maltose-dependent degron, maltose-responsive promoter, stabilized constructs and their use in the production of non-catabolic compounds

Abstract

The present invention relates to the use of a maltose-dependent degradation determinant for controlling the stability of a protein of interest fused thereto at the post-translational level. The present invention also relates to the use of a maltose-dependent degradation determinant in combination with a maltose-responsive promoter for controlling gene expression at the transcriptional level and protein stability at the post-translational level. The present invention also relates to the use of a stabilizing construct for coupling the expression of a protein affecting cell growth with the production of a non-catabolic compound. The present invention also relates to the use of synthetic maltose-responsive promoters. The present invention further provides compositions and methods for the production of non-catabolic compounds in a genetically modified host cell using a maltose-dependent degradation determinant, a maltose-responsive promoter, and a stabilizing construct, alone or in various combinations.

Description

[0001] 1. Cross-references to related applications

[0002] This application claims and enjoys priority to U.S. Provisional Patent Application No. 62 / 184,793, filed June 25, 2015, and U.S. Provisional Patent Application No. 62 / 266,436, filed December 11, 2015, both of which are incorporated herein by reference. Invention Field

[0003] This invention generally relates to maltose-dependent degradation determinants, maltose-responsive promoters, and stabilization constructs. It also relates to their use in controlling gene expression, protein stability, and the generation of non-catabolite compounds through genetically modified host cells. 3. Background of the Invention

[0005] The emergence of synthetic biology has brought the hope of using renewable resources to ferment biofuels, chemicals, and biomaterials on an industrial scale and with high quality. For example, functional non-natural biological pathways have been successfully constructed in microbial hosts for the production of precursors to the antimalarial drug artemisinin (see, for example, Martin et al., Nat Biotechnol 21: 796-802 (2003)); fatty acid-derived fuels and chemicals (e.g., fatty esters, fatty alcohols, and waxes; see, for example, Steen et al., Nature 463: 559-562 (2010)); polyketide synthases for the preparation of cholesterol-lowering drugs (see, for example, Ma et al., Science 326: 589-592 (2009)); and polyketide compounds (see, for example, Kodumal, Proc Natl Acad Sci USA 101: 15573-15578 (2004)). However, the commercial success of synthetic biology will largely depend on whether the production cost of renewable products is competitive with or better than that of their corresponding non-renewable products.

[0006] Strain stability is likely a major driver of industrial fermentation costs because it affects the duration for which continuous fermentation can operate effectively. Strain stability generally refers to the ability of microorganisms to maintain favorable characteristics for the production of non-catabolite fermentation products (e.g., high yield (grams of compound obtained per gram of substrate) and productivity (grams obtained per liter of fermentation broth per hour) over extended culture periods. Genetic stability, in particular, refers to the tendency of the generating microbial community to maintain relatively stable expected allele frequencies of genes associated with product production over time; this genetic stability plays a major role in the sustained production of the product.

[0007] For non-catabolite fermentation of products other than biomass (which, by definition, consume metabolic energy and carbon sources to prevent those carbon sources from being used to generate more cells), the basis of instability is twofold: evolutionary mutation and selectivity. First, loss-of-product mutations arise spontaneously and randomly. Second, the growth rate or "adaptive" advantage of cells with decreased product yield leads to low-productivity cells impacting the final cell population, thus reducing overall culture performance. This phenomenon can be termed "strain degradation."

[0008] Brazilian fuel ethanol achieves very high ethanol yields from sugar during long fermentation processes, approximately 90% of the maximum theoretical yield. This is partly due to the ethanol produced through catabolism: each molecule of sugar generates 2 molecules of ATP (adenosine triphosphate), and redox equilibrium is reached in the absence of oxygen. Mutants that do not produce ethanol are less suited to the low-oxygen conditions of the fermenter and do not affect the cell population. This allows industrial ethanol fermentation to reuse most of the yeast biomass throughout the cycle, resulting in minimal sugar conversion to yeast cell biomass and directing almost all sugar towards ethanol production. This expanded propagation and reuse of biomass improves the efficiency of ethanol production: operating expenses are reduced because less sugar is converted to biomass in each cycle (i.e., increased yield); and capital expenditures are reduced because fewer and smaller fermenters are needed to build the biomass for inoculation.

[0009] In contrast, the production of acetyl-CoA-derived hydrocarbons (e.g., isoprene, fatty acids, and polyketides) is generally non-catabolic in nature; they typically require a net input of ATP (adenosine triphosphate), NADPH (nicotinamide adenine dinucleotide phosphate), and a carbon source, and usually require large amounts of oxygen to help balance the redox system. This environment encourages the evolution of products towards lower-level products, with higher biomass production being more advantageous for the genotype and leading to higher strain degradation rates.

[0010] One way to reduce the negative selection pressure of generating non-catabolite products is to block their formation during processes that generate unwanted products, such as during fermentation phases where biomass must be generated to maximize fermenter productivity. Therefore, there is a need in the art for on / off switches that can control the timing of non-catabolite compound generation during fermentation. There is also a need in the art for methods and compositions that reduce strain degradation rates and stabilize the generation of non-catabolite compounds during fermentation. The compositions and methods provided by this invention meet these needs, and they can be used for applications beyond the fermentation environment. Invention Abstract

