heterologous non-catabolic compound.

[0023] Provided herein are methods for producing a heterologous non-catabolic compound in a genetically modified host cell, the method comprising:

[0024] (a) culturing a population of genetically modified host cells in a culture medium comprising a carbon source, including maltose, wherein the host cells comprise one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for making a heterologous non-catabolic compound, wherein expression of the one or more enzymes is negatively regulated by the activity of a maltose-responsive promoter, wherein the presence of maltose in the culture medium limits the amount of the heterologous non-catabolic compound produced by the host cells; and

[0025] (b) culturing the population or a subpopulation thereof in a culture medium comprising a carbon source, wherein maltose is absent or at a sufficiently low amount such that the maltose-responsive promoter is no longer effective, and increasing the yield of the heterologous non-catabolic compound produced by the host cells.

[0026] In some embodiments, the maltose-responsive promoter is operably linked to a heterologous nucleic acid encoding a transcriptional regulator that down-regulates expression of one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway, and the maltose in step (a) increases expression of the transcriptional regulator. In some embodiments, the transcriptional regulator is Gal80p, the host cell further comprises Gal4p, and each of the one or more heterologous nucleic acids encoding one or more enzymes of the enzyme pathway is operably linked to a Gal4p-responsive promoter selected from the group consisting of pGAL1, pGAL7, and pGAL10. In some embodiments, the maltose-responsive promoter comprises a sequence selected from the group consisting of pMAL1 (SEQ ID NO: 12), pMAL2 (SEQ ID NO: 13), pMAL11 (SEQ ID NO: 14), pMAL12 (SEQ ID NO: 15), pMAL31 (SEQ ID NO: 16), and pMAL32 (SEQ ID NO: 17). In some embodiments, the maltose-responsive promoter sequence comprises pMAL32 (SEQ ID NO: 17).

[0027] In some embodiments, the culture medium of step (a) comprises at least 0.1% (w / v) maltose. In some embodiments, the culture medium of step (a) comprises 0.25-3% (w / v) maltose. In some embodiments, the culture medium of step (b) comprises no more than 0.08% (w / v) maltose. In some embodiments, during the culturing period of step (b), the heterologous non- catabolic compound produced by the population of genetically modified host cells is improved compared to the heterologous non-catabolic compound achieved in a fermentation process in which the expression of the one or more enzymes of the enzyme pathway is not limited by the activity of the maltose-responsive promoter.

[0028] Also provided herein are methods for producing a heterologous isoprenoid in a genetically modified host cell, the method comprising:

[0029] (a) culturing a population of genetically modified host cells in a culture medium comprising a carbon source, including maltose, wherein the host cells comprise:

[0030] (i) one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway, each operably linked to a Gal4p-responsive promoter selected from the group consisting of pGAL 1, pGAL 7, and pGAL10; and

[0031] (ii) a nucleic acid encoding Gal4p; and

[0032] (iii) a nucleic acid encoding Gal80p operably linked to a maltose-responsive promoter; wherein maltose in the culture medium limits the amount of heterologous isoprenoid produced by the host cell; and

[0033] (b) culturing the population or subpopulation thereof in a culture medium comprising a carbon source in the absence or at a sufficiently low amount of maltose such that the maltose-responsive promoter is no longer operative, and increasing the yield of the heterologous non- assimilation compound produced by the host cell.

[0034] In some embodiments, the non-assimilation compound produced in step (a) of the methods described herein is less than 50%, 40%, 30%, 20%, or 10% of the non-assimilation compound produced in step (b). In some embodiments, the culturing of step (a) is for a period of time of at least 12, 24, 36, 48, 60, 72, 84, 96, or more than 96 hours. In some embodiments, the culturing of step (a) is for a period of time sufficient for the population to reach a cell density (OD 600 ) of 0.01 to 400. In some embodiments, the culturing of step (b) is for a period of time of 3-20 days. In some embodiments, the production of the non-assimilation compound is measured in terms of yield (grams of non-assimilation compound produced per gram of carbon substrate) or productivity (grams of non-assimilation compound produced per liter of culture medium per hour). In some embodiments, the methods further comprise recovering the non-assimilation compound.

[0035] In another aspect, provided herein is a fermentation composition produced by the fermentation methods described herein. In some embodiments, the fermentation composition comprises a population of genetically modified host cells in a culture medium comprising a carbon source, wherein the host cells comprise one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for making a heterologous non-assimilation compound, wherein expression of the one or more enzymes is positively regulated by activity of a microaerophilic-responsive promoter. In some embodiments, the microaerophilic-responsive promoter is operably linked to the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway, wherein expression of the one or more enzymes of the enzyme pathway is increased under microaerophilic fermentation conditions. In some embodiments, the microaerophilic-responsive promoter is operably linked to a heterologous nucleic acid encoding a transcriptional regulator that positively regulates expression of the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway, wherein expression of the transcriptional regulator is increased under microaerophilic fermentation conditions. In some embodiments, the transcriptional regulator is Gal4p, and each of the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway is operably linked to a Gal4p-responsive promoter selected from the group consisting of pGALl, pGAL7, and pGALlO.

[0036] In some embodiments, the fermentation composition comprises a population of genetically modified host cells in a culture medium comprising a carbon source, wherein the host cells comprise: (a) one or more heterologous nucleic acids encoding one or more enzymes of a mevalonate (MEV) pathway, each operably linked to a Gal4p-responsive promoter, and (b) a nucleic acid encoding Gal4p, operably linked to a microaerophilic-responsive promoter. In some embodiments, the host cells further comprise a functional disruption of Gal80p. In some embodiments, the microaerophilic-responsive promoter is a mutated DAN1 promoter. In some embodiments, the mutated DAN1 promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In some embodiments, the mutated DAN1 promoter sequence comprises SEQ ID NO: 1. In some embodiments, the mutated DAN1 promoter sequence comprises SEQ ID NO: 2. In some embodiments, the culture medium comprises a dissolved oxygen concentration of 100%. In some embodiments, the culture medium comprises a dissolved oxygen concentration of less than about 20%, less than about 15%, less than about 10%, or less than about 5%. In some embodiments, the culture medium comprises a dissolved oxygen concentration of about 0%.

[0037] In some embodiments, the fermentation composition comprises a population of genetically modified host cells in a culture medium comprising a carbon source, wherein the host cells comprise one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for making a heterologous non-catabolic compound, wherein expression of the one or more enzymes is positively modulated by activity of a maltose-responsive promoter. In some embodiments, the maltose-responsive promoter is operably linked to the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway, wherein expression of the one or more enzymes of the enzyme pathway is reduced in the presence of maltose. In some embodiments, the maltose-responsive promoter is operably linked to a heterologous nucleic acid encoding a transcriptional regulator that negatively modulates expression of the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway, wherein expression of the transcriptional regulator is increased in the presence of maltose. In some embodiments, the transcriptional regulator is Gal80p, the host cells further comprise Gal4p, and the one or more heterologous nucleic acids encoding the one or more enzymes of the enzyme pathway are each operably linked to a Gal4p-responsive promoter selected from the group consisting of pGAL1, pGAL7, and pGAL10.

[0038] In some embodiments, the fermentation composition comprises a population of genetically modified host cells in a medium comprising a carbon source, wherein the host cells comprise: (a) one or more heterologous nucleic acids encoding one or more enzymes of the mevalonate (MEV) pathway, each operably linked to a Gal4p-responsive promoter, (b) a nucleic acid encoding Gal4p; and (c) a nucleic acid encoding Gal80p, operably linked to a maltose-responsive promoter. In some embodiments, the maltose-responsive promoter comprises a sequence selected from the group consisting of pMALl (SEQ ID NO: 12), pMAL2 (SEQ ID NO: 13), pMALl l (SEQ ID NO: 14), pMALl2 (SEQ ID NO: 15), pMAL31 (SEQ ID NO: 16), and pMAL32 (SEQ ID NO: 17). In some embodiments, the maltose-responsive promoter sequence comprises pMAL32 (SEQ ID NO: 17). In some embodiments, the medium comprises at least 0.1% (w / v) maltose. In some embodiments, the medium comprises 0.25-3% (w / v) maltose. In some embodiments, the medium comprises no more than 0.08% maltose.

[0039] In some embodiments, the host cell is selected from the group consisting of a fungal cell, a bacterial cell, a plant cell, and an animal cell. In some embodiments, the host cell is a yeast cell. In some embodiments, the non-catabolic compound is selected from the group consisting of an amino acid, a fatty acid, an isoprenoid, and a polyketide.

[0040] In some embodiments, the host cell is capable of producing a isoprenoid and includes at least one heterologous nucleic acid encoding an isoprenoid pathway enzyme selected from the group consisting of: (a) an enzyme that condenses two molecules of acetyl-CoA to form acetoacetyl-CoA; (b) an enzyme that condenses acetoacetyl-CoA and another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA); (c) an enzyme that converts HMG-CoA to mevalonic acid; (d) an enzyme that converts mevalonic acid to mevalonate 5-phosphate; (e) an enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; (f) an enzyme that converts mevalonate 5-pyrophosphate to IPP; (g) an enzyme that converts IPP to DMAPP; (h) a polyisoprenoid synthase that condenses IPP and / or DMAPP molecules to form a polyisoprenoid compound comprising more than five carbons; (i) an enzyme that condenses IPP and DMAPP to form GPP; (j) an enzyme that condenses two molecules of IPP and one molecule of DMAPP; (k) an enzyme that condenses IPP and GPP to form FPP; (1) an enzyme that condenses IPP and DMAPP to form GGPP; and (m) an enzyme that condenses IPP and FPP to form GGPP.

[0041] In some embodiments, the host cell further comprises a heterologous nucleic acid encoding a enzyme that modifies a polyisoprenoid selected from the group consisting of: geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, a-pinene synthase, β-pinene synthase, sabinene synthase, γ-terpinene synthase, terpinolene synthase, amorphadiene synthase, a-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchoulol synthase, nootkatone synthase, abietadiene synthase.

[0042] In some embodiments, the host cell includes a plurality of heterologous nucleic acids encoding all of the enzymes of the mevalonate pathway. In some embodiments, the isoprenoid is selected from the group consisting of hemiterpenes, monoterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes. In some embodiments, the isoprenoid is a C5-C 20 isoprenoid. In some embodiments, the isoprenoid is a sesquiterpene. In some embodiments, the isoprenoid is selected from the group consisting of abietadiene, amorphadiene, carene, a-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

[0043] In some embodiments, the host cell is capable of producing a polyketide and comprises at least one heterologous nucleic acid encoding a polyketide synthase, wherein the polyketide synthase is selected from the group consisting of: (a) an enzyme that condenses at least one of acetyl-coenzyme A and malonyl-coenzyme A with an acyl carrier protein; (b) an enzyme that condenses a first reactant selected from the group consisting of acetyl-coenzyme A and malonyl-coenzyme A with a second reactant selected from the group consisting of malonyl-coenzyme A or methylmalonyl-coenzyme A to form a polyketide product; (c) an enzyme that reduces a β-keto chemical group of a polyketide compound to a β-hydroxyl group; (d) an enzyme that dehydrates an alkane chemical group of a polyketide compound to produce an α-β unsaturated alkene; (e) an enzyme that reduces an α-β-double bond of a polyketide compound to a saturated alkane; and (f) an enzyme that hydrolyzes a polyketide compound from an acyl carrier protein.

[0044] In some embodiments, the polyketide compound is a lipid having at least one of antibacterial, antifungal, and antitumor activity. In some embodiments, the polyketide compound is selected from the group consisting of a macrolide, an antibiotic, an antifungal, a cell growth inhibiting compound, an anticholesterolemic compound, an antiparasitic compound, an anticoccidial compound, an animal growth promoter, and an insecticide.

[0045] In some embodiments, the host cell is capable of producing a fatty acid and comprises at least one heterologous nucleic acid encoding a fatty acid synthase, wherein the fatty acid synthase is selected from the group consisting of: (a) an enzyme that covalently links at least one of acetyl-coenzyme A and malonyl-coenzyme A to an acyl carrier protein (ACP); (b) an enzyme that condenses acetyl ACP and malonyl ACP to form acetoacetyl ACP; (c) an enzyme that reduces the double bond of acetoacetyl ACP with NADPH to form a hydroxyl group in D-3-hydroxybutyryl dehydratase-ACP; (d) an enzyme that dehydrates D-3-hydroxybutyryl dehydratase-ACP to establish a double bond between the β- and γ-carbons to form a crotonyl-ACP; (e) an enzyme that reduces crotonyl-ACP with NADPH to form butyryl-ACP; and (f) an enzyme that hydrolyzes a C 16 acyl compound from an acyl carrier protein to form palmitate. In some embodiments, the fatty acid is selected from the group consisting of palmitate, palmitoyl coenzyme A, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. 4. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 A schematic of the mevalonate ("MEV") pathway for producing isopentenyl diphosphate ("IPP") is provided.

[0047] Figure 2 A schematic is provided for the conversion of IPP and dimethylallyl pyrophosphate ("DMAPP") to geranyl pyrophosphate ("GPP"), farnesyl pyrophosphate ("FPP"), and geranylgeranyl pyrophosphate ("GGPP").

[0048] Figure 3 Strain degeneration (i.e., the decline in production of the non- assimilated compound over time) is shown for a population of yeast host cells capable of producing the non- assimilated compound, farnesene.

[0049] Figure 4 A schematic is provided for an exemplary GAL-regulon-based hypoxic switch for controlling heterologous non- assimilated compound production in a host cell.

[0050] Figure 5 A schematic is provided for an exemplary GAL-regulon-based maltose switch for controlling heterologous non- assimilated compound production in a host cell.

[0051] Figure 6 The results provided demonstrate that a host cell capable of producing the isoprenoid farnesene and comprising a MEV pathway positively regulated by a microaerobic- responsive promoter ("low 02 switch") produces very low amounts of farnesene under high 02 conditions (shaker plates), and the yield substantially increases to a level comparable to that of a non-switchable parental strain in which the MEV pathway is constitutively expressed under low 02 conditions (shaker flasks with low RPM).

[0052] Figure 7 The results provided demonstrate that a host cell capable of producing the isoprenoid farnesene and comprising a MEV pathway positively regulated by a low 02 switch shows improved stability of farnesene production over long fermentation runs when the build-up phase of the fermentation is performed under aerobic conditions (resulting in a "off" state) compared to the yield of a constitutively producing strain producing farnesene throughout the build-up phase.

[0053] Figure 8 The results provided demonstrate that a host cell capable of producing the isoprenoid farnesene and comprising a MEV pathway negatively regulated by a maltose-responsive promoter ("maltose switch") produces very low amounts of farnesene in the presence of maltose (1.3%), and the yield substantially increases to a level close to that of a non-switchable parental strain in which the MEV pathway is constitutively expressed in the absence of maltose.

[0054] Figure 9The results presented demonstrate that host cells capable of producing isoprenoid famesene and comprising the MEV pathway positively regulated by a microaerobically responsive promoter ("low 02 switch") or negatively regulated by a maltose-responsive promoter ("maltose switch") have an increased growth rate during the "off state" of compound production compared to the famesene produced constitutively by the parent strain.

[0055] Figure 10 The results presented demonstrate that host cells capable of producing isoprenoid famesene and comprising the MEV pathway negatively regulated by a maltose switch show increased stability of famesene production over long fermentation runs when the build-up phase of the fermentation is performed in the presence of maltose (thus creating an "off state") compared to the yield of a constitutively producing strain producing famesene throughout the build-up phase.

[0056] Figure 11 The results presented demonstrate the sensitivity of the maltose-sensitive promoter pMAL11 (A) to different amounts of maltose and mixed feed in the medium, and (B) the switchability from "off state" to "on state" in the absence of maltose after repression by maltose in the "off state".

[0057] Figure 12 The results presented demonstrate the maltose-sensitive promoter pMAL12, (A) the sensitivity to different amounts of maltose and mixed feed in the medium, and (B) the switchability from "off state" to "on state" in the absence of maltose after repression by maltose in the "off state".

[0058] Figure 13 The results presented demonstrate the maltose-sensitive promoter pMAL31, (A) the sensitivity to different amounts of maltose and mixed feed in the medium, and (B) the switchability from "off state" to "on state" in the absence of maltose after repression by maltose in the "off state".

[0059] Figure 14 The results presented demonstrate the maltose-sensitive promoter pMAL32, (A) the sensitivity to different amounts of maltose and mixed feed in the medium, and (B) the switchability from "off state" to "on state" in the absence of maltose after repression by maltose in the "off state". 5. Specific embodiments

[0060] 5.1 Definitions

[0061] As used herein, the term "endogenous" refers to a substance or process that can occur naturally in a host cell.

[0062] As used herein, the phrase "functionally disrupt" or "functional disruption" of a target gene (e.g., of a target protein) means to alter the target gene in such a way as to reduce the activity of the protein encoded by the target gene in a host cell. Similarly, for example, "functionally disrupt" or "functional disruption" of a target protein means to alter the target protein in such a way as to reduce the activity of the protein in a host cell. In some embodiments, the activity of the target protein encoded by the target gene in the host cell is eliminated. In other embodiments, the activity of the target protein encoded by the target gene in the host cell is reduced. Functional disruption of a target gene can be achieved by deleting all or part of the gene, such that gene expression is eliminated or reduced or such that the activity of the gene product is eliminated or reduced. Functional disruption of a target gene can also be achieved by mutation of a gene regulatory element (e.g., the promoter of the gene), such that expression is eliminated or reduced, or by mutation of the gene coding sequence, such that the activity of the gene product is eliminated or reduced. In some embodiments, the functional disruption of the target gene results in removal of the entire open reading frame of the target gene.

[0063] As used herein, the term "genetically modified" refers to a host cell comprising a heterologous nucleotide sequence.

[0064] As used herein, the term "heterologous" refers to those that are not normally found in nature. The term "heterologous compound" refers to a compound produced by a cell that does not normally produce the compound, or produced at a level that is not normally produced by the cell.

[0065] As used herein, the phrase "heterologous enzyme" refers to an enzyme that is not normally found in a given cell in nature. Enzymes encompassed by the term are:

[0066] (a) foreign to the given cell (i.e., encoded by a nucleotide sequence that does not naturally occur in the host cell or in the particular environment of the host cell); and

[0067] (b) naturally occurring in the host cell (e.g., the enzyme is encoded by a nucleotide sequence that is endogenous to the cell), but produced in an unnatural amount in the host cell (e.g., more or less than naturally found).

[0068] As used herein, the phrase "operably linked" refers to functional linkage between a nucleic acid sequence and its associated regulatory elements such that the linked promoter and / or regulatory region controls expression of the coding sequence.

[0069] As used herein, the term "yield" generally refers to the amount of non- catabolic compound produced by a genetically modified host cell provided by the present disclosure. In some embodiments, yield is expressed as the production rate of non-catabolic compound by the host cell. In other embodiments, yield is expressed as the productivity of non-catabolic compound by the host cell.

[0070] As used herein, the term "productivity" refers to the yield of non-catabolic compound produced by a host cell expressed as the amount of non-catabolic compound produced (by weight) per volume of fermentation medium per hour of time as the host cell is cultured.