[0011] This invention provides compositions and methods for controlling the stability of any protein fused with a maltose-dependent degron. In one embodiment, the maltose-dependent degron is obtained by modifying a protein known to bind to maltose (e.g., MBP or maltose-binding protein) that becomes unstable when not bound to maltose. Therefore, the maltose-dependent degron provided by this invention is stable depending on its binding to its ligand (e.g., maltose). In some embodiments, the maltose-dependent degron can be used in combination with a maltose-responsive promoter to simultaneously control the timing of protein expression and stability. For example, the expression of enzymes in biosynthetic pathways for the production of non-catabolite compounds can be directly or indirectly controlled by manipulating the maltose content in the culture medium using the maltose-dependent degron and the maltose-responsive promoter. Therefore, in some embodiments, the same molecular effector (e.g., maltose) can be used to provide post-transcriptional and post-translational control of gene expression. The present invention also provides compositions and methods utilizing stabilizing constructs that provide favorable growth conditions for original strains producing high product yields and unfavorable growth conditions for spontaneously mutant cells that have become low-yielding or non-yielding. As a result, the original strains producing high product yields overgrow beyond the mutant cells and stabilize the production of the desired product (e.g., non-catabolite compounds) during fermentation. In the compositions and methods provided by the present invention, the maltose-dependent degradation determinant, the maltose-responsive promoter, and the stabilizing construct can be used alone, in combination, or in combination thereof.

[0012] On one hand, the present invention provides compositions and methods for regulating the stability of any target protein using a maltose-dependent degradation determinant as a switch. In specific embodiments, the maltose-dependent degradation determinant is fused to a target protein that influences the generation of desired products (such as non-catabolite compounds) in genetically modified host cells. In some embodiments, one or more mutations are introduced into a maltose-binding protein (MBP) to convert it into a maltose-dependent degradation determinant (also called a maltose-binding degradation determinant), which is more stable when bound to maltose than when not bound to maltose. In the absence of maltose, both the maltose-dependent degradation determinant and any protein fused to it are unstable or inactive, leading to faster degradation of the fused protein. Therefore, the stability of any target protein fused to the maltose-dependent degradation determinant can be controlled by manipulating the maltose content.

[0013] In some embodiments, the stability of the fusion protein in the host cell can have downstream effects on target molecules, such as enzymes in biosynthetic pathways that generate non-catabolite compounds during fermentation. For example, if the fusion protein contains a transcriptional regulator fused to a maltose-dependent degradation determinant frame, its stability can be controlled by adding or removing maltose from the culture medium. In such embodiments, the maltose-dependent degradation determinant and the maltose content in the culture medium can act as a switch to control the generation of non-catabolite compounds. Simultaneously, the maltose-dependent degradation determinant is useful during fermentation; it can be used in any environment where the stability of the target protein and / or the generation of target molecules needs to be regulated.

[0014] Therefore, in one aspect, the present invention provides a method for regulating protein stability, wherein the method includes contacting maltose with a fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame. In some embodiments, the fusion protein is more stable when the maltose-dependent degradation determinant is contacted with maltose compared to when the maltose-dependent degradation determinant is not contacted with maltose. In some embodiments, the method further comprises removing maltose from contact with the fusion protein. In some embodiments, the half-life of the fusion protein after removing maltose from contact with the fusion protein is at least about 50% shorter than the half-life of a fusion protein comprising a wild-type maltose-binding protein fused within the target protein frame.

[0015] In some embodiments, the method further includes providing a host cell containing a heterologous nucleic acid encoding a fusion protein containing a maltose-dependent degradation determinant fused to a target protein frame, and culturing the host cell expressing the fusion protein in a maltose-containing medium.

[0016] In some embodiments, the fusion protein directly or indirectly regulates the levels of one or more target molecules. In some embodiments, the host cell further comprises a biomolecule that interacts with the fusion protein in the host cell to regulate the levels of the one or more target molecules. In some embodiments, the target protein is a transcriptional regulator. In some embodiments, the biomolecule is a transcriptional regulator.

[0017] In some embodiments, the method includes providing a host cell, the host cell further comprising one or more heterologous nucleic acids encoding one or more target molecules, the levels of which are regulated by a target protein fused to the maltose-dependent degradation determinant frame. In some embodiments, the one or more target molecules are enzymes in a biosynthetic pathway that generate one or more non-catabolite compounds. In some embodiments, the one or more target molecules are non-catabolite compounds generated by the enzymes in the biosynthetic pathway. In some embodiments, the target protein fused to the maltose-dependent degradation determinant frame comprises Gal80p. In some embodiments, the heterologous nucleic acid sequence encoding the fusion protein is integrated into the genome of the host cell.

[0018] In some embodiments, the method of regulating protein stability includes providing a host cell containing a heterologous nucleic acid encoding the fusion protein operatively linked to a maltose-responsive promoter. In some embodiments, the method of regulating protein stability includes providing a host cell containing a heterologous nucleic acid encoding the fusion protein, the fusion protein being operatively linked to an endogenous promoter of a gene encoding the target protein.

[0019] In some embodiments, methods for modulating protein stability include providing a host cell containing a heterologous nucleic acid encoding a fusion protein, said fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame, wherein said maltose-dependent degradation determinant comprises an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 28, and comprising one or more variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, methods for modulating protein stability may employ any of the maltose-dependent degradation determinants described herein.

[0020] In another aspect of the invention, in the methods and compositions provided by the invention, a maltose-responsive promoter is used in combination with a maltose-dependent degradation determinant. More specifically, the maltose-responsive promoter can be operatively linked to a nucleic acid encoding a fusion protein comprising a target protein fused within the frame of a maltose-dependent degradation determinant. By combining maltose-dependent transcriptional control with post-translational control, the compositions and methods provided by the invention can impose very robust and stringent control over the expression and stability of any gene product.

[0021] Therefore, the method provided by the present invention comprises: (a) culturing a genetically modified host cell population in a medium containing a carbon source, the carbon source comprising maltose, wherein the genetically modified host cells contain a maltose-responsive promoter operatively linked to a heterologous nucleic acid encoding a fusion protein, the fusion protein containing a target protein fused within a maltose-dependent degradation determinant frame; and (b) culturing the host cell population or a subset thereof in a medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount compared to the medium in step (a). In some embodiments, during step (a), transcription of the heterologous nucleic acid is activated in the presence of maltose, and the fusion protein encoded therefrom is stabilized. When maltose is absent or present in a sufficiently low amount during step (b), the activity of the maltose-responsive promoter and the stability of the fusion protein are reduced compared to step (a). The timing of these two culture phases of the method can be controlled, for example, by adding or removing maltose from the medium.

[0022] In some embodiments, the host cell further comprises a biomolecule that interacts with the fusion protein in the host cell to regulate the levels of one or more target molecules. In some embodiments, the target protein is a transcriptional regulator. In some embodiments, the biomolecule is a transcriptional activator. In some embodiments, the host cell further comprises a heterologous nucleic acid encoding one or more target molecules, the levels of which are regulated by the target protein. In some embodiments, one or more target molecules are enzymes of a biosynthetic pathway, the enzymes of which are positively regulated by the maltose-responsive promoter and the activity of the fusion protein, the fusion protein being stable in the presence of maltose. In some embodiments, one or more target molecules are enzymes of a biosynthetic pathway, the enzymes of which are negatively regulated by the maltose-responsive promoter and the activity of the fusion protein, the fusion protein being stable in the presence of maltose.

[0023] In some implementations, the method for providing dual transcriptional and post-translational control can be carried out using any of the maltose-dependent degradation determinants described in this invention. In some embodiments, the method of providing dual transcription and translation may be performed using a maltose-responsive promoter comprising sequences selected from the group consisting of: pMAL1 (SEQ ID NO: 29), pMAL2 (SEQ ID NO: 30), pMAL11 (SEQ ID NO: 31), pMAL12 (SEQ ID NO: 32), pMAL31 (SEQ ID NO: 33), pMAL32 (SEQ ID NO: 34), pMAL32_v1 (SEQ ID NO: 78), pGMAL_v5 (SEQ ID NO: 35), pGMAL_v6 (SEQ ID NO: 36), pGMAL_v7 (SEQ ID NO: 37), pGMAL_v9 (SEQ ID NO: 38), pGMAL_v10 (SEQ ID NO: 39), pGMAL_v11 (SEQ ID NO: 40), pGMAL_v12 (SEQ ID NO: 78). NO: 41), pGMAL_v13 (SEQ ID NO: 42), pGMAL_v14 (SEQ ID NO: 43), pGMAL_v15 (SEQ ID NO: 44), pGMAL_v16 (SEQ ID NO: 45), pGMAL_v17 (SEQ ID NO: 46), pGMAL_v18 (SEQ ID NO: 47), pG2MAL_v1 (SEQ ID NO: 48), pG2MAL_v2 (SEQ ID NO: 49), pG2MAL_v3 (SEQ ID NO: 50), pG2MAL_v5 (SEQ ID NO: 51), pG2MAL_v6 (SEQ ID NO: 52), pG2MAL_v7 (SEQ ID NO: 53), pG2MAL_v8 (SEQ ID NO: 54), pG2MAL_v9 (SEQ ID NO: 55), pG2MAL_v10 (SEQ ID NO: 56), pG7MAL_v2 (SEQ ID NO: 57), pG7MAL_v4 (SEQ ID NO: 58), pG7MAL_v6 (SEQ ID NO: 59), pG7MAL_v8 (SEQ ID NO: 60), pG7MAL_v9 (SEQ ID NO: 61), pG172_MAL_v13 (SEQ ID NO: 62), pG271_MAL_v12 (SEQ ID NO: 63), pG721_MAL_v11 (SEQ ID NO: 64),pG712_MAL_v14 (SEQ ID NO: 65), which retains the functional portion of the promoter, or a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it. In some embodiments, other synthetic maltose-responsive promoters described in this invention can be used in methods providing dual transcriptional and post-translational control.

[0024] On the other hand, the present invention provides a fermentation method for generating heterologous non-catabolite compounds from genetically modified host cells. In some embodiments, the method comprises two phases: a construction phase during which the generation of non-catabolite compounds is greatly reduced (the “off” phase) while cell biomass accumulates; and a generation phase during which the generation of non-catabolite compounds is turned on (the “on” phase). Thus, the negative selection pressure associated with the generation of non-catabolite compounds is mitigated during the fermentation phase where generation is not required (i.e., the construction phase). Reducing or eliminating the generation of non-catabolite compounds during the construction phase results in: (i) an increased growth rate of the cells during the construction phase; and (ii) increased generation stability of the strain during the generation phase. This leads to a longer duration of non-catabolite compound generation, thereby increasing the overall yield and / or productivity of the strain. Advantageously, the “off” and “on” states of non-catabolite compound generation in the fermentation method provided by the present invention can be controlled by readily available, affordable, and industrially relevant conditions.

[0025] The term "off" phase as used in this invention does not necessarily mean that the production of non-catabolite compounds in genetically modified host cells is zero or close to zero during this phase. Rather, the term "off" phase is associated with the "on" phase because the amount of non-catabolite compounds produced during the "off" phase is significantly reduced compared to the "on" phase (e.g., more than about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% less than the amount of non-catabolite compounds produced during the "on" phase).