[0071] As used herein, the term "promoter" refers to a synthetic or naturally-occurring nucleic acid capable of conferring, activating or enhancing expression of a DNA coding sequence. A promoter can include one or more specific transcriptional regulatory sequences to further enhance expression and / or to alter the spatial expression and / or temporal expression of the coding sequence. A promoter can be located 5' (upstream) of the coding sequence under its control. The distance between a promoter and a coding sequence to be expressed can be approximately the same as the distance between a promoter and the native nucleic acid sequence that it controls. Variations in this distance can be accommodated without loss of promoter function, as is known in the art. A regulatable promoter as used herein generally allows for transcription of a nucleic acid sequence encoding a transcriptional regulatory factor (e.g., an activator such as pGal4 or a repressor such as pGal80) when in a permissive environment (e.g., under microaerobic fermentation conditions, or in the presence of maltose), but ceases transcription of the nucleic acid sequence encoding the transcriptional regulatory factor when in a non-permissive environment (e.g., under aerobic fermentation conditions, or in the absence of maltose).

[0072] The phrase "strain stability" generally refers to the stability of a genetically modified host cell described herein to produce a heterologous compound over an extended fermentation period. In particular, stability refers to the maintenance of good production characteristics (i.e., high yield (grams of compound per grams of substrate) and / or productivity (grams per liter of fermentation medium per hour) of the non-catabolic fermentation product by the microorganism over an extended fermentation time (e.g., 3 to 20 days). Genetic stability, which refers to the tendency for the frequency of a given allele of a gene involved in product formation to change little to not at all over time in a population of production microorganisms, plays a major role in the continued output of product.

[0073] The term "yield" refers to the amount of non-catabolic compound produced by a host cell expressed as the amount of non-catabolic compound produced (by weight) per amount of carbon source consumed by the host cell.

[0074] 5.2 Use of oxygen-sensitive promoters in conjunction with microaerobic fermentation as a switch to produce a heterologous compound

[0075] In some embodiments, the methods and compositions provided herein utilize an oxygen-sensitive promoter to drive expression of a heterologous enzyme capable of affecting non- catabolic compound production in a genetically modified host cell under microaerobic fermentation conditions. When fermentation of the host cell is conducted under aerobic fermentation conditions, non-catabolic compound production is substantially reduced or turned off; when the fermentation conditions are microaerobic, non-catabolic compound production is turned on or increased. Thus, the genetically modified cells described herein are capable of using low oxygen conditions as a switch to produce non-catabolic compounds. In particular, the timing of non-catabolic compound production is controlled to occur only when production is desired, redirecting carbon flow into cell maintenance and biomass during non-production phases. This more efficient use of carbon greatly reduces the metabolic burden on the host cell, increases the stability of heterologous genes, reduces strain degeneration, and contributes to better overall health and viability of the cell. Thus, the methods and genetically modified host cells provided herein utilize low oxygen fermentation conditions as a switch to achieve "off' and "on" phases of an improved fermentation process for producing heterologous non-catabolic compounds.

[0076] In a first step (i.e., the "build" phase, step (a)), the genetically modified host cell is grown in an aerobic condition (i.e., where a non-limiting amount of oxygen is provided) in a growth or "build" medium. In a second step (i.e., the "production" phase, step (b)), fermentation is conducted under microaerobic conditions, which act as a non-genetic switch to substantially increase production of non-catabolic compounds. Initial growth under fully aerobic conditions ensures that the energy needs of the cell are met, while biomass of the cell is rapidly increased. Thereafter, switching to microaerobic conditions allows for synthesis of non-catabolic products.

[0077] 5.2.1 Oxygen-sensitive DAN1 promoter

[0078] In some embodiments, the oxygen-sensitive promoter used to regulate expression of enzymes capable of effecting non-catabolic compounds in the methods provided herein is the DAN1 promoter, and homologs and variants thereof (SEQ ID NOs: 1-11). In some embodiments, the DAN1 promoter is from S. cerevisiae. The wild-type DAN1 promoter (SEQ ID NO: 1) is inactive under aerobic conditions but has high activity under anaerobic conditions. See, e.g., Kwast et al., J Bacteriol. 184(1): 250-265 (2002); Piper et al., J Biol Chem 277(40): 37001-37008 (2002); and ter Linde et al., J Bacteriol. 181(24): 7409-7413 (1999).

[0079] The DAN / TIR genes of S. cerevisiae are upregulated among a large group of genes during the adaptation to anaerobic growth (see, Lai et al., MoI Cell Biol. 25(10):4075-4091 (2005); Sertil et al., Gene 192(2): 199-205 (1997); and Tai et al., J Biol Chem. 280(1): 437-447 (2005), which encode cell wall mannoproteins that play a significant role in cell wall permeability. The kinetics of expression of these genes range from 30 minutes to 3 hours after the onset of anaerobiosis (see, Abramova et al., J Bacteriol. 183(9): 2881-2887 (2001)). It can be seen that a complex program of cell wall remodeling occurs during adaptation to anaerobiosis, as evidenced by the fact that the major aerobic cell wall mannoproteins encoded by CWP1 and CWP2 are replaced by their anaerobic counterparts encoded by the DAN / TIR genes under these conditions.

[0080] Stringent anaerobiosis is necessary for efficient induction of the wild-type DAN1 promoter, which is achieved by bubbling the culture with nitrogen gas to deplete oxygen (see, Cohen et al., Nucleic Acids Res 29(3):799-808 (2001)). However, Nevoigt et al. have developed a series of mutants of the DAN1 promoter (SEQ ID NOS: 1-10) that are inducible under conditions involving simple elimination or reduction of aeration. See, Nevoigt et al., Biotechnology and Bioengineering 96(3):550-558 (2007); and U.S. Patent Application No. 2007 / 0178505, the contents of which are incorporated by reference in their entirety.

[0081] Accordingly, in some embodiments, the DAN1 promoter used in the methods provided herein are those that have been described in U.S. Patent Application No. 2007 / 0178505, and which include the promoters comprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the DAN1 promoter used in the methods provided herein comprises SEQ ID NO: 1, 2, 3, 4, 5, or 6. In some embodiments, the DAN1 promoter used in the methods provided herein comprises SEQ ID NO: 1 or 2. In some embodiments, the DAN1 promoter used in the methods provided herein comprises SEQ ID NO: 1. In some embodiments, the DAN1 promoter used in the methods provided herein comprises SEQ ID NO: 2.

[0082] In another embodiment, a DAN1 promoter for use in the methods provided herein comprises a mutation in one or more of the following positions of SEQ ID No: 11 : 1-56; 66-139; 148-232; 245-283; 290-293; 301-302; 310; 322-326; 334-347; 357-371; 380-450; or 458-551. According to this aspect, and in one embodiment, the mutation is at position: 4, 7, 15, 18, 19, 21, 22, 26, 28, 36, 40, 53, 56, 60, 63, 66, 74, 75, 78, 86, 99, 122, 132, 135, 136, 149, 153, 162, 164, 165, 171, 172, 176, 187, 196, 198, 201, 205, 207, 211, 216, 226, 228, 233, 234, 237, 241, 260, 269, 274, 277, 280, 281, 285, 296, 299, 303, 307, 308, 310, 313, 322, 327, 331, 332, 337, 338, 343, 344, 346, 366, 368, 373, 375, 376, 381, 384, 386, 390, 391, 392, 396, 397, 402, 404, 422, 427, 428, 429, 432, 434, 439, 445, 467, 469, 470, 477, 480, 490, 492, 508, 511, 514, 518, 528, or a combination thereof. In one embodiment, the mutation at these positions can be to any nucleotide other than the wild-type nucleotide, while in another embodiment, the mutation at each position is to a specific nucleotide as described below.

[0083] In another embodiment, the DAN1 promoter for use in the methods provided herein comprises a sequence comprising the following substitutions: (a) replacing T with C at nucleotide position 4, 15, 19, 36, 53, 56, 60, 66, 74, 75, 78, 86, 99, 132, 136, 176, 201, 205, 207, 216, 226, 228, 269, 277, 281, 285, 299, 303, 310, 327, 331, 332, 375, 376, 390, 428, 434, 467, 477, 480, 508, 511, or a combination thereof, of the sequence set forth in SEQ ID NO: 11; (b) replacing A with G at nucleotide position 7, 18, 26, 40, 122, 135, 149, 153, 162, 164, 165, 171, 172, 187, 196, 211, 233, 234, 237, 241, 260, 274, 280, 308, 313, 322, 337, 343, 344, 346, 366, 368, 381, 384, 386, 396, 397, 402, 404, 422, 427, 429, 432, 445, 470, 490, 492, or a combination thereof; (c) replacing C with A at nucleotide position 21; (d) replacing A with C at nucleotide position 237, 338, 469, 514, 518; (e) replacing C with T at nucleotide position 28, 296, 307, 373, 392, 528, or a combination thereof; (f) replacing G with A at nucleotide position 22, 63, 391, 439, or a combination thereof; (g) replacing T with G at nucleotide position 198; or any combination thereof.

[0084] In another embodiment, the DAN1 promoter useful in the methods provided herein comprises a mutation in a promoter portion that is structurally or functionally homologous to a portion of the mutated DAN1 promoter described herein. In another embodiment, the promoter useful in the methods provided herein comprises a mutation in a promoter that is homologous to the DAN1 promoter. In one embodiment, the homologous promoter or portion thereof is derived from S. cerevisiae sequences, while in another embodiment, they are derived from other Saccharomyces species, while in another embodiment, they are derived from Saccharomycetaceae, while in another embodiment, they are derived from Saccharomycetales, while in another embodiment, they are derived from Saccharomycetes, while in another embodiment, they are derived from Saccharomycotina, while in another embodiment, they are derived from Ascomycota, while in another embodiment, they are derived from fungal species. In another embodiment, the promoter homologous to the DAN1 promoter displays similar oxygen dependence to the DAN1 promoter. One of skill in the art would be able to determine the oxygen dependence of a promoter using routine methods in the art. Determination of homologous promoters or promoter regions is routinely made by one of skill in the art using tools known in the art such as sequence alignment.

[0085] In one embodiment, the homologous promoter of DAN1 is DAN2, DAN3, DAN4, TIR1, TIR2, TIR3, or TIR4. In another embodiment, the homologous promoter of DAN1 is CYC1, CYC7, ANB1, COX5b, ERG11, MOX1, MOX2, MOX4 / UPC2, ROX7 / MOT3, or ROX1 promoter.

[0086] In one embodiment, the mutation can be in a promoter portion that corresponds to an anaerobic response element binding site, which in one embodiment is AR1 or AR2, while in another embodiment, the mutation can be in a Mot3 or Rox1 binding site.

[0087] 5.2.1.1 Targets of DAN1 promoter regulation

[0088] In some embodiments, the methods provided herein utilize a genetically modified host cell comprising one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for production of a heterologous non-catabolic compound. In some embodiments, expression of the one or more enzymes is under direct control of a mutant DAN1 promoter. That is, each of the one or more heterologous nucleic acid sequences encoding one or more enzymes of the enzyme pathway is operably linked (i.e., located 3') to a mutant DAN1 promoter, and the mutant DAN1 promoter drives expression of each of the one or more heterologous nucleic acids under microaerobic conditions.

[0089] In other embodiments, expression of one or more enzymes of the enzyme pathway is indirectly regulated by the mutant DAN1 promoter. For example, indirect regulation of one or more enzymes of the pathway can be achieved by operably linking the mutant DAN1 promoter to a single heterologous transcriptional regulator whose expression in turn directly regulates expression of one or more enzymes (e.g., all members) of the pathway.

[0090] The GAL regulon in yeast provides an exemplary regulatory network of activators, repressors, and promoters that can be utilized in combination with the mutant DAN1 promoters described herein. Yeast can utilize galactose as a carbon source to import galactose and metabolize it inside the cell via expression of the GAL genes. The GAL genes include the structural genes GAL1, GAL2, GAL7, and GAL10 genes (which encode galactokinase, galactose permease, a-D-galactose-1-phosphate uridyltransferase, and uridine diphosphate galactose-4-epimerase, respectively) and the regulatory genes GAL4, GAL80, and GAL3. For expression of the GAL1, GAL2, GAL7, and GAL10 genes, the GAL4 gene product is a positive regulator (i.e., an activator) and the GAL80 gene product is a negative regulator (i.e., a repressor). Gal4p activates transcription by binding to upstream activation sequences (UAS) such as those of the GAL structural genes, i.e., within the pGAL1, pGAL7, and pGAL10 promoters. In the absence of galactose, little expression of the structural proteins (Gal1p, Gal2p, Gal7p, and Gall0p) is normally detected due to the interaction of Gal80p with Gal4p and prevention of Gal4p transcriptional activity. However, in the presence of galactose, Gal3p interacts with Gal80p, relieving the repression of Gal4p by Gal80p. This allows Gal4p to bind genes (such as the GAL1, GAL2, GAL7, and GAL10 gene products) downstream of the sequence for expression.

[0091] Thus, in some embodiments, one or more GAL4-activated promoters (e.g., pGALl, pGAL7, and / or pGALlO) are operably linked to and used to drive expression of one or more enzymes of an enzyme pathway for production of a heterologous non-catabolic compound, and expression of a GAL4 gene product is driven by a mutant DANl promoter as described herein. Thus, expression of one or more enzymes of the pathway is induced by Gal4p under microaerobic conditions. In some such embodiments, expression of the GAL80 gene is reduced or eliminated using known techniques for gene disruption, such that Gal80p is no longer present to negatively regulate Gal4p activity, independent of oxygen conditions of fermentation. In some embodiments, the native pGAL4 promoter is replaced with a heterologous nucleic acid comprising a mutant DANl promoter. In some embodiments, the host cell comprises a heterologous nucleic acid comprising a nucleic acid encoding Gal4p operably linked to a heterologous nucleic acid comprising a mutant pDANl promoter. In one embodiment, the mutant DANl promoter is operably linked to a coding sequence for Gal4p, and a coding sequence for one or more enzymes (e.g., all members) of an enzyme pathway for production of a heterologous non-catabolic compound is operably linked to a GAL4-responsive promoter. In some embodiments, the GAL4-responsive promoter is pGALl. In some embodiments, the GAL4-responsive promoter is pGAL7. In some embodiments, the GAL4-responsive promoter is pGALlO.

[0092] 5.2.2 Oxygen Requirements for Aerobic and Microaerobic Conditions

[0093] In certain embodiments of the methods provided herein, the cells are cultured or maintained under conditions that are not oxygen-limited, i.e., aerobic conditions, during the build-up phase, and then are cultured or maintained under oxygen-limited conditions, i.e., microaerobic or anaerobic conditions.

[0094] Maintaining fully aerobic conditions can be challenging, particularly in large scale processes, due to limitations in mass transfer and the relatively low solubility of oxygen in aqueous solutions. For example, if air is used to sparge the tank, the solubility of oxygen in water at 20°C is 9 mg / L. If pure oxygen is used instead of air, the solubility increases to 43 mg / L. In either case (sparging with air or pure oxygen), these amounts of oxygen are depleted by an active and concentrated microbial population in seconds, unless oxygen is continuously supplied. In comparison, the amount of other nutrients used by the cells during the same period of time (seconds, e.g., less than a minute) is negligible compared to the large concentrations. We have discovered that host cells producing heterologous non-catabolic compounds are able to tolerate some period of oxygen limitation and still produce high levels of isoprenoid compounds. This flexibility allows for a more economical process by allowing savings in tank design, lower energy costs for aeration, and the like.

[0095] Oxygen limitation occurs when the specific growth rate of the host cell is less than the maximum specific growth rate, at which point oxygen is not limiting (e.g., is supplied in excess). The specific growth rate is the rate of cell growth per unit of biomass per unit of time and has units of the inverse of time (1 / t). The maximum specific growth rate of a cell in a culture relates to the effect of substrate concentration on growth rate, in this case oxygen. Generally, cells grow slowly at low levels of substrate and as the level of substrate in the culture medium increases, the rate of cell growth increases. However, the rate of cell growth does not continue to increase indefinitely and at high levels of substrate, a given increase in the amount of substrate results in an increasingly smaller increase in the rate of cell growth. Thus, the growth rate eventually reaches a limit, which is often referred to as the maximum specific growth rate.

[0096] Theoretical treatment of the relationship between growth rate in a culture is well known to those of skill in the art and is referred to as the Monod equation. See, e.g., Pirt, Principles of Microbe and Cell Cultivation, Wiley, NY, 1975, pages 4-10. In this theoretical treatment, the maximum specific rate is an asymptotic limit that is never reached until an infinite level of substrate is achieved. However, in practice, it can be considered that the maximum specific growth rate is achieved when the conditions under consideration (e.g., substrate levels, such as oxygen) support the fastest initial growth rate. For example, in a fed-batch reactor, the initial conditions of excess supply of all substrates (e.g., nutrients and oxygen) required for growth and fermentation at the optimal temperature for the host cell are treated as conditions for maximum growth rate. See, e.g., Lee et al. (1996) Trends Biotechnol. 14:98-105 and Korz et al. (1995) J Biotechnology 39:59-65. The maximum specific growth rate is also sometimes referred to as unrestricted growth.

[0097] In one approach, oxygen limitation is quantified by the oxygen concentration in the culture medium, and is expressed in terms of dissolved oxygen concentration (DOC). The DOC in the culture medium can be less than about 20%, less than about 15%, less than about 10%, and less than about 5%. In other embodiments, the DOC is about 0% or below the level of detection. However, because cells consume oxygen relatively quickly, a DOC of zero can mean that the cells are cultured under anaerobic conditions (no oxygen) or that the cells consume oxygen as fast as it is supplied. In another approach, the oxygen use by the cells is expressed in terms of oxygen uptake rate (OUR; the rate at which the cells consume oxygen per liter of culture medium) to distinguish between these two possibilities. Suitable oxygen uptake rates include less than about 50 millimoles, less than about 40 millimoles, less than about 30 millimoles, less than about 20 millimoles per liter of culture medium, or less than about 10 millimoles per liter of culture medium. Alternatively, when a normalized value with respect to cell density is preferred, the specific oxygen uptake rate (SOUR, i.e., OUR divided by cell density) can be used. The amount of microorganism per liter of fermentation broth or the density of microorganism can be measured by measuring the weight of microorganism isolated from a given volume of fermentation broth. A commonly used measurement is the dry cell weight per liter of fermentation medium. Another method that can be used to monitor fermentation as it proceeds is to measure the optical density of the culture medium. A commonly used method is to measure the optical density at a wavelength of 600 nm, referred to as OD600, or OD. The OD can be correlated to the density of a particular type of organism within a particular medium, but the specific correlation between OD and amount of microorganism per volume will not generally apply across all types of organisms in all types of media. By measuring OD and dry cell weight over a range of cell densities, a calibration curve can be generated. In some cases, these correlations can be used in different fermentations of the same or similar microorganism in the same or similar medium. Suitable specific oxygen uptake rates include less than about 30 millimoles, less than about 25 millimoles, less than about 20 millimoles, less than about 15 millimoles, less than about 10 millimoles, or less than about 5 millimoles per gram of dry cell weight per hour.

[0098] The culture medium can be maintained with a dissolved oxygen content during the cultivation process in accordance with the build phase or production phase to maintain cell growth and to maintain cell metabolism for the production of non-catabolic compounds as desired. The oxygen concentration of the culture medium can be monitored using known methods, such as via the use of an oxygen electrode. Oxygen can be added to the culture medium using methods known in the art, by agitation, shaking or sparging. While it has been described herein with respect to sparging the culture medium with air, other sources of oxygen can also be used. It is particularly useful to use a gas charge that contains a higher volume fraction of oxygen than that found in ambient air. In addition, such gas charges can include other gases that have no negative impact on the culture.