[0026] In some embodiments, the "off" and "on" states of non-catabolite compound generation in the fermentation culture can be controlled by the amount of maltose in the culture medium along with a maltose-responsive promoter using a gene expressing a pathway enzyme that influences the generation of heterologous non-catabolite compounds. Advantageously, by coupling pathway gene expression to a maltose-sensitive promoter, the generation of heterologous non-catabolite compounds can be turned on or off by controlling the amount of maltose in the feedstock. For example, the maltose-responsive promoter can be wired as an "on" switch to induce the generation of the heterologous non-catabolite compounds in the presence of maltose. Alternatively, the maltose-responsive promoter can be wired as an "off" switch to induce the expression of a negative regulator of the biosynthetic pathway in the presence of maltose.

[0027] In some embodiments, the "on" and "off" states of non-catabolite compound generation in the fermentation culture can be controlled by the amount of maltose in the culture medium along with a maltose-dependent degradation determinant that regulates the expression stability of pathway enzymes that influence the generation of heterologous non-catabolite compounds. Advantageously, by coupling pathway gene expression to the maltose-dependent stability of the maltose-dependent degradation determinant, the generation of heterologous non-catabolite compounds can be turned on or off by controlling the amount of maltose in the feedstock. For example, a maltose-dependent degradation determinant fused with a suitable fusion partner (e.g., a transcriptional regulator) can be wired as an "on" switch to induce the generation of the heterologous non-catabolite compounds in the presence of maltose. Alternatively, a maltose-dependent degradation determinant fused with a suitable fusion partner can be wired as an "off" switch to induce the expression of a negative regulator of the biosynthetic pathway that generates the compound in the presence of maltose.

[0028] In some embodiments, the "off" and "on" states of non-catabolite compound generation in the fermentation culture can be controlled by the amount of maltose in the culture medium, along with the use of maltose-responsive promoters and maltose-dependent degradation determinants, which can regulate the gene expression of pathway enzymes affecting the generation of heterologous non-catabolite compounds. By simultaneously combining transcriptional and post-translational control of the same small-molecule effector maltose, the timing and expression levels of pathway enzymes can be tightly controlled. Applying two layers of control to genes that are tightly regulated during long-term fermentation can also reduce the potential for strain degeneration.

[0029] Therefore, the present invention provides a method for generating heterologous non-catabolite compounds in genetically modified host cells, wherein the method comprises: (a) culturing the genetically modified host cell population in a culture medium containing a carbon source comprising maltose, wherein the genetically modified host cells comprise: (i) one or more heterologous nucleic acids encoding one or more enzymes for a biosynthetic pathway for preparing the heterologous non-catabolite compound; and (ii) a heterologous nucleic acid encoding a fusion protein comprising a maltose-dependent degradation determinant within a maltose-dependent degradation determinant frame. The method involves fusing a target protein, wherein the heteronucleotide is operatively linked to a maltose-responsive promoter, wherein in the presence of maltose, transcription of the heteronucleotide encoding the fusion protein is activated, and the fusion protein encoded therein is stabilized, and wherein the stabilized fusion protein regulates the expression of one or more enzymes, which in turn control the amount of the heterologous non-catabolite compound produced by the host cell; and (b) culturing the host cell population or a subpopulation thereof in a medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount compared to the medium in step (a). In some embodiments, the maltose-responsive promoter activity and fusion protein stability in step (b) are reduced compared to step (a), resulting in a change in the amount of the heterologous non-catabolite compound produced by the host cell compared to step (a). Using this method, any maltose-dependent degradation determinant and / or maltose-responsive promoter described in this invention can be used.

[0030] Furthermore, the present invention provides a method for generating non-catabolite compounds in genetically modified host cells. In some embodiments, the method includes: (a) culturing a population of genetically modified host cells in a medium containing a carbon source comprising maltose, wherein the host cells comprise: (i) one or more heterologous nucleic acids encoding one or more enzymes of a biosynthetic pathway, each of the one or more heterologous nucleic acids being operatively linked to a Gal4p-responsive promoter; (ii) a nucleic acid encoding Gal4p; and (iii) a nucleic acid encoding a fusion protein comprising Gal80p fused to a maltose-dependent degradation determinant frame, the nucleic acid being operatively linked to a maltose-responsive promoter, wherein the maltose in the medium limits the amount of heterologous non-catabolite compounds generated by the host cells; and (b) culturing the host cell population or a subpopulation thereof in a medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount such that the amount of heterologous non-catabolite compounds generated by the host cells is increased compared to step (a). When performing this method, any maltose-dependent degradation determinant and / or maltose-responsive promoter described in this invention may be used.

[0031] In some embodiments, the Gal4p-responsive promoter used in the method of the present invention is selected from the group consisting of pGAL1, pGAL2, pGAL7, pGAL10, pGCY1, pGAL80, and synthetic pGAL promoters.

[0032] In some embodiments, the culture step (a) is performed for at least about 12, 24, 36, 48, 60, 72, 84, 96, or about 96 hours. In some embodiments, the culture step (a) is performed sufficiently to allow the host cell population to reach a cell density (OD) between about 0.01 and 400. 600 The culture medium in step (a) contains at least about 0.1% (w / v) of the maltose. In some embodiments, the culture medium in step (a) contains about 0.25% to 3% (w / v) of maltose. In some embodiments, the culture medium in step (b) contains no more than about 0.08% (w / v) of maltose. In some embodiments, during the generation of the heterologous non-catabolite compound, the genetically modified host cell population or subpopulation is cultured for about 3 to 20 days during step (b).

[0033] In some embodiments, the amount of the heterologous noncatabolite compound generated by the genetically modified host cell population during the culture duration of step (b) is improved compared to that obtained during fermentation, wherein the expression of the one or more enzymes of the biosynthetic pathway is not limited by the activity of the maltose-responsive promoter and the fusion protein. In some embodiments, the amount of the noncatabolite compound generated during step (a) is less than about 50%, 40%, 30%, 20%, or 10% of the amount of the noncatabolite compound generated during step (b). In some embodiments, the noncatabolite compound provided by the method is selected from the group consisting of amino acids, fatty acids, isoprene-like compounds, and polyketides.

[0034] In some embodiments, the genetically modified host cell provided by the method of the present invention is capable of generating heteroprene and contains at least one heteronucleotide encoding an isoprene pathway enzyme, said isoprene pathway enzyme being 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 with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA); (c) an enzyme that converts HMG-CoA to mevalonate; (d) an enzyme that converts mevalonate 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) an enzyme capable of condensing IPP and / or DMAPP. The enzymes that form polyisoprene synthases containing five or more carbon atoms include: (i) an enzyme that condenses IPP with DMAPP to form GPP; (j) an enzyme that condenses two IPP molecules with one DMAPP molecule; (k) an enzyme that condenses IPP with GPP to form FPP; (l) an enzyme that condenses IPP with DMAPP to form GGPP; and (m) an enzyme that condenses IPP with FPP to form GGPP.

[0035] In some embodiments, the host cell further comprises a heterologous nucleic acid encoding an enzyme modified with a polyisoprene group, the enzyme being selected from the group consisting of geraniol synthase, linalool synthase, limonene synthase, myrcene synthase, ocimene synthase, α-pinene synthase, β-pinene synthase, sapinene synthase, γ-terpinene synthase, terpinene oil synthase, azadirachtin synthase, α-farnesene synthase, β-farnesene synthase, farnesol synthase, nerolidol synthase, patchouliol synthase, nocarbamate synthase, and abietadiene synthase.

[0036] In some embodiments, the host cell contains multiple heterologous nucleic acids encoding all enzymes of the mevalonate pathway. In some embodiments, the isoprene is selected from the group consisting of sesquiterpenes, monoterpenes, diterpenes, triterpenes, tetraterpenes, and polyterpenes. In some embodiments, the isoprene is C5-C6. 20 Isoprene. In some embodiments, the isoprene is a sesquiterpene. In some embodiments, the isoprene is selected from the group consisting of abietadiene, azadirachtin, carene, α-farnesene, β-farnesene, farnesol, geraniol, geraniylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchouliol, β-pinene, sabinene, γ-terpinene, terpinene, and valencene.

[0037] In some embodiments, the host cell is capable of generating polyketide compounds and contains at least one heterologous nucleic acid encoding a polyketide compound synthase, wherein the polyketide compound synthase is selected from the group consisting of: (a) an enzyme that condenses at least one of acetyl-CoA and malonyl-CoA with an acyl carrier protein; (b) an enzyme that condenses 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 methylmalonyl-CoA to form a polyketide compound product; (c) an enzyme that reduces β-ketone chemical groups on the polyketide compound to β-hydroxy groups; (d) an enzyme that dehydrates alkane chemical groups in the polyketide compound to form α,β-unsaturated alkenes; (e) an enzyme that reduces α,β-double bonds in the polyketide compound to saturated alkanes; and (f) an enzyme that hydrolyzes the polyketide compound from an acyl carrier protein.

[0038] In some embodiments, the polyketide compound is a lipid having at least one of antibiotic, antifungal, and antitumor activities. In some embodiments, the polyketide compound is selected from the group consisting of macrolides, antibiotics, antifungals, cell growth inhibitors, anticholinergics, antiparasitics, anticoccidials, animal growth promoters, and insecticides.

[0039] In some embodiments, the host cell is capable of generating fatty acids and contains 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-CoA and malonyl-CoA 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 in acetoacetyl-ACP using NADPH to form a hydroxyl group in D-3-hydroxybutyryl hydroxylase-ACP; (d) an enzyme that dehydrates D-3-hydroxybutyryl hydroxylase-ACP to form a double bond between the β- and γ-carbons to form crotonyl-ACP; (e) an enzyme that reduces crotonyl-ACP using NADPH to form butyryl-ACP; and (f) an enzyme that hydrolyzes a C16 acyl compound from an acyl carrier protein to form palmitic acid. In some embodiments, the fatty acid is selected from the group consisting of palmitic acid, palmitoyl-CoA, palmitoleic acid, sapienic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[0040] On the other hand, the present invention provides a recombinant host cell comprising a heterologous nucleic acid encoding a fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame, wherein the fusion protein is more stable when the maltose-dependent degradation determinant is in contact with maltose compared to when the maltose-dependent degradation determinant is not in contact with maltose. In some embodiments, the heterologous nucleic acid is operatively linked to a maltose-responsive promoter. In some embodiments, the host cell further comprises one or more heterologous nucleic acids encoding one or more enzymes for a biosynthetic pathway for the preparation of heterologous non-catabolite compounds. In some embodiments, the host cell is selected from the group consisting of fungal cells, bacterial cells, plant cells, and animal cells. In some embodiments, the host cell is a yeast cell.

[0041] On the other hand, the present invention provides a fermentation composition comprising the recombinant host cells of the present invention in a culture medium containing maltose.

[0042] On the other hand, the present invention provides isolated nucleic acid molecules encoding maltose-dependent degradation determinants. In some embodiments, the isolated nucleic acid molecules encode maltose-dependent degradation determinants comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 28, wherein the maltose-dependent degradation determinant comprises one or more variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant comprising an amino acid sequence having at least about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 28, wherein (a) compared to SEQ ID NO: 2 or SEQ ID NO: 28, the maltose-dependent degradation determinant comprises one or more variant amino acid residues; and (b) the one or more variant amino acid residues are located at positions 7, 10, 11, 21, 24, 28, 42, 43, 64, 68, 83, 88, 92, 95, 98, 101, 110, 117, 134, 135, 136, 149, 168, 177, 186, 187, 193, At least one of 198, 210, 216, 217, 229, 236, 237, 242, 263, 291, 304, 321, 322, 339, 351, 357, 367, 370, and 374, wherein the position of one or more variant amino acid residues corresponds to the amino acid position of SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the isolated nucleic acid molecule is cDNA.