[0099] In some embodiments, microaerobic conditions are achieved by sparging the culture with nitrogen, e.g., high purity nitrogen (99.8%). In some embodiments, microaerobic conditions are achieved by culturing the cells in a gas-tight vessel, e.g., screw-cap sample bottles and flasks, etc. Because residual dissolved oxygen is consumed during cell growth, these conditions sharply decrease but do not completely deplete oxygen availability during the course of cell growth. In some embodiments, microaerobic conditions can be achieved by mixing air with a carrier gas in appropriate amounts. Alternatively, a gas can be sparged at a suitably low flow rate. Oxygen electrodes or any other suitable device can be used to monitor the oxygen level in the fermentation medium, and the flow rate of the gas mixture adjusted to ensure that the oxygen level in the fermentation fluid is maintained at a constant level. In addition to changes in inlet gas flow rate or inlet gas composition, microaerobic conditions can also be created by reducing the agitation rate (and thus oxygenation of large cultures) or by adding more feed to increase cell density (and thus higher oxygen demand), or a combination thereof.

[0100] In some embodiments, the host cell is supplied with sugar to control the dissolved oxygen (d02) to keep the d02concentration at undetectable levels for most of the fermentation. In some embodiments, oxygen can be supplied via sparging of compressed gas and mechanical agitation of the fermentation medium. In some embodiments, oxygen is supplied at a rate ranging from about 50 to 200 mmol O2 / L / h, e.g., about 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mmol O2 / L / h. In particular embodiments, oxygen is supplied at a rate of about 110 mmol O2 / L / h. To take advantage of all this available oxygen, sugar is fed fast enough to ensure that all available d02in the medium is converted to H2O via its metabolism to generate enough NADH. To ensure this, sugar is fed at a slightly faster rate than the stoichiometric requirement for O2so that some ethanol is produced. Periodically, sugar is not fed to induce the culture to re-consume the ethanol produced, a process that also requires the culture to consume O2. Once the ethanol is depleted, the d02concentration spikes rapidly, signaling that the culture is depleted of oxidizable carbon. This d02spike is measured by a probe, and the process restarts with the feeding of sugar, returning the d02to undetectable levels.

[0101] Assuming a well-mixed environment with no spatial gradients and neglecting a dilution term that can be neglected (because the O2going into the tank can be neglected compared to the O2going into the gas phase), the first order differential equation describing the change in dissolved oxygen in the fermentor is as follows:

[0102]

[0103] where:

[0104] d02 is the dissolved oxygen concentration in the reactor at time t

[0105] k1 is the vapor-liquid mass transfer coefficient for O2

[0106] a is the ratio of foam surface area to liquid volume

[0107] d02sat is the equilibrium concentration of O2 in the liquid phase, which corresponds to the temperature, pressure, and gas phase O2 concentration of the process

[0108] q O2 is the specific oxygen consumption rate (mmol / g dw / h)

[0109] [X] is the biomass concentration in the reactor at time t

[0110] This equation can be simplified to highlight that there are two elements that affect the dissolved oxygen concentration: the oxygen transfer rate (OTR), which is a function of the mass transfer characteristics of the culture medium k1, the surface area for gas transport a, and the driving force for mass transfer (d02sat - d02); and the oxygen uptake rate (OUR), which is a function of the biomass concentration [X] and how fast the biomass consumes O2 q O2 . So now it is simply:

[0111]

[0112] These equations can be used to help understand the temporal profile of d02.

[0113] 5.3 Use of a maltose-responsive promoter in combination with a maltose operon as an on

[0114] - off switch for the production of heterologous compounds

[0115] In some embodiments, the methods and compositions provided herein utilize a maltose-responsive promoter in combination with manipulation of the maltose content of the fermentation medium to directly or indirectly modulate the expression of heterologous enzymes capable of effecting the production of non- catabolic compounds in a genetically modified host cell.

[0116] In one embodiment, when the host cell is fermented in the presence of maltose (e.g., at least 0.1% maltose), production of the non-cognate compound is substantially reduced or turned off, and when the amount of maltose in the fermentation medium is reduced or eliminated, production of the non-cognate compound is turned on or increased. Thus, in some embodiments, the genetically modified cells described herein comprise a heterologous biosynthetic pathway gene regulated by a maltose-responsive promoter, enabling the use of maltose in the fermentation medium as a switch for production of the non-cognate compound. Controlling the timing of non-cognate compound production occurs only when production is desired, redirecting carbon flow into cell maintenance and biomass during the non-production phase. This more efficient use of carbon greatly reduces the metabolic burden on the host cell, improving cell growth, increasing the stability of the heterologous genes, reducing strain degeneration, and contributing to better overall health and viability of the cell.

[0117] In some embodiments, the fermentation process comprises a two-step process that utilizes maltose as a switch to achieve the "off and "on" phases. In the first step, where production of the unwanted compound is not desired (i.e., the "build-up" phase, step (a)), the genetically modified host cell is grown in a growth or "build-up" medium that comprises maltose in an amount sufficient to induce expression of the genes under the control of the maltose-responsive promoter, and the induced gene products act to negatively regulate production of the non-cognate compound. In the second step (i.e., the "production" phase, step (b)), fermentation is carried out in a medium comprising a carbon source, wherein maltose is absent or present in an amount low enough such that the maltose-responsive promoter is no longer active, and production of the host cell's heterologous non-cognate compound is turned on or increased.

[0118] In other embodiments, the maltose-responsive promoter is operably linked to one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway, and the presence of an activating amount of maltose in the medium increases expression of the one or more enzymes of the enzyme pathway. In this way, the maltose-responsive promoter can be wired to function as a positive regulator of production of the non-cognate compound.

[0119] 5.3.1 Maltose-responsive Promoters

[0120] In preferred embodiments, the maltose-responsive promoters useful in the methods and compositions provided herein promote transcription of operably linked DNA coding sequences in the presence of maltose. In some embodiments, the maltose-responsive promoters useful for modulating expression of enzymes capable of effecting non-catabolic compounds in the methods and compositions provided herein are any maltose-responsive promoters known in the art. In some embodiments, the maltose-responsive promoter is selected from the group consisting of pMALl (SEQ ID NO: 12), pMAL2 (SEQ ID NO: 13), pMALl l (SEQ ID NO: 14), pMALl 2 (SEQ ID NO: 15), pMAL31 (SEQ ID NO: 16), and pMAL32 (SEQ ID NO: 17). In particular embodiments, the maltose-sensitive promoter is pMAL32 (SEQ ID NO: 17).

[0121] Other useful maltose-responsive promoters useful in the methods and compositions provided herein can be derived from the regulatory network of the maltose fermentation system of S. cerevisiae. Maltose fermentation in Saccharomyces species requires the presence of at least one of five unlinked MAL loci: MALl, MAL2, MAL3, MAL4, and MAL6. Each of these loci is composed of a complex of genes involved in maltose metabolism; the complex includes maltase, maltose permease, and activators of these genes. At the MAL6 locus, the activator is encoded by the MAL63 gene. Mal63p is a DNA-binding transcriptional activator required for maltose-dependent induction of the MAL structural genes that encode maltose permease and maltase.

[0122] The MAL activator intermediate complex is stable in the absence of the inducer maltose, but addition of maltose causes the inducible MAL activator to be released from the complex in an active form capable of DNA binding and transcriptional activation. See, e.g., Ran, F. and Michels., C. A., J. Biol. Chem. 285(18): 13850-13862 (2010). The binding site for the MAL63 protein in the divergently transcribed MAL61-62 promoter has been characterized as the upstream activating sequence for the MAL genes. See, e.g., Ni, B. and Needleman, R., "Identification of the Upstream Activating Sequence of MAL and the Binding Sites for the MAL63 Activator of Saccharomyces cerevisiae," Molecular and Cellular Biology 10(7): 3797-3800 (1990), the contents of which are incorporated by reference in their entirety.

[0123] Other useful maltose-responsive promoters useful in the methods and compositions provided herein can be derived from the regulatory network of the maltose / maltodextrin metabolism system of Escherichia coli. The ma1T nucleic acid encodes Ma1T, which is a four-maltose-responsive promoter (P PQ , P EFG , P KBM , and P SThe malT and mal promoter combination results in a tightly regulated expression system that has been shown to act as a strong promoter inducible by the addition of maltose. See, e.g., Schleif, "Two Positively Regulated Systems, ara and mal," pp. 1300-1309 in Escherichia coli and Salmonella Cellular and Molecular Biology, Second Edition, Neidhardt et al., eds., ASM Press, Washington, D.C., 1996; and Boos, W. and Shuman, H., "Maltose / Maltodextrin System of Escherichia coli: Transport, Metabolism and Regulation," Microbiology and Molecular Biology Reviews, 62(1): 204-229 (1998)), the contents of which are incorporated by reference in their entirety.

[0124] Other useful maltose-responsive promoters useful in the methods and compositions provided herein include those described in Berkner et al., "Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarious," Extremophiles 14: 249-259 (2010); and U.S. Patent No. 5,824,545.

[0125] 5.3.1.1 Target of maltose-responsive promoter regulation

[0126] In some embodiments, the methods provided herein utilize a genetically modified host cell comprising one or more heterologous nucleic acids encoding one or more enzymes of an enzyme pathway for producing a heterologous non-catabolic compound. In some embodiments, expression of the one or more enzymes is under direct control of a maltose-responsive promoter described herein. That is, each of the one or more heterologous nucleic acid sequences encoding one or more enzymes of an enzyme pathway is operably linked (i.e., positioned 3') to a maltose-responsive promoter, and the maltose-responsive promoter drives expression of each of the one or more heterologous nucleic acids in the presence of maltose.

[0127] In other embodiments, expression of one or more enzymes of the enzyme pathway is indirectly regulated by a maltose-responsive promoter. For example, indirect regulation of one or more enzymes of the pathway can be achieved by operably linking a maltose-responsive promoter to a single heterologous transcriptional regulator whose expression in turn directly regulates expression of one or more enzymes (e.g., all members) of the pathway. As detailed above, the GAL regulon in yeast provides an exemplary regulatory network of activators, repressors, and promoters that can be utilized in combination with the maltose-responsive promoters described herein.

[0128] In some embodiments, one or more GAL4-activating promoters (e.g., pGALl, pGAL7, and / or pGALlO) are operably linked to and used to drive expression of one or more enzymes of an enzyme pathway for producing a heterologous non-catabolic compound. In some embodiments, the host cell further comprises a nucleic acid encoding GAL4. In some embodiments, the GAL4 gene product is constitutively expressed, i.e., under the control of a constitutive promoter. In some embodiments, the host cell further comprises a nucleic acid encoding GAL80 under the control of a maltose-responsive promoter described herein, and expression of the GAL80 gene product is induced in the presence of maltose. Gal80p in turn interacts with Gal4p and prevents Gal4p transcriptional activity. When maltose is removed or sufficiently depleted such that expression of GAL80 is no longer induced, Gal4p is relieved of Gal80p repression and is free to activate expression of one or more enzymes of an enzyme pathway for producing a heterologous non-catabolic compound.

[0129] In other embodiments, the native pGAL4 promoter is replaced with a heterologous nucleic acid comprising a maltose-responsive promoter. In some embodiments, the host cell comprises a heterologous nucleic acid comprising a nucleic acid encoding Gal4p operably linked to a heterologous nucleic acid comprising a maltose-responsive promoter. In one embodiment, the maltose-responsive promoter is operably linked to the coding sequence for Gal4p, and the coding sequence for one or more enzymes (e.g., all members) of an enzyme pathway for producing a heterologous non-catabolic compound is operably linked to a GAL4-responsive promoter such that expression of the one or more enzymes is induced in the presence of maltose. In some embodiments, the GAL4-responsive promoter is pGALl. In some embodiments, the GAL4-responsive promoter is pGAL7. In some embodiments, the GAL4-responsive promoter is pGALlO.

[0130] 5.3.2 Repressed and non-repressed amounts of maltose

[0131] As shown below, maltose is a disaccharide formed from 2 glucose molecules. It has the chemical formula C 12 H 22 O 11and a molecular weight of 343 g / mol.

[0132]

[0133] In some embodiments of the methods provided herein, one can be able to determine the "inducing" amount of maltose (i.e., the amount of maltose sufficient to induce expression of a coding sequence operably linked to a maltose-responsive promoter) and the "non-inducing" amount of maltose (i.e., the amount of maltose below which it does not induce expression of a coding sequence operably linked to a maltose-responsive promoter) for use in the methods provided herein for any genetically modified host cell capable of producing a heterologous non-catabolic compound. In some embodiments, the non-inducing amount of maltose is determined by performing a gene expression curve (i.e., a maltose titration) in the presence of increasing amounts of maltose in a culture medium to be used in a fermentation process. For example, a population of genetically modified host cells can be divided into multiple subpopulations and cultured in parallel, with each subpopulation cultured in a culture medium comprising a different (e.g., increasing) amount of maltose (including no maltose), and reporter gene expression or yield of non-catabolic compound is determined after a defined period of time.

[0134] In some embodiments in which a maltose-responsive promoter is wired to achieve a "off' state for production of a non-catabolic compound in the presence of maltose, the maltose titration comprises at least two maltose concentrations, whereby the yield of non-catabolic compound by the host cell stabilizes at a minimum, i.e., no further decrease in compound production is observed as the maltose concentration is increased. In some embodiments, the "repressing" amount of maltose is at least the minimum amount of maltose at which the yield of non-catabolic compound by the host cell stabilizes at its minimum (e.g., zero). Such an amount can also be referred to as the "saturation" or "optimal" amount of maltose for repressing production of a non-catabolic compound by a particular host cell. In some such embodiments, the "repressing" amount of maltose can include any maltose concentration at which the yield of non-catabolic compound has already decreased relative to the "on" state, even if there is low-level production of the compound. In some embodiments, in such a configuration of the on-off switch, the non-repressing amount of maltose is any amount of maltose that is lower than the maltose "repressing" amount. In some embodiments, the non-repressing amount of maltose is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100-fold less than the repressing amount of maltose. In one particular embodiment, the non-repressing amount of maltose is less than 0.8% (w / v) of the culture medium. In another particular embodiment, the non-repressing amount of maltose is less than 0% (w / v) of the culture medium.

[0135] In particular embodiments, as described above, a repressing amount of maltose is an optimal or saturating amount for a given host cell, and a non-repressing amount is no maltose. In another particular embodiment, a repressing amount of maltose is at least 0.25%, and a non-repressing amount is no maltose. In another particular embodiment, a repressing amount of maltose is an amount of maltose from 0.25% to 3%, and a non-repressing amount is no maltose. In another particular embodiment, a repressing amount of maltose is at least 3%, and a limiting amount is no maltose.

[0136] In some embodiments in which a maltose-responsive promoter is wired to achieve a "off state of production of a non-dissimilated compound in the presence of maltose, a repressing amount of maltose in the culture medium is at least 0.1% (weight maltose per volume of culture medium). In some embodiments, a repressing amount of maltose in the culture medium is at least 0.25%. In some embodiments, a repressing amount of maltose in the culture medium is at least 0.5%. In some embodiments, a repressing amount of maltose in the culture medium is at least 0.75%. In some embodiments, a repressing amount of maltose in the culture medium is at least 1.0%. In some embodiments, a repressing amount of maltose in the culture medium is at least 1.25%. In some embodiments, a repressing amount of maltose in the culture medium is at least 1.5%. In some embodiments, a repressing amount of maltose in the culture medium is at least 1.75%. In some embodiments, a repressing amount of maltose in the culture medium is at least 2.0%. In some embodiments, a repressing amount of maltose in the culture medium is at least 2.25%. In some embodiments, a repressing amount of maltose in the culture medium is at least 2.5%. In some embodiments, a repressing amount of maltose in the culture medium is at least 2.75%. In some embodiments, a repressing amount of maltose in the culture medium is at least 3.0%. In some embodiments, a repressing amount of maltose in the culture medium is at least 3.25%. In some embodiments, a repressing amount of maltose in the culture medium is at least 3.5%. In some embodiments, a repressing amount of maltose in the culture medium is at least 3.75%. In some embodiments, a repressing amount of maltose in the culture medium is at least 4.0%. In some embodiments, a repressing amount of maltose in the culture medium is at least 4.25%. In some embodiments, a repressing amount of maltose in the culture medium is at least 4.5%. In some embodiments, a repressing amount of maltose in the culture medium is at least 4.75%. In some embodiments, a repressing amount of maltose in the culture medium is at least 5.0%. In some embodiments, a repressing amount of maltose in the culture medium is between 5% and 50%. In some embodiments, a repressing amount of maltose in the culture medium is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50%.

[0137] In some embodiments, a non-repressing amount of maltose is an amount that is at least 2-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold less than a repressing amount of maltose determined according to the methods described above. In some embodiments, a non-repressing amount of maltose is an amount that is at least 2-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold less than a saturating amount of maltose determined according to the methods described above. In some embodiments, a non-repressing amount of maltose is an amount that is less than 50%, less than 20%, less than 10%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.01%, or less than 0.001% of a repressing amount of maltose determined according to the methods described above. In some embodiments, a non-repressing amount of maltose is an amount that is less than 50%, less than 20%, less than 10%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of a saturating amount of maltose determined according to the methods described above. In a particular embodiment, a non-repressing amount of maltose is 0 mg / L (0%), i.e., no maltose. As such, in this particular embodiment, the host cell is cultured in a cell culture medium that does not contain an external source of maltose during the production phase.

[0138] In some embodiments in which a maltose-responsive promoter is wired to achieve an "on" state for production of a non-dissimilated compound in the presence of maltose, the maltose titration comprises at least two maltose concentrations, whereby the production of the non-dissimilated compound by the host cell tends to plateau at a maximum value, i.e., no further increase in compound production is observed as the maltose concentration is increased. In some embodiments, a "non-repressing" amount of maltose is at least the minimum amount of maltose at which the production of the non-dissimilated compound by the host cell tends to plateau at its maximum value. In this configuration of the switch, such an amount can also be referred to as a "saturating" or "optimal" amount of maltose for inducing production of the non-dissimilated compound by a particular host cell. In some such embodiments, a "inducing" amount of maltose can include any maltose concentration at which the production of the non-dissimilated compound has increased relative to the "off' state, even if production of the compound is suboptimal. In some embodiments, in this configuration of the switch, a non-inducing amount of maltose is any amount of maltose that is less than the "inducing" amount of maltose. In some embodiments, a non-inducing amount of maltose is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100-fold less than the inducing amount of maltose. In a particular embodiment, a non-inducing amount of maltose is less than 0.8% (w / v) of the culture medium. In another particular embodiment, a non-inducing amount of maltose is less than 0% (w / v) of the culture medium.

[0139] In particular embodiments, the inducing amount of maltose is at least 0.25% and the non-inducing amount is no maltose. In another particular embodiment, the inducing amount of maltose is 0.25% to 3% of the amount of maltose, and the non-inducing amount is no maltose. In another particular embodiment, the inducing amount of maltose is at least 3% and the limiting amount is no maltose.

[0140] In some embodiments in which the maltose-responsive promoter is wired to achieve an "on" state for production of the non-dissimilated compound in the presence of maltose, the inducing amount of maltose in the culture medium is at least 0.1% (weight maltose per volume of culture medium). In some embodiments, the inducing amount of maltose in the culture medium is at least 0.25%. In some embodiments, the inducing amount of maltose in the culture medium is at least 0.5%. In some embodiments, the inducing amount of maltose in the culture medium is at least 0.75%. In some embodiments, the inducing amount of maltose in the culture medium is at least 1.0%. In some embodiments, the inducing amount of maltose in the culture medium is at least 1.25%. In some embodiments, the inducing amount of maltose in the culture medium is at least 1.5%. In some embodiments, the inducing amount of maltose in the culture medium is at least 1.75%. In some embodiments, the inducing amount of maltose in the culture medium is at least 2.0%. In some embodiments, the inducing amount of maltose in the culture medium is at least 2.25%. In some embodiments, the inducing amount of maltose in the culture medium is at least 2.5%. In some embodiments, the inducing amount of maltose in the culture medium is at least 2.75%. In some embodiments, the inducing amount of maltose in the culture medium is at least 3.0%. In some embodiments, the inducing amount of maltose in the culture medium is at least 3.25%. In some embodiments, the inducing amount of maltose in the culture medium is at least 3.5%. In some embodiments, the inducing amount of maltose in the culture medium is at least 3.75%. In some embodiments, the inducing amount of maltose in the culture medium is at least 4.0%. In some embodiments, the inducing amount of maltose in the culture medium is at least 4.25%. In some embodiments, the inducing amount of maltose in the culture medium is at least 4.5%. In some embodiments, the inducing amount of maltose in the culture medium is at least 4.75%. In some embodiments, the inducing amount of maltose in the culture medium is at least 5.0%. In some embodiments, the inducing amount of maltose in the culture medium is between 5% and 50%. In some embodiments, the inducing amount of maltose in the culture medium is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50%.

[0141] In some embodiments, the non-repressing amount of maltose is an amount that is at least 2-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold less than the repressing amount of maltose determined according to the methods described above. In some embodiments, the non-repressing amount of maltose is an amount that is at least 2-fold, 10-fold, 100-fold, 1000-fold, 10,000-fold, or 100,000-fold less than the saturating amount of maltose determined according to the methods described above. In some embodiments, the non-repressing amount of maltose is an amount that is less than 50%, less than 20%, less than 10%, less than 1%, less than 0.5%, less than 0.2%, less than 0.1%, less than 0.01%, or less than 0.001% of the repressing amount of maltose determined according to the methods described above. In some embodiments, the non-repressing amount of maltose is an amount that is less than 50%, less than 20%, less than 10%, less than 1%, less than 0.1%, less than 0.01%, or less than 0.001% of the saturating amount of maltose determined according to the methods described above. In a particular embodiment, the non-repressing amount of maltose is 0 mg / L (0%), i.e., no maltose. As such, in this particular embodiment, the host cell is cultured during the production phase in a cell culture medium that does not contain an external source of maltose.

[0142] 5.3.3 Yield of non-catabolic products

[0143] In some embodiments of the fermentation methods provided herein, the yield of non-catabolic compounds during the construction phase (step (a) of the methods described above) is less than 50%, 40%, 30%, 20%, or 10% of the maximum non-catabolic compound yield of the genetically modified host cell (e.g., the amount of non-catabolic compound produced when the host cell is cultured during the production phase (step (b) of the methods described above)) using a microaerobically responsive promoter in combination with an oxygen condition manipulation or a maltose-responsive promoter in combination with a maltose condition manipulation.

[0144] The time period for conducting the construction phase and the production phase of the fermentation process can vary and will depend on factors such as the growth rate of the host cell, the intrinsic growth rate of the host cell, and the like; as well as other culture conditions, such as pH, temperature, depending on the specific requirements of the host cell, the fermentation, and the process. However, any duration of the construction phase is expected to result in some benefit to the final production rate of the fermentation, as some amount of the negative selection pressure associated with non-catabolic compound production is relieved in the "off" state.

[0145] In some embodiments, the construction phase is carried out for a period of time sufficient to produce a certain amount of cellular biomass capable of supporting the production of non-dissimilatory compounds during the production phase. In some embodiments, the construction phase is carried out for a period of time sufficient to allow the population present at inoculation to undergo multiple doublings until a desired cell density is reached. In some embodiments, the host cell population in the fermentation vessel or container where the construction phase is carried out is sufficient to achieve a cell density (OD) between 0.01 and 400. 600 The build phase is conducted within a specified timeframe. In some implementations, the build phase continues until an OD of at least 0.01 is reached. 600 In some implementations, the build phase proceeds until an OD of at least 0.1 is achieved. 600 In some implementations, the build phase proceeds until an OD of at least 1.0 is achieved. 600 In some implementations, the build phase proceeds until at least 10 ODs are reached. 600 In some implementations, the build phase proceeds until at least 100 ODs are achieved. 600 In some implementations, the build phase proceeds until an OD (Original Depth) between 0.01 and 100 is achieved. 600 In some implementations, the build phase proceeds until an OD (Optical Displacement) between 0.1 and 10 is achieved. 600 In some implementations, the build phase proceeds until an OD (Optical Displacement) between 1 and 100 is reached. 600 In other implementations, the build phase is carried out over a period of at least 12, 24, 36, 48, 60, 72, 84, 96 or more hours.

[0146] In some embodiments, the production phase is carried out for a period of time sufficient to produce the desired amount of the non-dissimilar compound. In some embodiments, the production phase is carried out for a period of at least 12, 24, 36, 48, 60, 72, 84, 96, or more than 96 hours. In some embodiments, the production phase is carried out for a period of time between 3 and 20 days. In some embodiments, the production phase is carried out for a period of time of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 days.

[0147] In a particular embodiment, the method for producing non-dissimilatory compounds includes fermenting genetically modified host cells under aerobic conditions sufficient to allow the growth and maintenance of genetically modified host cells; then providing microaerobic fermentation conditions sufficient to induce the production of non-dissimilatory compounds, and maintaining microaerobic conditions throughout the fermentation run.

[0148] In another embodiment, the method of producing a non-catabolic compound comprises culturing the host cell in separate build and production media. For example, the method can comprise culturing the genetically modified host cell in a build phase, wherein the cell is cultured under non-production conditions to produce an inoculum, then transferring the inoculum into a second fermentation medium under conditions suitable for inducing production of the compound, and maintaining steady state conditions in the second fermentation phase to produce a cell culture containing the non-catabolic product.

[0149] In some embodiments, the methods provided herein are sufficient to produce one or more non-catabolic compounds in an amount greater than about 10 grams per liter of fermentation medium. In some such embodiments, the non-catabolic derivative compound is produced in an amount of about 10 to about 50 grams, more than about 15 grams, more than about 20 grams, more than about 25 grams, or more than about 30 grams per liter of cell culture.

[0150] In some embodiments, the methods provided herein are sufficient to produce one or more non-catabolic compounds in an amount greater than about 50 milligrams per gram of cell dry weight. In some embodiments, the recombinantly produced non-catabolic compound is produced in an amount of about 50 to about 1500 milligrams, more than about 100 milligrams, more than about 150 milligrams, more than about 200 milligrams, more than about 250 milligrams, more than about 500 milligrams, more than about 750 milligrams, or more than about 1000 milligrams per gram of cell dry weight.

[0151] In some embodiments, the performance of the methods provided herein results in an increased production of non-catabolic compounds by the population of genetically modified host cells compared to the production resulting from a method that does not include a build phase during which the host cell is cultured under non-production conditions. In some embodiments, the performance of the methods results in an amount of one or more non-catabolic compounds produced per unit volume of cell culture that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold or more than the amount of non-catabolic compounds produced by a method that does not include a build phase during which the host cell is cultured under non-production conditions.

[0152] In some embodiments, implementation of the method results in an amount of one or more non-catabolic compounds produced that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold or more higher per unit of cell dry weight than the amount of non-catabolic compounds produced by a method that does not include a build phase during which the host cells are cultured under non-production conditions.

[0153] In some embodiments, implementation of the method results in an amount of one or more non-catabolic compounds produced that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold or more higher per unit of cell dry weight per unit of time than the amount of non-catabolic compounds produced by a method that does not include a build phase during which the host cells are cultured under non-production conditions.

[0154] In some embodiments, implementation of the method results in an amount of one or more non-catabolic compounds produced that is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75-fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 1000-fold or more higher per unit of cell dry weight per unit of time than the amount of non-catabolic compounds produced by a method that does not include a build phase during which the host cells are cultured under non-production conditions.

[0155] 5.3.4 Media and conditions

[0156] 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, e.g., Bailey et al., Biochemical Engineering Fundamentals, Second Edition, McGraw Hill, New York, 1986). Depending on the specific requirements of the host cell, fermentation, and process, consideration must be given to the appropriate medium, pH, temperature, and requirement for aerobic, microaerophilic, or anaerobic conditions.

[0157] The methods for producing non-catabolic compounds provided herein can be carried out in a suitable container, including but not limited to a cell culture plate, flask, or fermentor. Further, the methods can be carried out at any fermentation scale known in the art to support the industrial production of microbial products. Any suitable fermentor can be used, including a stirred tank fermentor, a gas lift fermentor, a bubble fermentor, or any combination thereof. In particular embodiments utilizing S. cerevisiae as the host cell, the strains can be cultured in a fermentor as detailed in Kosaric, et al., in Ullmann's Encyclopedia of Industrial Chemistry, Sixth Edition, Volume 12, pages 398-473, Wiley-VCH Verlag GmbH & Co. KDa A, Weinheim, Germany. Further, the methods can be carried out at any fermentation volume, for example from laboratory scale (e.g., 10 ml to 20 L) to pilot scale (e.g., 20 L to 500 L) to industrial scale (e.g., 500 L to > 500,000 L) fermentation.

[0158] In some embodiments, the culture medium used in the methods for producing non-catabolic compounds provided herein includes any medium in which the genetically modified microorganism capable of producing a non-catabolic compound can survive, i.e., support and maintain growth and viability. In some embodiments, the culture medium also facilitates the biosynthetic pathway necessary to produce the desired non-catabolic compound.

[0159] In some embodiments, the culture medium is an aqueous medium containing assimilable sources of carbon, nitrogen, and phosphate. 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 medium, and each of the required nutrients is maintained at a level substantially at the minimum level required for effective assimilation by the growing cells, e.g., based on the metabolic or respiratory functions of the cells to convert the carbon source into biomass in accordance with a pre-determined cell growth curve.

[0160] Suitable conditions and suitable media for culturing microorganisms are well known in the art. In some embodiments, a suitable culture medium is supplemented with one or more additional reagents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding a gene product is under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding a gene product is under the control of a repressible promoter), or a selection agent (e.g., an antibiotic selected for microorganisms comprising a genetic modification).

[0161] 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, 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.

[0162] The concentration of the carbon source (such as glucose) in the culture medium should promote cell growth, but should not be so high as to inhibit the growth of the microorganism being used. Typically, cultures are run with a carbon source (such as glucose) added at a level to achieve the desired level of growth and biomass, rather than at undetectable levels (detection limit of about <0.1 g / l). In other embodiments, the concentration of the 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. Additionally, the concentration of the 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 reference to the concentration of a culture component can refer to both the initial and / or ongoing concentration of the component. In some cases, it can be desirable to allow the culture medium to become carbon source depleted during the course of the culture.

[0163] Assimilable nitrogen sources that can be used in suitable culture media include, but are not limited to, simple nitrogen sources, organic nitrogen sources, and complex nitrogen sources. Such nitrogen sources include anhydrous ammonia, ammonium salts, and materials of animal, plant, and / or microbial origin. Suitable nitrogen sources include, but are not limited to, protein hydrolysates, microbial biomass hydrolysates, peptones, yeast extract, ammonium sulfate, urea, and amino acids. Typically, the concentration of the nitrogen source 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. However, the addition of nitrogen sources to the culture medium beyond certain concentrations is detrimental to the growth of the microorganism. As a result, the concentration of the nitrogen source 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 cases, it can be desirable to allow the culture medium to become nitrogen source depleted during the course of the culture.

[0164] The effective medium can contain other compounds such as inorganic salts, vitamins, trace metals, or growth promoters. Such other compounds can also be present in the carbon, nitrogen, or mineral sources in the effective medium or can be added specifically to the medium. The 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, phosphates such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, ammonium phosphate, and mixtures thereof. Typically, the concentration of phosphate in the 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. However, the addition of phosphate to the medium beyond certain concentrations is detrimental to the growth of the microorganism. Thus, the concentration of phosphate in the 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.

[0165] The suitable medium can also include a magnesium source, preferably in the form of a physiologically acceptable salt such as magnesium sulfate heptahydrate, although other magnesium sources can be used at concentrations that contribute similar amounts of magnesium. Typically, the concentration of magnesium in the 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. However, the addition of magnesium to the medium beyond certain concentrations is detrimental to the growth of the microorganism. Thus, the concentration of magnesium in the 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 cases, it can be desirable to allow the medium to become depleted of the magnesium source during the course of the cultivation.

[0166] In some embodiments, the medium can also include a biologically acceptable chelating agent such as trisodium citrate dihydrate. In such cases, the concentration of the chelating agent in the 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. However, the addition of the chelating agent to the medium beyond certain concentrations is detrimental to the growth of the microorganism. Thus, the concentration of the chelating agent in the 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.

[0167] The medium can also initially include a biologically acceptable acid or base to maintain the desired pH of the 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.

[0168] The 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 medium is in the range of about 5 mg / L to about 2000 mg / L, preferably in the range of about 20 mg / L to about 1000 mg / L, and more preferably in the range of about 50 mg / L to about 500 mg / L.

[0169] The medium can also include sodium chloride. Typically, the concentration of sodium chloride in the medium is in the range of about 0.1 g / L to about 5 g / L, preferably in the range of about 1 g / L to about 4 g / L, and more preferably in the range of about 2 g / L to about 4 g / L.

[0170] In some embodiments, the medium can also include trace metals. Such trace metals can be added to the medium as a stock solution, which can be prepared separately from the remainder of the medium for convenience. Typically, the amount of such trace metal solution added to the medium is greater than about 1 ml / L, preferably greater than about 5 mL / L, and more preferably greater than about 10 mL / L. However, the addition of trace metals to the medium beyond certain concentrations is detrimental to the growth of the microorganism. Thus, the amount of such trace metal solution added to the 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 as a stock solution, the various components can be added separately, each within the ranges specified above for the amount of component corresponding to the trace metal solution.

[0171] The medium can include other vitamins, such as biotin, calcium, pantothenate, inositol, pyridoxine-HCl, and thiamine-HCl. Such vitamins can be added to the medium in the form of a stock solution, which can be prepared separately from the remainder of the medium for convenience. However, the addition of vitamins to the medium beyond certain concentrations is detrimental to the growth of the microorganism.

[0172] The fermentation processes described herein can be carried out in conventional culture modes, including but not limited to batch, fed-batch, cell recycle, continuous, and semi-continuous. In some embodiments, the fermentation is carried out in a fed-batch mode. In such cases, some components of the culture medium are depleted during the course of the culture during the production phase of the fermentation. In some embodiments, the culture can be supplemented with relatively higher concentrations of such components at the beginning of the production phase, for example, to support growth and / or production of non-catabolic compounds for a period of time before additions are required. By making additions at the level of consumption by the culture, the preferred ranges of these components are maintained throughout the course of the culture. The levels of each component in the medium can be monitored by, for example, periodically sampling the medium and determining the concentrations. Alternatively, once standard culture protocols have been developed, additions can be made at regular intervals corresponding to known levels throughout the culture. As will be recognized by those skilled in the art, the rate of nutrient consumption increases during the course of the culture as the cell density of the culture medium increases. In addition, to avoid introducing foreign microorganisms into the culture medium, additions are made using sterile addition methods known in the art. In addition, a small amount of antifoam agent can be added during the course of the culture.

[0173] The temperature of the culture medium can be any temperature suitable for growth of the genetically modified cells and / or production of non-catabolic compounds. For example, the culture medium can be brought to and maintained at a temperature in the range of about 20°C to about 45°C, preferably in the range of about 25°C to about 40°C, and more preferably in the range of about 28°C to about 32°C, prior to inoculation of the culture medium with inoculum.

[0174] The pH of the culture medium can be controlled by adding an acid or a base to the culture medium. In such cases, when ammonia is used to control the pH, it also conveniently serves as a source of nitrogen in the culture medium. Preferably, the pH is maintained at about 3.0 to about 8.0, more preferably about 3.5 to about 7.0, and most preferably about 4.0 to about 6.5.

[0175] In some embodiments, the carbon source concentration of the culture medium, such as the maltose or glucose concentration, is monitored during the culturing period. The glucose concentration of the culture medium can be monitored using known techniques, such as, for example, using a glucose oxidase test or high pressure liquid chromatography, which can be used to monitor the glucose concentration in the supernatant (e.g., the cell-free medium components), while maltose levels can be similarly monitored. As previously described, the carbon source concentration should be kept below the level at which cell growth inhibition occurs. While such concentrations can vary between organisms, for glucose as the carbon source, cell growth inhibition occurs at glucose concentrations greater than about 60 g / L, and can be readily determined by trial. Thus, when using glucose as the carbon source, it is preferred that glucose be fed to the fermentor and maintained below the detection limit. Alternatively, the glucose concentration in the culture medium is maintained in the range of about 1 g / L to about 100 g / L, more preferably in the range of about 2 g / L to about 50 g / L, and still more preferably in the range of about 5 g / L to about 20 g / L. While the carbon source concentration can be maintained within the desired level by adding, for example, a substantially pure glucose solution, it can be acceptable and possibly preferable to maintain the carbon source concentration of the culture medium by adding an aliquot of the initial culture medium. It can be desirable to use an aliquot of the initial culture medium because the concentrations of other nutrients in the culture medium, such as nitrogen and phosphorous sources, can be simultaneously maintained. Likewise, the trace metal concentration in the culture medium can be maintained by adding an aliquot of a trace metal solution.

[0176] 5.3.5 Recovery of non-catabolic compounds

[0177] Once the non-catabolic compound is produced by the host cell, it can be recovered or isolated for subsequent use using any suitable isolation and purification methods known in the art. In some embodiments, the organic phase containing the non-catabolic compound is separated from the fermentation broth by centrifugation. In other embodiments, the organic phase containing the non-catabolic compound spontaneously separates from the fermentation broth. In other embodiments, the organic phase containing the non-catabolic compound is separated from the fermentation broth by adding a demulsifier and / or nucleating agent to the fermentation reaction. Exemplary examples of demulsifiers include flocculants and coagulants. Exemplary examples of nucleating agents include droplets of the non-catabolic compound itself and organic solvents such as dodecane, isopropyl myristate, and methyl oleate.

[0178] The non-catabolic compound produced in these cells can be present in the culture supernatant and / or associated with the host cell. In embodiments where the non-catabolic compound is associated with the host cell, recovery of the non-catabolic compound can involve methods of permeabilizing or lysing the cell. Alternatively or simultaneously, recovery processes can be used to recover the non-catabolic compound in the culture medium, including but not limited to chromatography, extraction, solvent extraction, membrane separation, electrodialysis, reverse osmosis, distillation, chemical derivatization, and crystallization.

[0179] In some embodiments, the non-dissimilar compound 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 chromatography techniques.

[0180] In some implementations, the recovered non-dissimilar compound is pure, for example, at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or more than 98% pure, where “pure” in the context of non-dissimilar compound means a non-dissimilar compound that is free from other non-dissimilar compounds, contaminants, etc.