[0043] In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant, said maltose-dependent degradation determinant comprising a subset selected from K7R, I10T, W11G, L21S, V24A, F28Y, D42V, K43E, A64T, F68S, D83G, D88N, P92T, W95R, V98I, N101I, A110T, I117V, P134S, A135T, L136M, M149I, Y168C, Y168N, Y177H, N186S, A187P, L193S, D198V, D210E, A216V, A217D, G229C, I236N. One or more variant amino acid residues of the group consisting of D237N, N242D, L263M, L291V, A304S, T321N, M322L, A339T, A351T, T357S, T367S, S370P, and N374S. The positions of these one or more variant amino acid residues correspond to the amino acid positions in SEQ ID NO: 2 or SEQ ID NO: 28.

[0044] In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant, which, compared to SEQ ID NO: 2 or SEQ ID NO: 28, comprises at least one set of variant amino acid residues, wherein the at least one set of variant amino acid residues is selected from the group consisting of the following variant amino acid residues: (a) I10T, V24A, D42V, K43E, D83G, P92T, M149I, Y168N, N186S, A216V, and T357S; (b) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, (c) N186S, A216V, and D237N; (d) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and A339T; (e) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and N242D; (f) I10T, V24A, D42V, A110T, M149I, and A216V; (c) I10T, V24A, D42V, K43E, D83G, and M149I. Y168N, N186S, and A216V; (g)L21S, A64T, L136M, Y177H, A187P, A304S, T321N, and A351T; (h) K7R, D83G, V98I, L193S, I236N, and N374S; (i) W11G, D88N, P134S, A135T, D210E, and M322L; (j) I117V, Y168N, G229C, L263M, T367S, and S370P; (k) F68S, W95R, N186S, and D198V; and (l) F28Y, K43E, N101I, Y168C, A217D, and L291V.

[0045] In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant, the maltose-dependent degradation determinant comprising a sequence selected from the group consisting of SEQ ID NOS: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, and 26. In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant, the maltose-dependent degradation determinant comprising a sequence selected from the group consisting of positions 1 to 365 of SEQ ID NOS: 16, 18, 20, 22, 24, and 26. In some embodiments, the isolated nucleic acid molecule encodes a maltose-dependent degradation determinant, the maltose-dependent degradation determinant comprising a sequence selected from the group consisting of positions 1 to 370 of SEQ ID NOS: 16, 18, 20, 22, 24, and 26.

[0046] In some embodiments, the isolated nucleic acid molecule comprises a sequence selected from the group consisting of SEQ ID NOS: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, and 25. In some embodiments, the isolated nucleic acid molecule comprises a sequence selected from positions 1 to 1098 of SEQ ID NOS: 11, 15, 17, 19, 21, 23, and 25. In some embodiments, the isolated nucleic acid molecule comprises a sequence selected from positions 1 to 1113 of SEQ ID NOS: 11, 15, 17, 19, 21, 23, and 25.

[0047] On the other hand, the present invention provides a fusion protein comprising a target protein fused within the maltose-dependent degradation determinant frame described in the present invention.

[0048] On the other hand, the present invention provides a DNA construct comprising a maltose-responsive promoter operatively linked to a nucleic acid encoding a fusion protein, the fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame, the maltose-dependent degradation determinant comprising an amino acid sequence encoded by the isolated nucleic acid sequence described in the present invention.

[0049] On the other hand, the present invention provides a synthetic maltose-responsive promoter comprising a pGAL promoter having at least one or all of the Gal4p binding sites, wherein the at least one or all of the Gal4p binding sites are replaced by one or more binding sites of the MAL transcription activator. In some embodiments, the synthetic maltose-responsive promoter comprises a sequence selected from the group consisting of: pGMAL_v5 (SEQ ID NO: 35), pGMAL_v6 (SEQ ID NO: 36), pGMAL_v7 (SEQ ID NO: 37), pGMAL_v9 (SEQ ID NO: 38), pGMAL_v10 (SEQ ID NO: 39), pGMAL_v11 (SEQ ID NO: 40), pGMAL_v12 (SEQ ID NO: 41), pGMAL_v13 (SEQ ID NO: 42), pGMAL_v14 (SEQ ID NO: 43), pGMAL_v15 (SEQ ID NO: 44), pGMAL_v16 (SEQ ID NO: 45), pGMAL_v17 (SEQ ID NO: 46), pGMAL_v18 (SEQ ID NO: 47), and pG2MAL_v1 (SEQ ID NO: 48). pG2MAL_v2 (SEQ ID NO: 49), pG2MAL_v3 (SEQ ID NO: 50), pG2MAL_v5 (SEQ ID NO: 51), pG2MAL_v6 (SEQ ID NO: 52), pG2MAL_v7 (SEQ ID NO: 53), pG2MAL_v8 (SEQ ID NO: 54), pG2MAL_v9 (SEQ ID NO: 55), pG2MAL_v10 (SEQ ID NO: 56), pG7MAL_v2 (SEQ ID NO: 57), pG7MAL_v4 (SEQ ID NO: 58), pG7MAL_v6 (SEQ ID NO: 59), pG7MAL_v8 (SEQ ID NO: 60), pG7MAL_v9 (SEQ ID NO: 61), pG172_MAL_v13 (SEQ ID pG271_MAL_v12 (SEQ ID NO: 63), pG721_MAL_v11 (SEQ ID NO: 64), pG712_MAL_v14 (SEQ ID NO: 65), the portion of which retains promoter function, or the sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with it.In some embodiments, other synthetic maltose-responsive promoters provided by the present invention comprise: (a) a core promoter containing a transcription start site; and (b) one or more MAL transcription activator binding sites, wherein, under uninduced conditions in the absence of maltose, the promoter activity of the synthetic maltose-responsive promoter is less than that of a natural maltose-responsive promoter from which the one or more MAL transcription activator binding sites are derived.