[0181] 5.4. Genetically modified microorganisms

[0182] This article describes genetically modified microorganisms (e.g., genetically modified Saccharomyces cerevisiae cells) that produce heterologous acetyl-CoA-derived (non-dissimilatory) compounds. Compared to parental microorganisms lacking the genetic modifications described herein, genetically modified microorganisms produce greater quantities of one or more compounds biosynthesized from acetyl-CoA.

[0183] Methods for genetically modifying microorganisms using expression vectors or chromosomally integrated constructs, for example, to achieve elevated production of one or more non-catabolic compounds in the host cell, are well known in the art. See, for example, Sherman, F., et al., Methods Yeast Genetics, Cold Spring Harbor Laboratory, N.Y. (1978); Guthrie, C, et al. (eds.) Guide To Yeast Genetics and Molecular Biology Vol. 194, Academic Press, San Diego (1991); Sambrook et al., 2001, Molecular Cloning - A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY; the disclosures of which are incorporated herein by reference. Additionally, inhibition of gene expression can be achieved by deletion, mutation, and / or gene rearrangement, for example, which results in elevated production of one or more non-catabolic compounds in the cell. It can also be performed using antisense RNA, siRNA, miRNA, ribozymes, triplex DNA, and transcriptional and / or translational inhibitors. Additionally, transposons can be employed to disrupt gene expression, for example, by inserting it between a promoter and coding region, or between two adjacent genes to inactivate one or both genes.

[0184] In some embodiments, the increased production of non-dissimilated compounds in the cell is achieved by expressing particular proteins (e.g., proteins involved in the biosynthetic pathways described above) using expression vectors. Generally, expression vectors are recombinant polynucleotide molecules that include a replication signal and expression control sequences (e.g., a promoter and terminator operably linked to a nucleotide sequence encoding a polypeptide). Expression vectors useful for expressing polypeptide-encoding nucleotide sequences include viral vectors (e.g., retroviruses, adenoviruses, and adeno-associated viruses), plasmid vectors, and cosmids. Exemplary examples of expression vectors suitable for use in yeast cells include, but are not limited to, CEN / ARS and 2μ plasmids. Exemplary examples of promoters suitable for use in yeast cells include, but are not limited to, the promoter of the TEF1 gene of K. lactis, the promoter of the PGK1 gene of S. cerevisiae, the promoter of the TDH3 gene of S. cerevisiae, repressible promoters (e.g., the promoter of the CTR3 gene of S. cerevisiae), and inducible promoters, such as the galactose-inducible promoters of S. cerevisiae (e.g., the promoters of the GAL 1, GAL7, and GAL 10 genes).

[0185] Expression vectors and chromosomal integration constructs can be introduced into microbial cells by any method known to those of skill in the art without limitation. See, e.g., Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1292-3 (1978); Cregg et al., Mol. Cell. Biol. 5: 3376-3385 (1985); U.S. Patent No. 5,272,065; 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., 1999, Current Protocols in Molecular Biology, John Wiley & Sons, NY. Exemplary techniques include, but are not limited to, spheroplasting, electroporation, PEG 1000-mediated transformation, and lithium acetate or lithium chloride-mediated transformation.

[0186] 5.4.1 Host Cells

[0187] Cells useful in the methods and compositions provided herein include any cell capable of naturally or recombinantly producing a non-terpene compound (e.g., a polyketide, a fatty acid, the like). In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell. In some embodiments, the cell is an Escherichia coli cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a Chinese hamster ovary (CHO) cell, a COS-7 cell, a mouse fibroblast cell, a mouse embryonal carcinoma cell, or a mouse embryonic stem cell. In some embodiments, the cell is an insect cell. In some embodiments, the cell is an S2 cell, a Schneider cell, an S12 cell, a 5B1-4 cell, a Tn5 cell, or an Sf9 cell. In some embodiments, the cell is a unicellular eukaryotic organism cell.

[0188] In some embodiments, the cell is a mycelial bacterial cell. In some embodiments, the mycelial bacterial cell is of the class actinomycete. In particular embodiments, the mycelial bacterial cell is of the genus Streptomyces, e.g., Streptomyces ambofaciens, Streptomyces avermitilis, Streptomyces azureus, Streptomyces cinnamonensis, Streptomyces coelicolor, Streptomyces curacoi, Streptomyces erythraeus, Streptomyces fradiae, Streptomyces galilaeus, Streptomyces glaucescens, Streptomyces hygroscopicus, Streptomyces lividans, Streptomyces parvulus, Streptomyces peucetius, Streptomyces rimosus, Streptomyces roseofulvus, Streptomyces thermotolerans, Streptomyces violaceoruber.

[0189] In another embodiment, the cell is a fungal cell. In a more particular embodiment, the cell is a yeast cell. Yeasts useful in the methods and compositions provided herein include yeasts that have been deposited with a microorganism depository (e.g., IFO, ATCC, etc.) and belong to the following 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, Eremosynnema, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hohemiopsis, Hanseniaspora, Hansenula,Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiops, Wikenomyces, Yamadazyma, Yarrowia, Zygocapsa, Zygoascus, Zygowilliopsis, and Zygozyma, among others.

[0190] In particular embodiments, the yeast useful in the methods and compositions provided herein include Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (formerly Saccharomyces lactis), Kluveromyces marxianus, Arxula adeninivorans, or Hansenula polymorpha (now Pichia angusta). In some embodiments, the microorganism is a strain of Candida, such as Candida lipolytica, Candida guilliermondii, Candida kefyr, Candida tropicalis, or Candida utilis.

[0191] In particular embodiments, the cell is a Saccharomyces cerevisiae cell. In some embodiments, the strain of Saccharomyces cerevisiae is selected from the group consisting of Baker's Yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the strain of Saccharomyces cerevisiae is selected from the group consisting of PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1. In particular embodiments, the strain of Saccharomyces cerevisiae is PE-2. In another particular embodiment, the strain of Saccharomyces cerevisiae is CAT-1. In another particular embodiment, the strain of Saccharomyces cerevisiae is BG-1.

[0192] In some embodiments, the cell is a haploid microbial cell. In other embodiments, the cell is a diploid microbial cell. In some embodiments, the cell is heterozygous. In other embodiments, the cell is homozygous for all but its mating type allele (i.e., if the cell were to sporulate, the resulting four haploid microbial cells would be genetically identical except for their mating type alleles, which would be mating type a in two haploid cells and mating type a in the other two haploid cells).

[0193] In some embodiments, the cell is a cell suitable for industrial fermentation (e.g., bioethanol fermentation). In particular embodiments, the cell is adapted to survive conditions of high solvent concentration, high temperature, expanded substrate utilization, nutrient limitation, osmotic stress due, acidity, sulfite and bacterial contamination, or combinations thereof, which are recognized stress conditions in industrial fermentation environments.

[0194] Exemplary non-catabolic compound-producing cells, such as recombinant isoprenoid-, polyketide-, and fatty acid-producing cells, and methods for producing such cells are provided below.

[0195] 5.5 Isoprenoid Production

[0196] In some embodiments, the non-catabolic compound is an isoprenoid. Isoprenoids are derived from isopentenyl pyrophosphate (IPP), which can be biosynthesized by enzymes of the mevalonate-dependent ("MEV") pathway or the 1-deoxy-D-xylulose 5-phosphate ("DXP") pathway.

[0197] 5.5.1 MEV Pathway

[0198] In some embodiments of the methods provided herein, the genetically modified microorganism comprises one or more heterologous nucleotide sequences encoding one or more enzymes of the MEV pathway, which achieves increased production of one or more isoprenoid compounds compared to the ungenetically modified parent cell.

[0199] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme capable of condensing two molecules of acetyl-CoA to form acetoacetyl-CoA (e.g., an acetyl-CoA thiolase). Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_000913 region: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).

[0200] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., HMG-CoA synthase) that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_001145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).

[0201] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., HMG-CoA reductase) that can convert HMG-CoA to mevalonate. Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NM_206548; Drosophila melanogaster), (NC_002758, locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces KO3988), (AF542543; Nicotiana attenuate), (AB037907; Kitasatospora griseola), (AX128213, provides sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC_001145: complement 115734.118898; Saccharomyces cerevisiae).

[0202] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., HMG-CoA synthase) that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_001145. complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).

[0203] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA to mevalonate. Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. complement 712315.713670; Saccharomyces cerevisiae).

[0204] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert mevalonate 5-phosphate to mevalonate 5-pyrophosphate (e.g., phosphomevalonate kinase). Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. complement 712315.713670; Saccharomyces cerevisiae).

[0205] In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding more than one enzyme of the MEV pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding two enzymes of the MEV pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme that can convert HMG-CoA to mevalonate and an enzyme that can convert mevalonate to mevalonate 5-phosphate. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding three enzymes of the MEV pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding four enzymes of the MEV pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the MEV pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding six enzymes of the MEV pathway.

[0206] In some embodiments, the isoprenoid-producing cell further comprises a heterologous nucleotide sequence encoding an enzyme that can convert IPP produced via the MEV pathway to its isomer dimethylallyl pyrophosphate ("DMAPP"). DMAPP can be condensed and modified via the action of various additional enzymes to form simple and more complex isoprenoids. Figure 2 ).

[0207] 5.5.2 DXP pathway

[0208] In some embodiments of the methods provided herein, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding one or more enzymes of the DXP pathway that effect increased production of one or more isoprenoid compounds as compared to a non-genetically modified parent cell.

[0209] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme capable of condensing two molecules of acetyl-CoA to form acetoacetyl-CoA (e.g., an acetyl-CoA thiolase). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_000913 REGION: 2324131.2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).

[0210] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme capable of condensing pyruvate with D-glyceraldehyde 3-phosphate to form 1-deoxy-D-xylulose-5-phosphate (e.g., a 1-deoxy-D-xylulose-5-phosphate synthase). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (AF035440; Escherichia coli), (NC_002947, locus tag PP0527; Pseudomonas putida KT2440), (CP000026, locus tag SPA2301; Salmonella enterica Paratyphi, see ATCC 9150), (NC_007493, locus tag RSP_0254; Rhodobacter sphaeroides 2.4.1), (NC_005296, locus tag RPA0952; Rhodopseudomonas palustris CGA009), (NC_004556, locus tag PD1293; Xylella fastidiosa Temeculal), and (NC_003076, locus tag AT5G11380; Arabidopsis thaliana).

[0211] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 1-deoxy-D-xylulose-5-phosphate to 2C-methyl-D-erythritol-4-phosphate (e.g., 1-deoxy-D-xylulose-5-phosphate reductoisomerase). Illustrative examples of nucleotide sequences include, but are not limited to: (AB013300; Escherichia coli), (AF148852; Arabidopsis thaliana), (NC_002947, locus tag PP1597; Pseudomonas putida KT2440), (AL939124, locus tag SC05694; Streptomyces coelicolor A3(2)), (NC_007493, locus tag RSP_2709; Rhodobacter sphaeroides 2.4.1), and (NC_007492, locus tag Pfl_1107; Pseudomonas fluorescens PfO-1).

[0212] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 2C-methyl-D-erythritol-4-phosphate to 4-diphosphocytidyl-2C-methyl-D-erythritol (e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol synthase). Illustrative examples of nucleotide sequences include, but are not limited to: (AF230736; Escherichia coli), (NC_007493, locus tag RSP_2835; Rhodobacter sphaeroides 2.4.1), (NC_003071, locus tag AT2G02500; Arabidopsis thaliana), and (NC_002947, locus tag PP1614; Pseudomonas putida KT2440).

[0213] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 4-diphosphocytidyl-2C-methyl-D-erythritol to 4-diphosphocytidyl-2C-methyl-D-erythritol-2-phosphate (e.g., 4-diphosphocytidyl-2C-methyl-D-erythritol kinase). Illustrative examples of nucleotide sequences include, but are not limited to: (AF216300; Escherichia coli) and (NC_007493, locus tag RSP_1779; Rhodobacter sphaeroides 2.4.1).

[0214] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 4-diphosphocytid-2C-methyl-D- erythritol-2-phosphate to 2C-methyl-D-erythritol 2,4-cyclodiphosphate (2C-methyl-D- erythritol 2,4-cyclodiphosphate synthase). Exemplary examples of nucleotide sequences include, but are not limited to: (AF230738; Escherichia coli), (NC_007493, locus tag RSP_6071; Rhodobacter sphaeroides 2.4.1), and (NC_002947, locus tag PP1618; Pseudomonas putida KT2440).

[0215] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1- hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (e.g., 1-hydroxy-2-methyl-2-(E)- butenyl-4-diphosphate synthase). Exemplary examples of nucleotide sequences include, but are not limited to: (AY033515; Escherichia coli), (NC_002947, locus tag PP0853; Pseudomonas putida KT2440), and (NC_007493, locus tag RSP_2982; Rhodobacter sphaeroides 2.4.1).

[0216] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate to IPP or its isomer DMAPP (e.g., isopentyl / dimethylallyl diphosphate synthase). Exemplary examples of nucleotide sequences include, but are not limited to: (AY062212; Escherichia coli), and (NC_002947, locus tag PP0606; Pseudomonas putida KT2440).

[0217] In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding more than one enzyme of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding two enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding three enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding four enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding six enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the DXP pathway. In some embodiments, the isoprenoid-producing cell comprises one or more heterologous nucleotide sequences encoding seven enzymes of the DXP pathway.

[0218] In some embodiments, "cross talk" (or interference) between the host cell's own metabolic processes and those involved in IPP production is minimized or eliminated altogether. For example, when a host microorganism relies solely on the DXP pathway to synthesize IPP, cross talk is minimized or eliminated altogether, and the MEV pathway is introduced to provide additional IPP. Such a host organism would not be equipped to alter expression of MEV pathway enzymes or to process intermediates associated with the MEV pathway. Organisms that rely solely or predominantly on the DXP pathway include, for example, E. coli.

[0219] In some embodiments, the host cell produces IPP via the MEV pathway, either alone or in combination with the DXP pathway. In other embodiments, the host's DXP pathway is rendered functionally inoperative, such that the host cell produces IPP via the heterologously introduced MEV pathway alone. The DXP pathway can be rendered functionally inoperative by rendering gene expression inoperative or by inactivating the function of one or more DXP pathway enzymes.

[0220] In some embodiments, the isoprenoid produced by the cell is a C5 isoprenoid. These compounds are derived from one isoprene unit and are also known as hemiterpenes. An exemplary example of a hemiterpene is isoprene. In other embodiments, the isoprenoid is a C 10 isoprenoid. These compounds are derived from two isoprene units and are also known as monoterpenes. Exemplary examples of monoterpenes are limonene, citranellol, geraniol, menthol, perillyl alcohol, linalool, thujone, and myrcene. In other embodiments, the isoprenoid is a C 15Isoprenoids. These compounds are derived from three isoprene units, also known as a sesquiterpene. Illustrative examples of sesquiterpenes are Periplanone B, gingkolide B, amorphadiene, artemisinic acid, kaurene, norcane, epi-cedrol, epi- aristolane, farnesol, gossypol, sanonin, periplanone, p-menthane, and patchoulol (also known as patchouli alcohol). In other embodiments, the isoprenoid is a C 20 Isoprenoid. These compounds are derived from four isoprene units, also known as a diterpene. Illustrative examples of diterpenes are euphadiene, eleutherobin, paclitaxel, prostratin, pseudopterosin, and taxadiene. In other embodiments, the isoprenoid is a C 20+ Isoprenoid. These compounds are derived from more than four isoprene units, and include: triterpenoids (C 30 isoprenoid compounds) such as arbruside E, didemnin B, testosterone, progesterone, cortisone, digitoxin, and squalene; tetraterpenes (C 40 isoprenoid compounds) such as beta-carotene; and polyterpenes (C 40+ isoprenoid compounds) such as polyisoprene. In some embodiments, the isoprenoid is selected from the group comprising abietadiene, amorphadiene, carene, a-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene. The isoprenoid compounds also include, but are not limited to, carotenoids (such as lycopene, a- and β-carotene, a- and β-cryptoxanthin, bixin, zeaxanthin, astaxanthin, and lutein), steroid compounds, and compounds consisting of isoprenoids modified with other chemical groups, such as mixed terpene-alkaloids and coenzyme Q-10.

[0221] In some embodiments, the isoprenoid-producing cell further comprises a heterologous nucleotide sequence encoding an enzyme capable of converting IPP produced via the MEV pathway to DMAPP (e.g., an IPP isomerase). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_000913, 3031087.3031635; Escherichia coli) and (AF082326; Haematococcus pluvialis).

[0222] In some embodiments, the isoprenoid-producing cell further comprises a heterologous nucleotide sequence encoding a polyisoprene synthase capable of condensing IPP and / or DMAPP molecules to form a polyisoprenoid compound comprising more than five carbons.

[0223] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme capable of condensing one molecule of IPP with one molecule of DMAPP to form one molecule of geranyl pyrophosphate ("GPP") (e.g., a GPP synthase). Illustrative examples of nucleotide sequences encoding these enzymes include, but are not limited to: (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsis thaliana), (AE016877, locus AP11092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ips pini), (DQ286930; Lycopersicon lycopersicum), (AF182828; Mentha piperita), (AF182827; Mentha piperita), (MPI249453; Mentha piperita), (PZE431697, locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Peltandra virginica), (AY351862; Vitis vinifera), and (AF203881, locus AAF12843; Zymomonas mobilis).

[0224] In some embodiments, the isoprenoid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme capable of condensing two molecules of IPP with one molecule of DMAPP, or adding an IPP molecule to a GPP molecule, to form farnesyl pyrophosphate ("FPP") (e.g., a FPP synthase). Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (ATU80605; Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, locus AAL95523; Fusobacterium nucleatum subsp. polymorphum ATCC 25586), (GFFPPSGEN; Gibberella fujikuroi), (CP000009, locus AAW60034; Gluconacetobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1; Parthenium argentatum), (PAFPS2; Parthenium argentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomyces cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, locus AAT87386; Streptococcus pyogenes), (CP000017, locus AAZ51849; Streptococcus pyogenes), (NC_008022, locus YP_598856; Streptococcus pyogenes MGAS10270), (NC_008023, locus YP_600845; Streptococcus pyogenes MGAS2096), (NC_008024, locus YP_602832; Streptococcus pyogenes MGAS10750), (MZEFPS; Zea mays), (AE000657, locus AAC06913; Aquifex aeolicus VF5), (NM_202836; Arabidopsis thaliana), (D84432, locus BAA12575; Bacillus subtilis), (U12678, locus AAC28894; Bradyrhizobium japonicum USDA 110), (BACFDPS; Geobacillus caldoxylos), (NC_002940, locus NP_873754; Haemophilus ducreyi 35000HP), (L42023, locus AAC23087; Haemophilus influenzae Rd KW20), (J05262; Homo sapiens), (YP_395294; Lactobacillus sakei subsp. sakei 23K), (NC_005823, locus YP_000273;Leptospira interrogans serovar Copenhageni (AB 003187; Micrococcus luteus), (NC_002946, locus YP_208768; Neisseria gonorrhoeae FA 1090), (U00090, locus AAB91752; Rhizobium NGR234), (J05091; Saccharomyces cerevisiae), (CP000031, locus AAV93568; Silicibacter pomeroyi DSS-3), (AE008481, locus AAK99890; Streptococcus pneumoniae R6), and (NC_004556, locus NP_779706; Xylella fastidiosa Temeculal).