[0050] On the other hand, the present invention provides a vector comprising a DNA construct containing a maltose-responsive promoter operatively linked to a nucleic acid encoding a fusion protein, the fusion protein containing a target protein fused within a maltose-dependent degradation determinant frame, wherein the maltose-responsive promoter is selected from natural maltose-responsive promoters and synthetic maltose-responsive promoters provided by the present invention.

[0051] On the other hand, the present invention provides a method for gene expression comprising: (a) culturing host cells containing a DNA construct comprising a pGMAL promoter operatively ligated to a target gene in a maltose-free medium; and (b) adding maltose to the medium to activate or increase the activity of the pGMAL promoter, wherein the pGMAL promoter comprises a pGAL promoter having at least one or all GAL4p binding sites, the at least one or all GAL4p binding sites being replaced by one or more binding sites of a MAL transcription activator. In some embodiments, other synthetic maltose-responsive promoters of the present invention may be operatively ligated to a target gene in the gene expression method.

[0052] On the other hand, the present invention provides a method for generating heterologous non-catabolite compounds in genetically modified host cells, the method comprising: (a) culturing a population of genetically modified host cells in a culture medium containing a carbon source, the carbon source comprising maltose, wherein the host cells contain one or more heterologous nucleic acids encoding one or more enzymes for a biosynthetic pathway for the preparation of the heterologous non-catabolite 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 culture medium limits the amount of heterologous non-catabolite compound generated by the host cells; and (b) culturing the host cell population or a subpopulation thereof in a culture medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount such that the activity of the maltose-responsive promoter is lower than that in step (a), and the amount of the heterologous non-catabolite compound generated by the host cells increases. In some embodiments, the maltose-responsive promoter comprises the pGMAL promoter provided by the present invention.

[0053] On the other hand, the present invention provides compositions and methods for stabilizing the generation of heterologous noncatabolite compounds using stabilization constructs that minimize the negative effects of spontaneous mutations that increase positive selection pressure to reduce the generation of noncatabolite compounds. In some embodiments, a method for generating heterologous noncatabolite compounds includes culturing genetically modified cells in a culture medium, the genetically modified cells comprising: (a) a heterologous nucleic acid encoding an enzyme of a biosynthetic pathway for generating the heterologous noncatabolite compound, wherein the heterologous nucleic acid is operatively linked to a first promoter; (b) a nucleic acid encoding a protein affecting cell growth, wherein the nucleic acid is operatively linked to a second promoter; and (c) a nucleic acid encoding a transcriptional regulatory factor. In these embodiments, both the first promoter and the second promoter are regulated by the same transcriptional regulatory factor. Therefore, the negative effects of functional disruption of the transcriptional regulatory factor (e.g., caused by spontaneous mutation) encode the expression of both the heterologous nucleic acid encoding the enzyme of the biosynthetic pathway and the nucleic acid encoding the protein affecting cell growth. Reducing the expression of the nucleic acids encoding the proteins that affect cell growth will provide unfavorable conditions for growth and prevent such cells from dominating the cell population during long-term fermentation.

[0054] In some embodiments, the non-catabolite compound is itself the target protein, rather than an enzyme in the biosynthetic pathway used to generate the heterologous non-catabolite compound. In these embodiments, the transcriptional regulatory factor co-regulates the expression of the heterologous nucleic acid encoding the target protein and the nucleic acid encoding proteins that affect cell growth.

[0055] In some embodiments, the transcriptional regulatory factors that regulate the first and second promoters are regulatory proteins of the GAL regulator. For example, the genetically modified host cell may contain heterologous nucleic acids encoding the transcriptional activator Gal4p and / or the transcriptional repressor Gal80p. In some embodiments, both the first and second promoters are naturally derived pGAL promoters or pGAL synthetic promoters. In some embodiments, the heterologous nucleic acids encoding the proteins affecting cell growth and the heterologous nucleic acids encoding enzymes for biosynthetic pathways to generate non-catabolite compounds are chromosomally integrated into the genome of the genetically modified host cell.

[0056] In some embodiments, the heterologous nucleic acid encoding a protein affecting cell growth is an essential gene that is absolutely necessary for life in the genetically modified host cell under any culture medium or condition. In other embodiments, the heterologous nucleic acid encoding a protein affecting cell growth is a conditionally essential gene that is necessary for cell growth when the host cell is grown in a culture medium lacking essential compounds. These include, for example, one or more biosynthetic genes encoding one or more enzymes in biosynthetic pathways for the production of amino acids, nucleotides, or fatty acids. In some embodiments, the conditionally essential gene encodes an enzyme in the biosynthetic pathway for the production of lysine. In other embodiments, the conditionally essential gene encodes an enzyme in the biosynthetic pathway for the production of methionine.