[0225] In some embodiments, the isoprenoid-producing cell further comprises a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate ("GGPP"). Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845; Arabidopsis thaliana), (NZ_AAJM01000380, locus ZP_00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sq1563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, locus ZP_00144509; Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN; G. fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucor circinelloides f. Iusitanicus), (AB016044; Mus musculus), (AABX01000298, locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, locus ZP_00943566; Ralstonia solanacearum UW551) (AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongatus), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, locus YP_461832; Syntrophus aciditrophicus SB), (NC_006840, locus YP_204095; Vibrio fischeri ES114), (NM_112315; Arabidopsis thaliana), (ERWCRTE; Pantoea agglomerans), (D90087, locus BAA14124; Pantoea ananatis), (X52291, locus CAA36538; Rhodobacter capsulatus), (AF195122, locus AAF24294; Rhodobacter sphaeroides), and (NC_004350, locus NP_721015; Streptococcus mutans UA159).

[0226] In some embodiments, the isoprenoid-producing cell further comprises a heterologous nucleotide sequence encoding an enzyme that can modify a polyisoprene to form a hemiterpene, a monoterpene, a sesquiterpene, a diterpene, a triterpene, a tetraterpene, a polyterpene, a steroid compound, a carotenoid, or a modified isoprenoid compound.

[0227] In some embodiments, the heterologous nucleotide encodes a caryophyllene synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AF461460, REGION 43.1926; Picea abies) and (AF527416, REGIN: 78.1871; Salvia officinalis).

[0228] In some embodiments, the heterologous nucleotide encodes a geraniol synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AJ457070; Cinnamomum micranthum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora).

[0229] In some embodiments, the heterologous nucleotide encodes a linalool synthase. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, locus AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana), (AF154124; Artemisia annua), (AF067603; Asarum maximum), (AF067602; Asarum maximum), (AF067601; Asarum maximum), (U58314; Asarum maximum), (AY840091; Solanum lycopersicum), (DQ263741; Lavandula angustifolia), (AY083653; Mentha x piperita), (AY693647; Ocimum basilicum), (XM_463918; Oryza sativa), (AP004078, locus BAD07605; Oryza sativa), (XM_463918, locus XP_463918; Oryza sativa), (AY917193; Perilla citriodora), (AF271259; Perilla frutescens), (AY473623; Picea abies), (DQ195274; Picea glauca), and (AF444798; Perilla frutescens var. crispa cultivar No. 79).

[0230] In some embodiments, the heterologous nucleotide encodes a limonene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (+)-limonene synthase (AF514287, region: 47.1867; Citrus limon) and (AY055214, region: 48.1889; Agastache rugosa) and (-)-limonene synthase (DQ195275, region: 1.1905; Picea sitchensis), (AF006193, region: 73.1986; Abies procera), and (MHC4SLSP, region: 29.1828; Mentha crispa).

[0231] In some embodiments, the heterologous nucleotide encodes a myrcene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (U87908; Abies procera), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (NM_127982; Arabidopsis thaliana TPS10), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercus ilex).

[0232] In some embodiments, the heterologous nucleotide encodes a myrcene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (U87908; Abies procera), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (NM_127982; Arabidopsis thaliana TPS10), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies), and (AJ304839; Quercus ilex).

[0233] In some embodiments, the heterologous nucleotide encodes an alpha-pinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (+) alpha-pinene synthase (AF543530, region: 1.1887; Pinus taeda), (-) alpha-pinene synthase (AF543527, region: 32.1921; Pinus taeda), and (+) / (-) alpha-pinene synthase (AGU87909, region: 61 11892; Abies procera).

[0234] In some embodiments, the heterologous nucleotide encodes a beta-pinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (-) beta-pinene synthase (AF276072, region: 1.1749; Artemisia annua) and (AF514288, region: 26.1834; Citrus limon).

[0235] In some embodiments, the heterologous nucleotide encodes a sabinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: AF051901, region: 26.1798, from Salvia officinalis.

[0236] In some embodiments, the heterologous nucleotide encodes a gamma-terpinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AF514286, region: 30.183 from Citrus limon) and (AB110640, region 1.1803, from Citrus sinensis).

[0237] In some embodiments, the heterologous nucleotide encodes a terpinolene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AY693650 from Ocimum basilicum) and (AY906866, region: 10.1887, from Pseudotsuga taxifolia).

[0238] In some embodiments, the heterologous nucleotide encodes a amorphadiene synthase. An illustrative example of a suitable nucleotide sequence is SEQ ID NO. 37 of U.S. Patent Publication No. 2004 / 0005678.

[0239] In some embodiments, the heterologous nucleotide encodes an alpha-farnesene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: DQ309034, from Pyrus communis cultivar d'Anjou (Pyrus; gene name AFS1), and AY182241, from Malus domestica (apple; gene AFS1). Pechouus et al., Planta 219(1): 84-94 (2004).

[0240] In some embodiments, the heterologous nucleotide encodes a beta-farnesene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: Accession No. AF024615 from Mentha piperita (mint; gene Tspall), and AY835398 from Artemisia annua. Picaud et al., Phytochemistry 66(9): 961-967 (2005).

[0241] In some embodiments, the heterologous nucleotide encodes a farnesol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128: 149-158 (2006).

[0242] In some embodiments, the heterologous nucleotide encodes a nerolidol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to AF529266 from Zea mays (maize; gene tpsl).

[0243] In some embodiments, the heterologous nucleotide encodes a pogostol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to AY508730 region: 1.1659 from Pogostemonis herba.

[0244] In some embodiments, the heterologous nucleotide encodes a nootkatol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to AF441124 region: 1.1647 from Citrus sinensis and AY917195 region: 1.1653 from Perilla frutescens.

[0245] In some embodiments, the heterologous nucleotide encodes a abietadiene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to (U50768; Picea sitchensis) and (AY473621; Picea abies).

[0246] 5.6 Production of Polyketides

[0247] In some embodiments, the non-catalytic compound is a polyketide. Polyketides are synthesized by sequential reactions catalyzed by an assembly of enzymatic activities known as polyketide synthases (PKSs), which are large multi-enzyme protein complexes containing coordinated groups of active sites. The biosynthesis of polyketides proceeds stepwise, starting from simple 2-, 3-, 4-carbon building blocks (such as acetyl-CoA, propionyl-CoA, butyryl-CoA and their activated derivatives, malonyl-CoA, methylmalonyl-CoA and ethylmalonyl-CoA), mainly through decarboxylative condensations of malonyl-CoA-derived units via Claisen condensations. PKS genes are usually arranged in one operon in bacteria and in gene clusters in eukaryotes. Three types of polyketide synthases are characterized: Type I polyketide synthases are large, highly modular proteins, which are subdivided into two classes: 1) iterative PKSs, which use domains in a cyclic fashion and 2) modular PKSs, which contain individual module sequences and do not repeat domains. Type II polyketide synthases are polymers of single-function proteins, and Type III polyketide synthases do not use acyl carrier protein domains.

[0248] Unlike the biosynthesis of fatty acids, in which each successive chain elongation step is followed by ketoreduction, dehydration, and enoyl reduction in a fixed order, individual chain elongation intermediates of polyketide biosynthesis undergo all, some, or no functional group modification, resulting in a large number of chemically distinct products. Additional complexity is caused by the use of different starter units and chain elongation units and the generation of new stereoisomers.

[0249] The complete polyketide synthesis sequence is directed by the polyketide synthase as follows (in N-terminal to C-terminal order): initiation or loading of the initial carbon building block onto the acyl carrier protein, elongation of the growing macrocyclic lactone chain catalyzed by the extender modules, and terminal module elongation, which catalyzes release of the synthesized macrocyclic lactone. Component domains or independent enzyme functions are active in this biosynthesis, including acyl- transferases for loading initiators, extenders, and intermediate acyl units; acyl carrier proteins hold the growing macrocyclic lactone as a thioester; β-keto-acyl synthases catalyze chain elongation; β-keto reductases are responsible for the first reduction to an alcohol function; dehydratases remove water to give unsaturated thioesters; enoyl reductases catalyze the final reduction to full saturation; and thioesterases catalyze release and cyclization of the macrocyclic lactone.

[0250] In some embodiments, the genetically engineered microorganism useful in the methods disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can condense at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein, such as an acyl- transferase. In some embodiments, the genetically engineered microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can condense a first reactant selected from the group consisting of acetyl-CoA and malonyl-CoA with a second reactant selected from the group consisting of malonyl-CoA or methyl-malonyl-CoA to form a polyketide product, such as a β-keto-acyl synthase.

[0251] In some embodiments, the polyketide-producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can reduce a β-keto chemical group on a polyketide to form a β-hydroxy group, such as a β-keto reductase.

[0252] In some embodiments, the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can dehydrate an alkane chemical group of a polyketide to produce an α-β-unsaturated alkene, such as a dehydratase.

[0253] In some embodiments, the polyketide-producing cell disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can reduce an α-β-double bond of a polyketide to produce a saturated alkane, such as an enoyl-reductase.

[0254] In some embodiments, the polyketide-producing cell disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme that can hydrolyze a polyketide from an acyl carrier protein, such as a thioesterase.

[0255] In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising an AT catalytic domain. In some embodiments, the polyketide-producing cell comprises more than one heterologous nucleotide sequence encoding an enzyme comprising an AT catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising a CLF catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding an enzyme comprising ACP activity. In some embodiments, the polyketide-producing cell comprises more than one heterologous nucleotide sequence encoding an enzyme comprising ACP activity.

[0256] In particular embodiments, the polyketide-producing cell comprises a minimal aromatic PKS system, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic domain, an enzyme comprising an AT catalytic domain, an enzyme comprising a CLF catalytic domain, and an enzyme comprising ACP activity, respectively. In particular embodiments, the polyketide-producing cell comprises a minimal modular PKS system, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic domain, an enzyme comprising an AT catalytic domain, and an enzyme comprising ACP activity, respectively. In another particular embodiment, the polyketide-producing cell comprises a modular aromatic PKS system for de novo synthesis of a polyketide, e.g., heterologous nucleotide sequences encoding an enzyme comprising a KS catalytic domain, one or more enzymes comprising an AT catalytic domain, and one or more enzymes comprising ACP activity, respectively.

[0257] In some embodiments, the polyketide-producing cell comprises a minimal PKS system, e.g., a minimal aromatic PKS system or a minimal modular PKS system, further comprising additional catalytic activities that contribute to the production of the final product polyketide. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding enzymes comprising a cyclase (CYC) catalytic domain, which facilitates cyclization of the nascent polyketide backbone. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding enzymes comprising a ketoreductase (KR) catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding enzymes comprising an aromatase (ARO) catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding enzymes comprising an enoyl reductase (ER) catalytic domain. In some embodiments, the polyketide-producing cell comprises one or more heterologous nucleotide sequences encoding enzymes comprising a thioesterase (TE) catalytic domain. In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding enzymes comprising full ACP synthase activity, which results in pantetheinylation of the ACP.

[0258] In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences conferring post-synthetic polyketide modification activities. In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding enzymes comprising glycosylase activity, which results in post-synthetic modification of the polyketide, e.g., when a polyketide with antibacterial activity is desired. In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding enzymes comprising hydroxylase activity. In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding enzymes comprising epoxidase activity. In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding enzymes comprising methylase activity.

[0259] In some embodiments, the polyketide-producing cell further comprises one or more heterologous nucleotide sequences encoding biosynthetic enzymes, including, but not limited to, at least one polyketide synthesis pathway enzyme, and enzymes that can modify acetyl-CoA compounds to form polyketide products, such as macrolide compounds, antibiotics, antifungal agents, cell growth inhibiting compounds, anticholesterolemic compounds, antiparasitic compounds, anticoccidial compounds, animal growth promoting agents, or pesticides. In some embodiments, the non- assimilated compound is a polyene. In some embodiments, the non- assimilated compound is a cyclic lactone. In some embodiments, the non- assimilated compound comprises a 14, 15, or 16 membered lactone ring. In some embodiments, the non- assimilated compound is selected from the group consisting of polyketide macrolides, antibiotics, antifungal agents, cell growth inhibitors, anticholesterolemic agents, antiparasitic agents, anticoccidial agents, animal growth promoting agents, and pesticides.

[0260] In some embodiments, the polyketide-producing cell comprises a heterologous nucleotide sequence, e.g., a sequence encoding a PKS enzyme and a polyketide-modifying enzyme, which is capable of producing a polyketide selected from, but not limited to, the following polyketides: avermectin (see, e.g., U.S. Pat. No. 5,252,474; U.S. Pat. No. 4,703,009; EP Pub. No. 118,367; MacNeil et al., 1993, "Industrial Microorganisms: Basic and Applied Molecular Genetics"; Baltz, Hegeman, & Skatrud, eds. (ASM), pp. 245-256, "A Comparison of the Genes Encoding the Polyketide Synthases for Avermectin, Erythromycin, and Nemadectin"; MacNeil et al., 1992, Gene 115: 119-125; and Ikeda and Omura, 1997, Chem. Res. 97: 2599-2609); candicidin (FR008) (see, e.g., Hu et al., 1994, MoI. Microbiol. 14: 163-172); capromycin, curamycin (see, e.g., Bergh et al., Biotechnol Appl Biochem. 1992 Feb; 15(1): 80-9); daunomycin (see, e.g., J Bacteriol. 1994 Oct; 176(20): 6270-80); epothilone (see, e.g., PCT Pub. No. 99 / 66028; and PCT Pub. No. 00 / 031247); erythromycin (see, e.g., PCT Pub. No. 93 / 13663; U.S. Pat. No. 6,004,787; U.S. Pat. No. 5,824,513; Donadio et al., 1991, Science 252: 675-9; and Cortes et al., Nov. 8, 1990, Nature 348: 176-8); FK-506 (see, e.g., Motamedi et al., 1998; Eur. J Biochem. 256: 528-534; and Motamedi et al., 1997, Eur. J Biochem. 244: 74-80); FK-520 (see, e.g., PCT Pub. No. 00 / 020601; and Nielsen et al., 1991, Biochem.30:5789-96); Griseusin (see, e.g., Yu et al., J Bacteriol. 1994 May; 176(9):2627-34); Lovastatin (see, e.g., U.S. Pat. No. 5,744,350); Frenolycin (see, e.g., Khosla et al., Bacteriol. 1993 Apr; 175(8):2197-204; and Bibb et al., Gene 1994 May 3; 142(1):31-9); Luteovorin (see, e.g., Sherman et al., EMBO J. 1989 Sep; 8(9):2717-25; and Bechtold et al., Mol Gen Genet. 1995 Sep 20; 248(5):610-20); Mandelamycin (see, e.g., Ichinose et al., Microbiology 2003 Jul; 149(Pt 7):1633-45); Monensin (see, e.g., Arrowsmith et al., Mol Gen Genet. 1992 Aug; 234(2):254-64); Nonactin (see, e.g., FEMS Microbiol Lett. 2000 Feb 1; 183(1): 171-5); Septamycin (see, e.g., Kitao et al., J Antibiot (Tokyo). 1980 Jul; 33(7):711-6); Nermackin (see, e.g., MacNeil et al., 1993, supra); Niddamycin (see, e.g., PCT Pub. No. 98 / 51695; and Kakavas et al., 1997, J. Bacteriol. 179:7515-7522); Neriolycin (see, e.g., Swan et al., 1994, Mol. Gen. Genet. 242:358-362; PCT Pub. No. 00 / 026349; Olano et al., 1998, Mol. Gen. Genet. 259(3):299-308; and PCT Pat. App. Pub. No. WO 99 / 05283); Oxatetracycline (see, e.g., Kim et al., Gene. 1994 Apr 8; 141(1): 141-2); Picromycin (see, e.g., PCT Pub. No. 99 / 61599; PCT Pub. No. 00 / 00620; Xue et al., 1998, Chemistry & Biology 5(11):661-667; Xue et al., October 1998, Proc. Natl. Acad. Sci.USA 95:1211112116); platensimycin (see, e.g., EP Pub. No. 791,656; and U.S. Pat. No. 5,945,320); rapamycin (see, e.g., Schwecke et al., August 1995, Proc. Natl. Acad. Sci. USA 92:7839-7843; and Aparicio et al., 1996, Gene 169:9-16); rifamycin (see, e.g., PCT Pub. No. WO 98 / 07868; and August et al., Feb. 13, 1998, Chemistry & Biology, 5(2):69-79); Sorangium (see, e.g., U.S. Pat. No. 6,090,601); Soraphen (see, e.g., U.S. Pat. No. 5,716,849; Schupp et al., 1995, J. Bacteriology 177:3673-3679); Spinocyn (see, e.g., PCT Pub. No. 99 / 46387); spiramycin (see, e.g., U.S. Pat. No. 5,098,837); tetrachlorosalicylanilide (see, e.g., U.S. Pat. No. 5,876,991; U.S. Pat. No. 5,672,497; U.S. Pat. No. 5,149,638; EP Pub. No. 791,655; EP Pub. No. 238,323; Kuhstoss et al., 1996, Gene 183:231-6; and Merson-Davies and Cundliffe, 1994, Mol. Microbiol. 13:349-355); and 6-methyl salicylate (see, e.g., Richardson et al., Metab Eng. 1999 Apr;1(2):180-7; and Shao et al., Biochem Biophys Res Commun. 2006 Jun 23;345(1):133-9).

[0261] 5.7 Production of fatty acids

[0262] In some embodiments, the non-catabolic compound is a fatty acid. Fatty acids are synthesized from acetyl-CoA and malonyl-CoA by a series of decarboxylative Claisen condensations catalyzed by fatty acid synthase. Like polyketide synthases, fatty acid synthases are not single enzymes but rather enzyme systems composed of multifunctional polypeptides in which substrates are passed from one functional domain to the next. Two major classes of fatty acid synthases have been characterized. Type I fatty acid synthases are common to mammals and fungi (although the structural organization of fungal and mammalian synthases differs) and CMN group bacteria (corynebacteria, mycobacteria, and nocardia). Type II synthases, found in archaea and eubacteria, are a series of discrete, single-function enzymes involved in fatty acid synthesis. The mechanisms of fatty acid elongation and shortening are the same in both classes of synthases, as the enzyme domains responsible for these catalytic events are largely homologous between the two classes.

[0263] Following each round of fatty acid chain elongation in the decarboxylative Claisen condensation, the 3-keto group is reduced to a fully saturated carbon chain by the sequential action of a keto reductase, a dehydratase, and an enoyl reductase. The growing fatty acid chain is shuttled between these active sites attached to an acyl carrier protein, and is finally released by the action of a thioesterase upon reaching a carbon chain length of 16 (palmitic acid).

[0264] In some embodiments, the genetically modified microorganism useful for the methods disclosed herein comprises a heterologous nucleotide sequence encoding a biosynthetic enzyme, including but not limited to at least one fatty acid synthesis pathway enzyme, and enzymes capable of modifying acetyl-CoA compounds to form fatty acid products such as palmitic acid, palmitoyl-CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. In some embodiments, the non-catabolic compound is a fatty acid selected from the group consisting of palmitic acid, palmitoyl-CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, a-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0265] In some embodiments, the fatty acid-producing cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., an acyltransferase) capable of covalently linking at least one of acetyl-CoA and malonyl-CoA to an acyl carrier protein.

[0266] In some embodiments, the genetically modified microorganism disclosed herein comprises a heterologous nucleotide sequence encoding an enzyme (e.g., a β-ketoacyl-ACP synthase) capable of condensing an acetyl chemical moiety and a malonyl chemical moiety (each bound to an acyl carrier protein (ACP)) to form acetoacetyl-ACP.

[0267] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can reduce the double bond in acetoacetyl-ACP with NADPH to form a hydroxyl group in D-3-hydroxybutyryl-hydroxylase-ACP (e.g., a β-ketoacyl-ACP reductase).

[0268] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can dehydrate D-3-hydroxybutyryl-hydroxylase-ACP to create a double bond between the β- and γ-carbons to form crotonoyl-ACP (e.g., a β-hydroxyacyl-ACP dehydratase).

[0269] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can reduce crotonoyl-ACP with NADPH to form butyryl-ACP (e.g., an enoyl ACP reductase).

[0270] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that can hydrolyze a C16 acyl compound from the acyl carrier protein to form palmitic acid (e.g., a thioesterase).

[0271] In some embodiments, the fatty acid producing cell comprises one or more heterologous nucleotide sequences encoding an acetyl-CoA synthase and / or a malonyl-CoA synthase to achieve increased production of one or more fatty acids compared to a parent cell that has not been genetically modified.

[0272] For example, to increase acetyl-CoA production, one or more of the following genes can be expressed in the cell: pdh, panK, aceEF (encoding the Elp dehydrogenase module and E2p dihydrolipoylamine acyltransferase module of the pyruvate and 2-oxoglutarate dehydrogenase complex), fabH, fabD, fabG, acpP, and fabF. Illustrative examples of nucleotide sequences encoding such enzymes include, but are not limited to: pdh (BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC76952), aceEF (AAC73227, AAC73226), fabH (AAC74175), fabD (AAC74176), fabG (AAC74177), acpP (AAC74178), fabF (AAC74179).

[0273] In some embodiments, elevated fatty acid levels can be achieved in a cell by attenuating or knocking out a gene encoding a protein involved in fatty acid degradation. For example, the expression level of fadE, gpsA, idhA, pflb, adhE, pta, poxB, ackA, and / or ackB can be attenuated or knocked out in an engineered host cell using techniques known in the art. Exemplary examples of nucleotide sequences encoding such proteins include, but are not limited to: fadE (AAC73325), gspA (AAC76632), IdhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta (AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430). The resulting host cell will have elevated levels of acetyl-CoA production when grown in a suitable environment.

[0274] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding an enzyme that converts acetyl-CoA to malonyl-CoA, such as the multi-subunit AccAB(3) protein. An exemplary example of a suitable nucleotide sequence encoding AccAB(3) includes, but is not limited to, accession number AAC73296, EC 6.4.1.2.

[0275] In some embodiments, the fatty acid producing cell comprises a heterologous nucleotide sequence encoding a lipase. Exemplary examples of suitable nucleotide sequences encoding a lipase include, but are not limited to, accession numbers CAA89087 and CAA98876.

[0276] In some embodiments, elevated fatty acid levels can be achieved in a cell by inhibiting PlsB, which can result in elevated levels of long chain acyl-ACP, which inhibits early steps in the fatty acid biosynthetic pathway (e.g., accABCD, fabH, and fabl). The expression level of PlsB can be attenuated or knocked out in an engineered host cell using techniques known in the art. An exemplary example of a suitable nucleotide sequence encoding PlsB includes, but is not limited to, accession number AAC77011. In particular embodiments, a PlsB D3 1 IE mutation can be used to increase the amount of acyl-CoA available in the cell.

[0277] In some embodiments, elevated production of monounsaturated fatty acids can be achieved in a cell by overexpressing sfa, which results in fabA repression. An exemplary example of a suitable nucleotide sequence encoding sfa includes, but is not limited to, accession number AAN79592.

[0278] In some embodiments, elevated fatty acid levels in cells can be achieved by modulating the expression of enzymes that control the length of fatty acid substrate chains (e.g., thioesterases). In some embodiments, fatty acid-producing cells have been modified to overexpress the tes or fat gene. Exemplary examples of suitable tes nucleotide sequences include, but are not limited to, accession numbers: (tesA: AAC73596, from *E. coli*, capable of producing C18:1 fatty acids) and (tesB: AAC73555, from *E. coli*). Exemplary examples of suitable fat nucleotide sequences include, but are not limited to: (fatB: Q41635 and AAA34215, from California Laurel, capable of producing C18:1 fatty acids). 12：0 Fatty acids), (fatB2: Q39513 and AAC49269, from the calyx spur flower, capable of producing C 8：0 -C 10：0 Fatty acids), (fatB3: AAC49269 and AAC72881, from the calyx spur, capable of producing C 14：0 -C 16：0 Fatty acids), (fatB: Q39473 and AAC49151, derived from camphor (Cinnamonum camphorum), can produce C 14：0 Fatty acids), (fatB[M141T]: CAA85388, from Arabidopsis thaliana, capable of producing C 16：1 Fatty acids), (fatA: NP 189147 and NP 193041, from Arabidopsis thaliana, capable of producing C 18：1 Fatty acids), (fatA: CAC39106, derived from slow-growing soybean rhizobia, which preferentially produces C...) 18：1 Fatty acids), (fatA: AAC72883, from the calyx spur flower, capable of producing C 18：1 Fatty acids), and (fatA1, AAL79361 from sunflower).

[0279] In some embodiments, thioesterase C is weakened by using techniques known in the art. 18 The expression or activity of [a substance] can achieve elevated C [level] in cells. 10 Fatty acid levels. Encoding thioesterase C 18 Exemplary examples of suitable nucleotide sequences include, but are not limited to, accession numbers AAC73596 and P0ADA1. In other embodiments, thioesterase C is enhanced using techniques known in the art. 10 The expression or activity of [a substance] can achieve elevated C [level] in cells. 10 Fatty acid levels. Encoding thioesterase C 10 An exemplary example of a suitable nucleotide sequence includes, but is not limited to, accession number Q39513.

[0280] In some embodiments, elevated levels of C 14 fatty acids in the cell can be achieved by attenuating the expression or activity of endogenous thioesterases that produce non-C 14 fatty acids, using techniques known in the art. In other embodiments, elevated levels of C 14 fatty acids in the cell can be achieved by increasing the expression or activity of thioesterases that use substrate C 14 -ACP, using techniques known in the art. An exemplary example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to, accession number Q39473.

[0281] In some embodiments, elevated levels of C 12 fatty acids in the cell can be achieved by attenuating the expression or activity of endogenous thioesterases that produce non-C 12 fatty acids, using techniques known in the art. In other embodiments, elevated levels of C 12 fatty acids in the cell can be achieved by increasing the expression or activity of thioesterases that use substrate C 12 -ACP, using techniques known in the art. An exemplary example of a suitable nucleotide sequence encoding such a thioesterase includes, but is not limited to, accession number Q41635.

[0282] 6. Example

[0283] 6.1 Example 1

[0284] This example describes an exemplary method for determining the cell density (OD 600 ) of a yeast cell culture.

[0285] Combine 8 μL of cell culture sample with 92 μL of Triton OD diluent (20 g / L Triton X-114, 200 mL / L PEG 200, 200 mL / L 100% ethanol, remainder water) in a clear 96-well plate, shake the solution at 1,000 RPM for 6 minutes, and determine the OD 600 at 600 nm on a M5 spectrophotometer (Molecular Devices, Sunnyvale, CA).

[0286] 6.2 Example 2

[0287] This example describes an exemplary Nile Red-based method useful for determining the farnesene titer of a yeast cell culture.

[0288] Ninety-eight μL of the cell culture sample was transferred into a 96-well black polystyrene flat-bottom assay plate and 2 μL of Nile Red (Invitrogen, Carlsbad, CA) dissolved at 100 μg / mL in DMSO was added to each well. Fluorescence levels were immediately measured on a M5 spectrophotometer with 500 nm excitation and 550 nm emission.

[0289] 6.3 Example 3

[0290] This example describes an exemplary gas chromatography (GC)-based method useful for determining the farnesene titer of a yeast cell culture.

[0291] The sample was extracted with methanol-heptane (1 :1 v / v) and the mixture centrifuged to remove cellular material. An aliquot of the methanol-heptane extract was diluted in n-heptane containing 0.001% t-caryohyllene, which acts as a retention time marker for monitoring successful injection and elution during the prescribed GC oven profile, and then injected onto a methyl silicone stationary phase using pulsed split injection. Farnesene was separated by boiling point using a GC equipped with flame ionization detection (FID).

[0292] 6.4 Example 4

[0293] This example demonstrates the phenomenon of strain degeneration that occurs when non-catabolic compound production is "on" during both the build-up and production phases of a fermentation process.

[0294] A 1 ml vial containing a heterologous enzyme (including MEV pathway enzymes (KARI, IDI, and ERG 20) was thawed and 0.5 ml of the cell culture was added to the vial. Figure 1Fermentation of a yeast strain capable of producing both an isoprenoid (IPP isomerase, FPP synthase, and farnesene synthase) and an exemplary non-catabolic compound (farnesene) was performed as follows. Frozen cell suspension of the yeast strain was transferred into a 250-ml Erlenmeyer flask containing 50 ml of BSM 2.0 (containing 2% sucrose and 10 mg / L D-calcium pantothenate) and grown in a shaker at 34°C, 200 RPM for 24 hours. The entire culture was then transferred into a 2.8 L Fernbach flask containing 850 ml of BSM 2.0 (containing 2.0% sucrose and 10 mg / L D-calcium pantothenate) and grown in a shaker at 34°C, 250 RPM for 24 hours. The entire culture was then transferred into a 2 L fermenter. The nutrient feed to the fermenter was an undefined Brazilian sugar cane syrup medium containing 10 mg / L D-calcium pantothenate delivered as an initial pulse equivalent to 14 g / L / h of sugar. The feed rate was then self-regulated based on the carbon demand of the fermenter as indicated by the rise in dissolved oxygen. The fermentation was run microaerobically at a constant temperature of 34°C, constant pH of 4.5 (controlled by addition of sodium hydroxide), and an initial oxygen transfer rate of 200 mmol O2 / L / h until the dissolved oxygen reached 0%, and then reduced to 100 mmol O2 / L / h for the remainder of the fermentation. Every 3 days, the volume of the tank was reduced to approximately 0.9 L to prevent overflow. At that time, trace metals and vitamins lost in the sugar cane syrup feed were replenished. The amount of farnesene produced and total sugar consumed by the cells were monitored daily, the ratio of these two values (i.e., product yield / sugar) was determined for each 72 hour period, and plotted as shown in Figure 3 .

[0295] 6.5 Example 5

[0296] The results provided in this example demonstrate that a host cell capable of producing a isoprenoid (farnesene) and containing the MEV pathway under positive regulation of a microaerobically responsive promoter ("low O2 switch") produces very low amounts of farnesene in high O2 conditions (shaker plates), and substantially higher levels in low O2 conditions (low RPM shake flasks) that match the production of the non-switchable parental strain that constitutively expresses the MEV pathway. The results are depicted in Figure 6 .

[0297] Yeast strains producing farnesene:

[0298] A yeast strain derived from a wild-type Saccharomyces cerevisiae strain (CEN.PK2) and expressing the genes of the mevalonate pathway under the control of the GAL promoter (IPP isomerase, FPP synthase, and farnesene synthase) was fermented as follows. Frozen cell suspension of the yeast strain was transferred into a 250-ml Erlenmeyer flask containing 50 ml of BSM 2.0 (containing 2% sucrose and 10 mg / L D-calcium pantothenate) and grown in a shaker at 34°C, 200 RPM for 24 hours. The entire culture was then transferred into a 2.8 L Fernbach flask containing 850 ml of BSM 2.0 (containing 2.0% sucrose and 10 mg / L D-calcium pantothenate) and grown in a shaker at 34°C, 250 RPM for 24 hours. The entire culture was then transferred into a 2 L fermenter. The nutrient feed to the fermenter was an undefined Brazilian sugar cane syrup medium containing 10 mg / L D-calcium pantothenate delivered as an initial pulse equivalent to 14 g / L / h of sugar. The feed rate was then self-regulated based on the carbon demand of the fermenter as indicated by the rise in dissolved oxygen. The fermentation was run microaerobically at a constant temperature of 34°C, constant pH of 4.5 (controlled by addition of sodium hydroxide), and an initial oxygen transfer rate of 200 mmol O2 / L / h until the dissolved oxygen reached 0%, and then reduced to 100 mmol O2 / L / h for the remainder of the fermentation. Every 3 days, the volume of the tank was reduced to approximately 0.9 L to prevent overflow. At that time, trace metals and vitamins lost in the sugar cane syrup feed were replenished. The amount of farnesene produced and total sugar consumed by the cells were monitored daily, the ratio of these two values (i.e., product yield / sugar) was determined for each 72 hour period, and plotted as shown in Figure 1) as a constitutive farnesene producing control. This non-switchable strain contains the following chromosomally integrated under the control of the GAL promoter: the mevalonate pathway genes from S. cerevisiae: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate decarboxylase; and six copies of the farnesene synthase mutant from Artemisinin annua. This non-switchable strain has the gal80 gene deleted and has an extra copy of GAL4 under the GAL4oc promoter, where the coding sequence of the GAL4 gene from S. cerevisiae is under the control of the "operable constitutive" version of its native promoter (PGAL4oc; see, e.g., Griggs & Johnston (1991) PNAS 88(19):8597-8601).

[0299] Farnesene production in this "non-switchable" strain was then made "switchable," i.e., repressible under aerobic conditions. A low O2 switchable strain was constructed on top of this constitutive strain by replacing the promoter of the multiple copies of GAL4 with the DAN1#1 promoter (SEQ ID NO: 1). A first copy of pDAN1#1 driving GAL4 was introduced by replacing pGAL4oc with pDAN1#1. The native GAL4 promoter was then replaced with pDAN1#1. This strain is referred to in Figure 6 as "low O2 switchable strain #1." Starting from this strain, a third and fourth copy of pDAN1#1 driving GAL4 was simultaneously integrated at the GAS2 locus. This second switchable strain, which has four copies of GAL4 under the control of the DAN1#1 promoter, is referred to in Figure 7 as "low O2 switchable strain #2."

[0300] Modulation of non-dissimilated compound production by changing oxygen conditions:

[0301] Both the non-switchable farnesene producing control strain and the low O2 switchable strain #1 were cultured under aerobic and microaerobic conditions, respectively, to assess farnesene production under fermentation conditions intended to act as "off" and "on" states.

[0302] For "off" or "high O2 conditions," colonies of both strains were inoculated into 360 uL BSM 2.0, 2% sucrose in a 96-well plate. The plate was shaken at high RPM in an ATR shaker for 3 days. From this preculture plate, 6 uL of culture was transferred to a production 96-well plate containing 360 uL BSM 2.0, 4% sucrose. The plate was shaken at high RPM in an ATR shaker for 2 days. Farnesene concentrations were determined by GC-FID.

[0303] For "open" or "low 02conditions", 50 ml of BSM 2.0, 4% media was injected into a 125 ml flask and inoculated with 0.1 OD of each strain. 50 ml is a common large media volume for 125 ml flask size and this also helps to reduce the oxygen transfer rate (OTR) into the flask to ensure the cultures become microaerophilic. Dissolved oxygen was measured via a probe and confirmed that both strains reached undetectable levels of dissolved oxygen within 24 hours. The concentration of famesene was determined by GC-FID after 120 hours. At 120 hours, the dissolved oxygen of both cultures was approximately the expected value for gas phase equilibrium, suggesting carbon depletion.

[0304] As shown in Figure 6 , low 02switchable strain #1 produced little famesene under high 02conditions compared to the constitutive non-switchable strain that produced high levels of famesene. Famesene production in low 02switchable strain #1 was induced higher in low 02conditions and exceeded the famesene production of the constitutive non-switchable strain. These results demonstrate that oxygen manipulation can be used to achieve tight "off and "on states for low 02switchable famesene producing strains.

[0305] 6.6 Example 6

[0306] The results provided in this example demonstrate that host cells that produce isoprenoids famesene and contain the MEV pathway under the control of a low 02switch positive regulator exhibit improved famesene production stability in long fermentation runs when the build phase of the fermentation is performed under aerobic conditions (thereby achieving an "off state) compared to production of a constitutive production strain that produces famesene throughout the build phase. The results are depicted in Figure 7 .

[0307] For both the non-switchable constitutive farnesene producing strain and the low O2 switchable strain #2, 1 ml vials of frozen cell suspension were thawed and transferred into 250-ml Erlenmeyer flasks containing 50 ml of BSM 3.0 (with 1.6% sucrose, 0.4% glucose as carbon source) and grown in a shaker at 34°C, 250 RPM for 24 hours. Then 2 ml was transferred from the flask into 50 ml of BSM 3.0 with 1.6% sucrose, 0.4% glucose as carbon source and grown in a shaker at 34°C, 250 RPM for 24 hours. Then 25 ml was transferred into an inoculum flask containing 225 ml of tank medium and transferred to a 0.5 L fermentor. The fermentor was fed with 650 g / L sucrose solution at an initial pulse equivalent to 10 g / L / h sugar. Then the feed rate was self-regulated based on the fermentor's demand for carbon as indicated by the dissolved oxygen rising. Once the dissolved oxygen reached 0%, the fermentation was run microaerobically at a constant temperature of 34°C, constant pH of 4.5 (controlled by addition of sodium hydroxide), and a maximum oxygen transfer rate of 110 mmol O2 / L / h. Daily, the volume of the tank was reduced to about 0.29 L to prevent overflow. At that time trace metals and vitamins were replenished. The total amount of farnesene produced and total sugar consumed were updated daily, the ratio of these two values (i.e. cumulative product yield / sugar) was determined for the interval from time=0 to time=t, and plotted as shown in Figure 7 Figure 6. The cumulative product yield of the non-switchable parent strain declined from its peak at 160 hours to about 83% of the peak yield of the switchable descendant strain at 300 hours. In comparison, the low O2 switchable strain #2 maintained its cumulative yield at >95% of its peak from 110 hours to 300 hours. Thus, these results demonstrate that a low O2 switch that turns off farnesene production under aerobic conditions during the build-up phase of a two-stage fermentation process results in increased production stability of farnesene production during the production phase.

[0308] 6.7 Example 7

[0309] This example provides results that demonstrate that a host cell capable of producing isoprenoid farnesene and comprising the MEV pathway under negative regulation by a maltose-responsive promoter ("maltose switch") produces very low amounts of farnesene in the presence of maltose (1.3%), and in the absence of maltose, produces a substantial increase to levels of production close to that of the non-switchable parent strain that constitutively expresses the MEV pathway. The results are depicted in Figure 8 .

[0310] Yeast strains producing farnesene:

[0311] A yeast strain capable of producing farnesene was used that was derived from a wild-type Saccharomyces cerevisiae strain (CEN.PK2) and expresses the mevalonate pathway genes (ERG 13, ERG 19, ERG 8, ERG 7, ERG 9, and ERG 10) under the control of a GAL promoter. Figure 1) as a constitutive farnesene-producing control. This non-switchable strain contains the following chromosomally integrated under the control of the GAL promoter: the mevalonate pathway genes from S. cerevisiae: acetyl-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate pyrophosphate decarboxylase; and six copies of the farnesene synthase mutant from A. cruenta. This non-switchable strain has the gal80 gene deleted and has an extra copy of GAL4 under the GAL4oc promoter, where the coding sequence of the GAL4 gene from S. cerevisiae is under the control of a "promoter-competent constitutive" version of its native promoter (PGAL4oc; see, e.g., Griggs & Johnston (1991) PNAS 88(19):8597-8601).

[0312] The famesene production in this "non-switchable" strain was then made "switchable," i.e., repressible in the presence of maltose. A copy of GAL80 was chromosomally integrated under the control of the maltose-responsive promoter pMAL32 (SEQ ID NO: 17), to create a maltose-switchable strain on top of the constitutive strain.

[0313] Non-dissimilated compound production was regulated by varying the maltose in the medium:

[0314] Both the non-switchable famesene-producing control strain and the maltose-switchable strain were cultured in media with and without maltose, respectively, to assess famesene production under fermentation conditions intended to act as "off" and "on" states.

[0315] For pre-culture conditions, both the non-switchable famesene-producing control strain and the maltose-switchable strain were cultured in sterile 96-well plates (1.1 ml working volume; Axygen) containing 360 ul Bird Seed Medium (BSM, originally described in van Hoek et al. (2000)) at 33.5°C, 80% humidity, and 1000 rpm (Infors Multitron; ATR Biotec) for approximately 72 hours. For famesene production experiments, the saturated cultures described above were diluted 1 / 25 in sterile 1.1 ml plates containing 145 μΙ_ BSM and 5 μΙ_ mineral oil. The carbon source was either 4% sucrose or a mixture of 2.7% sucrose and 1.3% maltose. After 72 hours of incubation, famesene extraction was performed by adding 600 μΙ_ isopropyl alcohol (IPA) to each well. After 30 minutes of incubation, 8 μΙ_ was transferred to a clear-bottom assay plate containing 192 μΙ_ IPA. Famesene concentration was measured by UV absorbance at 222 nm on a SpectraMax plate reader.

[0316] AsFigure 8 As shown, compared to constitutively non-switchable strains that produce high levels of farnesene, maltose-switchable strains produce very little farnesene in the presence of maltose. In the absence of maltose, farnesene production induction in maltose-switchable strains is higher and close to that in constitutively non-switchable strains. These results demonstrate that maltose manipulation can be used to achieve a tight "off" and "on" state for maltose-switchable, farnesene-producing strains.

[0317] 6.8 Example 8

[0318] The results provided in this example demonstrate that, compared to parental strains that constitutively produce farnesene, host cells capable of producing isoprene-like farnesenes and comprising the MEV pathway under positive regulation by a microaerophilic responsive promoter (“low O2 switch”) or negative regulation by a maltose-responsive promoter (“maltose switch”) exhibit improved growth rates during the compound production “off” state. The results are depicted in… Figure 9 .

[0319] For growth rate experiments, saturated cultures of low-O2 switchable strain #1, maltose switchable strain, and non-switchable constitutive farnesene-producing strain were diluted 1 / 25 in sterile 1.1 ml plates containing 360 μL of fresh defined-component medium (containing 3% (w / v) sucrose or a mixture of 2% sucrose and 1% maltose). OD was measured over an 8-hour period (2.5, 3.5, 6, and 8 hours) immediately following transfer to fresh medium. 600 Growth rates were calculated using a SpectraMax M5 plate reader (Molecular Devices). To eliminate any contribution of the farnesene emulsion to the OD signal, the culture was diluted in a solution of 20% (v / v) PEG20, 20% (v / v) ethanol, and 2% (v / v) Triton X-114. Growth rates were determined by applying linear regression to LN(OD) vs time.

[0320] like Figure 9 As shown, the maltose-on switchable strain exhibited improved growth in the "off" state (i.e., in the presence of maltose) compared to its "on" state, and showed significantly improved growth (143% vs. 100% relative growth rate) compared to the non-on constitutive farnesene-producing strain. Similarly, the low-O2 on switchable strain #1 showed significantly improved growth (167% vs. 100% relative growth rate) in the "off" state (i.e., under aerobic conditions) compared to the non-on constitutive farnesene-producing strain.

[0321] 6.9 Example 9

[0322] The results provided by this example demonstrate that host cells capable of producing isoprenoids, famesene, and comprising the MEV pathway under negative regulation by a maltose switch exhibit improved stability of famesene production in long fermentation runs when the build phase of the fermentation is performed in the presence of maltose (thereby achieving an "off state) as compared to the production of constitutive producing strains that produce famesene throughout the build phase. The results are depicted in Figure 10 .

[0323] Both the non-switchable famesene production control strain and the maltose switchable strain were first streaked on solid agar medium containing 2% dextrose and 1% maltose and grown at 30°C until colonies were visible. Seed flasks were prepared by inoculating single colonies into 15 ml tubes containing 3 ml of BSM 2% sucrose 1% maltose. After approximately 48 hours, all 3 ml was transferred into a 500 ml disposable shake flask containing 125 ml of 2% sucrose and 1% maltose BSM (seed flask medium). Cells were grown in a shaker at 30°C at 200 rpm until an OD600 between 4 and 7 was reached. Once the desired OD was reached, 36 ml of sterile 50% glycerol stock was added to 84 ml of culture, the suspension was aliquoted into seed flasks, and the seed flasks were slowly frozen to -80°C at a rate of approximately 1°C / min. Biomass build was achieved prior to fermentation by thawing one or more seed flasks in a 250 mL shake flask containing 50 mL of 2% sucrose and 1% maltose BSM (biomass build medium) and by growing the culture for 24 hours at 34°C and 200 RPM. A portion of this culture was then transferred into a 500 ml flask containing 100 ml of the same medium to achieve a starting OD600 of 0.1 and grown for an additional 24 hours. This 25 ml of culture was then used to inoculate a 0.5 L fermentor containing 225 ml of BSM medium (without any sugar). Cane sugar syrup (without any maltose) was dosed as needed and the fermentation was run for 13 days following a dosing regimen that maximized famesene yield.

[0324] The total amount of famesene produced and the total sugar consumed by the cells was updated daily, the ratio of these two values was determined for the interval from time=0 to time=t, and plotted as the normalized fermenter interval yield as shown in Figure 10 The normalized interval yield of the non-switchable parent strain continued to decline from its peak at 120 hours to approximately 20% below the peak yield of the switchable descendant strain at 300 hours. In comparison, the maltose switchable strain maintained the normalized interval yield at approximately 50% of its peak from 72 hours to 120 hours. Thus, these results demonstrate that the maltose switch resulting in the shut-off of famesene production in the presence of maltose during the build phase of a two-stage fermentation process results in increased production stability of famesene production during the production phase.

[0325] 6.10 Example 10

[0326] The results provided by this example demonstrate the sensitivity of several maltose-sensitive promoters described herein to different amounts of maltose in the culture medium and to mixed feedings, as well as the switchability of going from an "off state to an "on state after repression by maltose in the "off state. The results are depicted in Figures 11-14 .

[0327] For each of the maltose-responsive promoters pMALl l (SEQ ID NO: 14), pMAL12 (SEQ ID NO: 15), pMAL31 (SEQ ID NO: 16), and pMAL32 (SEQ ID NO: 17), two different reporter strains derived from the wild-type S. cerevisiae strain (CEN.PK2) were created by integrating at the ATG20 locus the following reporter constructs: (i) pMAL>GFP, a GFP-encoding sequence operably linked to a maltose-sensitive promoter, and (ii) pMAL>GAL80; PGAL1>GFP, a construct containing a GFP expression cassette operably linked to the GAL1 promoter; and a GAL80-encoding sequence operably linked to a maltose-sensitive promoter.

[0328] For pMAL>GFP and the switch strains with pGAL1>GFP, pre-cultures were diluted 50-fold in fresh culture medium containing the indicated mixtures of glucose and maltose, or sucrose and maltose. After an additional 24 hours of incubation, cultures were diluted in PBS solution to a final cell density ranging from 300-1000 cells / uL, and sorted on a Guava. Cells were first sorted by forward and side scatter to distinguish intact yeast cells and gate off much smaller debris and fine particles. Green fluorescent cells were identified using the output from the isogenic non-GFP expressing control strain as the non-fluorescent background signal. Bar graphs were made using Flowjo software.

[0329] As shown in Figures 11-14 (A), each of pMALl l, pMAL12, pMAL31, and pMAL32 was strongly activated by as little as 0.5% maltose, even when mixed with glucose or sucrose. In addition, as shown in Figures 11-14 (B), each of pMALl l, pMAL12, pMAL31, and pMAL32 was able to maintain a strong off state when wired as a "maltose on" switch (left panel), or a strong on state when wired as a "maltose off switch (right panel) when the host strain was subsequently cultured in media without maltose (4% sucrose).

[0330] All publications, patents, and patent applications cited in this specification are incorporated by reference herein as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Various modifications and changes can be made to the disclosed embodiments without departing from the scope and spirit of the disclosure. Although the present disclosure has been described in some detail for the purpose of clarity and understanding, one of ordinary skill in the art will readily appreciate that various changes can be made without departing from the spirit or scope of the disclosure. There are many alternate ways of implementing the processes, compositions, and methods described herein. The scope of the present disclosure should not be limited by the specific illustrative structures, methods, implementations, and implementations disclosed, but only by the structures, methods, implementations and implementation claims provided below.

Claims

1. A method for producing a heterologous non-dissimilar compound in genetically modified host cells, the method comprising: (a) A population of genetically modified host cells cultured in a medium containing a carbon source of maltose, wherein the host cells contain one or more heterologous nucleic acids encoding one or more enzymes of an enzymatic pathway for generating the heterologous nondissimilatory compound, wherein the expression of the one or more enzymes is negatively regulated by the activity of a maltose-responsive promoter, wherein the presence of maltose in the medium limits the amount of heterologous nondissimilatory compound produced by the host cells; and (b) The population or a subpopulation thereof is cultured in a medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount such that the maltose-responsive promoter is no longer active and the production of heterologous nondissimilatory compounds by the host cells is increased. The maltose-responsive promoter is operatively linked to a heteronucleotide encoding a transcriptional regulatory factor, which negatively regulates the expression of one or more heteronucleotides encoding one or more enzymes in the enzyme pathway, and the maltose in step (a) increases the expression of the transcriptional regulatory factor. The transcriptional regulatory factor is Gal80p, the host cell also contains Gal4p, and one or more heterologous nucleic acids encoding one or more enzymes of the enzyme pathway are each operatively linked to a Gal4p-responsive promoter.

2. The method of claim 1, wherein the Gal4p response promoter is selected from the group consisting of pGAL1, pGAL7 and pGAL10.

3. A method for producing heteroprene-like compounds in genetically modified host cells, the method comprising: (a) A population of genetically modified host cells cultured in a medium containing a carbon source containing maltose, wherein the host cells comprise: (i) One or more heteronucleotides encoding one or more enzymes of the mevalonate (MEV) pathway, each of the one or more heteronucleotides being operatively linked to a Gal4p-responsive promoter selected from the group consisting of pGAL1, pGAL7 and pGAL10. (ii) Nucleic acid encoding Gal4p; and (iii) A nucleic acid encoding Gal80p, said nucleic acid being operatively linked to a maltose-responsive promoter; wherein maltose in the culture medium limits the amount of heteroprene produced by said host cells; and (b) The population or its subpopulation is cultured in a medium containing a carbon source, wherein maltose is absent or in a sufficiently low amount such that the maltose-responsive promoter is no longer active and the production of heterologous non-dissimilatory compounds by the host cell is increased.

4. The method of any one of claims 1 to 3, wherein the maltose-responsive promoter comprises a sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:

17.

5. The method of claim 4, wherein the maltose-responsive promoter sequence comprises SEQ ID NO:

17.

6. The method of claim 1, wherein the culture medium in step (a) contains at least 0.1% (w / v) maltose.

7. The method of claim 1, wherein the culture medium in step (a) comprises 0.25%-3% (w / v) maltose.

8. The method of claim 1, wherein the culture medium in step (b) contains no more than 0.08% (w / v) maltose.

9. The method of claim 1, wherein the production of heterologous non-dissimilatory compounds by the population of the genetically modified host cells during the culture in step (b) is improved compared to the production of heterologous non-dissimilatory compounds achieved in a fermentation method in which the expression of one or more enzymes of an enzymatic pathway is not limited by the activity of the maltose-responsive promoter.

10. The method of claim 1, wherein the generation of the non-dissimilating compound during step (a) is less than 50%, 40%, 30%, 20%, or 10% of the generation of the non-dissimilating compound during step (b).

11. The method of claim 1, wherein the culture in step (a) is for a period of at least 12, 24, 36, 48, 60, 72, 84, 96 or more than 96 hours.

12. The method of claim 1, wherein the culture in step (a) is sufficient to bring the population to a cell density (OD) of 0.01-400. 600 (The time period) 13. The method of claim 1, wherein the culture in step (b) is for a period of 3 to 20 days.

14. The method of claim 1, wherein the host cell is selected from the group consisting of fungal cells, bacterial cells, plant cells and animal cells.

15. The method of claim 1, wherein the host cell is a yeast cell.

16. The method of claim 1, wherein the generation of the non-dissimilar compound is measured based on yield or productivity.

17. The method of claim 1, wherein the method further comprises recovering the non-dissimilar compound.

18. The method of claim 1, wherein the non-dissimilating compound is selected from the group consisting of amino acids, fatty acids, isoprene-like compounds, and polyketides.

19. The method of claim 1, wherein the host cell is capable of producing isoprene-like substances and comprises at least one heteronucleotide encoding an enzyme of the isoprene-like pathway, wherein the enzyme of the isoprene-like pathway is selected from the group consisting of: (i) An enzyme that condenses two molecules of acetyl-CoA to form acetyl-CoA; (ii) An enzyme that condenses acetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA; (iii) An enzyme that converts HMG-CoA into mevalonic acid; (iv) An enzyme that converts mevalonic acid to mevalonic acid 5-phosphate; (v) An enzyme that converts mevalonate 5-phosphate to mevalonate 5-pyrophosphate; (vi) An enzyme that converts mevalonate 5-pyrophosphate into IPP; (vii) An enzyme that converts IPP into DMAPP; (viii) Polyisoprene synthases capable of condensing IPP and / or DMAPP molecules to form polyisoprene compounds containing more than five carbons; (ix) An enzyme that condenses IPP and DMAPP to form GPP; (x) An enzyme that condenses two molecules of IPP and one molecule of DMAPP; (xi) An enzyme that condenses IPP and GPP to form FPP; (xii) An enzyme that condenses IPP and DMAPP to form GGPP; and (xiii) An enzyme that condenses IPP and FPP to form GGPP.

20. The method of claim 19, wherein the host cell further comprises a heterologous nucleic acid encoding an enzyme that modifies polyisoprene, said enzyme being selected from the group consisting of: geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, juniperene synthase, γ-terpinene synthase, terpinene oil synthase, amorpha diene synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouli alcohol synthase, nocarbamate synthase, and rosindiene synthase.

21. The method of claim 19, wherein the host cell comprises a variety of heterologous nucleic acids encoding all enzymes of the mevalonate pathway.

22. The method of claim 19, wherein the isoprene is selected from the group consisting of: hemiterpenes, monoterpenes, diterpenes, triterpenes, tetraterpenes and polyterpenes.

23. The method of claim 19, wherein the isoprene is C5-C. 20 Isoprene.

24. The method of claim 19, wherein the isoprene is a sesquiterpene.

25. The method of claim 19, wherein the isoprene is selected from the group consisting of: rosin diene, azadirachtin, carene, α-farnesene, β-farnesene, farnesol, geraniol, geraniylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchouli alcohol, β-pinene, sabinene, γ-terpinene, terpinene oil, and sesquiterpenes.

26. The method of claim 1, wherein the host cell is capable of producing polyketides and comprises at least one heteronucleotide encoding a polyketide synthase, wherein the polyketide synthase is selected from the group consisting of: (i) An enzyme that condenses at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein; (ii) An enzyme that condenses a first reactant selected from the group consisting of acetyl-CoA and malonyl-CoA with a second reactant selected from malonyl-CoA or methylmalonyl-CoA to form a polyketide product. (iii) An enzyme that reduces β-keto chemical groups on polyketide compounds to β-hydroxy groups; (iv) An enzyme that dehydrogenates alkane chemical groups in polyketides to produce α-β-unsaturated olefins; (v) An enzyme that reduces the α-β-double bond in polyketide compounds to saturated alkanes; and (vi) Enzymes that hydrolyze polyketide compounds from acyl carrier proteins.

27. The method of claim 26, wherein the polyketide is a lipid having at least one of antibacterial, antifungal, and antitumor activities.

28. The method of claim 26, wherein the polyketide is selected from the group consisting of macrolides, antibiotics, antifungals, cell growth inhibitors, anticholesterol compounds, antiparasitic compounds, anticoccidial compounds, animal growth promoters, and insecticides.

29. The method of claim 1, wherein the host cell is capable of producing fatty acids and comprises at least one heteronucleotide encoding a fatty acid synthase, wherein the fatty acid synthase is selected from the group consisting of: (i) An enzyme that covalently links at least one of acetyl-CoA and malonyl-CoA to an acyl carrier protein (ACP); (ii) An enzyme that condenses acetyl-ACP and malonyl-ACP to form acetoacetyl-ACP; (iii) Reduce the double bond in acetoacetyl-ACP with NADPH to form the hydroxyl group in D-3-hydroxybutyryl hydroxylase-ACP; (iv) Dehydrate D-3-hydroxybutyryl hydroxylase-ACP to produce a double bond between the β-carbon and γ-carbon of crotonyl-ACP; (v) An enzyme that reduces crotonyl-ACP with NADPH to form butyryl-ACP; and (vi) An enzyme that hydrolyzes C16 acyl compounds from acyl carrier proteins to form palmitic acid.

30. The method of claim 29, wherein the fatty acid is selected from the group consisting of: palmitic acid, palmitoyl-CoA, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

31. A fermentation composition comprising a population of genetically modified yeast host cells in a culture medium containing a carbon source, wherein the yeast host cells contain (a) One or more heteronucleotides, said heteronucleotides encoding one or more enzymes of an enzymatic pathway for the generation of heterologous non-dissimilatory compounds, wherein the expression of said one or more enzymes is positively regulated by the activity of a maltose-responsive promoter, and (b) Gal4p, wherein the maltose-responsive promoter is operatively linked to a heterologous nucleic acid encoding the product of the Gal80p gene and one or more enzymes of the enzyme pathway are each operatively linked to the Gal4p-responsive promoter, wherein the Gal4p-responsive promoter is selected from the group consisting of pGAL1, pGAL7 and pGAL10.

32. A fermentation composition comprising a population of genetically modified host cells in a culture medium containing a carbon source, wherein the host cells comprise: (a) One or more heteronucleotides encoding one or more enzymes of the mevalonate (MEV) pathway, each of the one or more heteronucleotides being operatively linked to a Gal4p-responsive promoter; (b) Nucleic acid encoding Gal4p; and (c) A nucleic acid encoding Gal80p, said nucleic acid being operatively linked to a maltose-responsive promoter.

33. The fermentation composition of claim 31, wherein the maltose-responsive promoter comprises a sequence selected from the group consisting of: SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO:

17.

34. The fermentation composition of claim 31, wherein the sequence of the maltose-responsive promoter comprises SEQ ID NO:

17.

35. The fermentation composition of claim 31, wherein the culture medium contains at least 0.1% (w / v) maltose.

36. The fermentation composition of claim 31, wherein the culture medium comprises 0.25%-3% (w / v) maltose.

37. The fermentation composition of claim 31, wherein the culture medium contains no more than 0.08% maltose.

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