[0057] In some embodiments, the culture method comprises two phases: (a) a cell biomass building phase, wherein the genetically modified host cell population is cultured in a medium that restricts the production of heterologous noncatabolite compounds; and (b) a generation phase, wherein the host cell population or a subpopulation thereof is cultured under conditions that promote the production of the heterologous noncatabolite compounds. During the cell biomass building phase, the expression of the regulators (e.g., nucleic acids encoding enzymes for biosynthetic pathways for the production of noncatabolite compounds and nucleic acids encoding conditionally essential gene products) in the genetically modified host cells is restricted, and essential compounds are supplemented in the medium to allow cell growth. During the generation phase, the genetically modified host cells are cultured in a medium lacking or containing sufficiently low amounts of essential compounds so that only cells capable of synthesizing the essential compounds can grow. This provides positive selection pressure on cells that maintain the expression of transcriptional regulatory factors, thus leading to the expression of the regulators during the generation phase.

[0058] On the other hand, the present invention provides compositions and methods for generating heterologous noncatabolite compounds, utilizing a combination of a fusion protein comprising a transcriptional regulatory factor fused to the maltose-dependent degradation determinant frame and a stabilizing construct. When the fusion protein is used in combination with the stabilizing construct described in this invention, the generation of heterologous noncatabolite compounds is further stabilized because both constructs can counteract any negative effects of spontaneous mutations. The stabilizing construct, which couples the cell growth of genetically modified host cells with the generation of noncatabolite compounds, mitigates any negative effects of spontaneous mutations at the transcriptional level. The fusion protein comprising a transcriptional regulatory factor fused to the maltose-dependent degradation determinant frame mitigates any negative effects of spontaneous mutations at the posttranslational level.

[0059] In some embodiments, a method for generating a heterologous noncatabolite compound includes culturing genetically modified host cells in a culture medium, the host cells comprising: (a) a heterologous nucleic acid encoding a fusion protein, the fusion protein comprising a transcriptional regulatory factor fused to a maltose-dependent degradation determinant frame; (b) one or more heterologous nucleic acids encoding one or more enzymes of a biosynthetic pathway for generating the heterologous noncatabolite compound, each of wherein the heterologous nucleic acids is operatively linked to a promoter regulated by the fusion protein; and (c) a nucleic acid encoding a protein affecting cell growth, wherein the nucleic acid is operatively linked to a promoter regulated by the fusion protein. In one embodiment, the fusion protein comprises a Gal80p fused to a maltose-dependent degradation determinant frame, and a Gal4p-responsive promoter operatively linked to the heterologous nucleic acid encoding one or more enzymes of the biosynthetic pathway and operatively linked to the nucleic acid encoding the protein affecting cell growth.

[0060] These and other embodiments of the present invention, as well as many of their features, will be described in more detail below and in conjunction with the accompanying drawings.

[0061] 5. Brief description of the attached drawings

[0062] Figures 1A and 1B show schematics illustrating the use of maltose-dependent stability of a fusion protein containing Gal80p fused within the maltose-dependent degradation determinant frame to negatively regulate the expression of genes in the biosynthetic pathway.

[0063] Figures 2A and 2B show schematics illustrating the use of maltose-dependent stability of a fusion protein containing Gal4p fused within the maltose-dependent degradation determinant frame to positively regulate the expression of genes in biosynthetic pathways.

[0064] Figures 3A and 3B show schematic diagrams illustrating the combination of a maltose-responsive promoter and a maltose-dependent degradation determinant to control the transcription of the fusion DNA construct and the post-translational stability of the fusion protein by manipulating the maltose content in the culture medium.

[0065] Figure 4 shows a schematic diagram illustrating the selection and screening of nucleic acids encoding maltose-dependent Gal80p fused within the MBP mutant frame.

[0066] Figure 5 shows a graph representing the production of farnesene by strains expressing a fusion protein comprising Gal80p fused to various MBP mutants obtained from the first layer mutagenesis. The X-axis represents the amount of farnesene produced by strains cultured in maltose-containing medium, and the Y-axis represents the amount of farnesene produced by strains cultured in maltose-free medium. Each MBP mutant strain obtained through the first layer mutagenesis is indicated by a square marker.

[0067] Figure 6 shows a schematic diagram of the second-layer optimization process, which illustrates competitive enrichment on the first-layer MBP mutant strains to improve their maltose-dependent switchability.

[0068] Figures 7A and 7B show the results of competitive selection for the combination of MBP mutations from the first layer of mutagenesis after one and three rounds of selection / anti-selection cycles. After three rounds of selection / anti-selection cycles in the second layer optimization process, strains with an improved "off" state were obtained.

[0069] Figure 8A shows a schematic diagram illustrating a cell sorting strategy to screen for MBP mutants with improved instability in the absence of maltose.

[0070] Figure 8B shows the relative GFP (green fluorescent protein) intensity of host cells expressing GFP fused with various MBP mutants when the host cells are cultured in media containing or without maltose.

[0071] Figure 8C shows the GFP fluorescence intensity (in original units) of host cells expressing GFP fused with various MBP mutants and wild-type MBP when the host cells are in maltose-containing medium (top panel) and maltose-free medium (bottom panel). The table at the bottom of Figure 8C shows the ratio of GFP fluorescence intensity of genetically modified host cells cultured in the presence of maltose to that in the absence of maltose.

[0072] Figures 9A to 9C show the maltose-dependent farnesene production levels in host cells expressing the fusion protein, which contains Gal80p fused to different MBP mutant frames.

[0073] Figure 9D shows a schematic diagram illustrating how to visualize and interpret the data represented by box plots in Figures 9A to 9C, 10, and 19. The box plots of these figures are created using... Generated by data visualization and analysis software. When measurements for different runs are very precise and close to each other, the bins, represented by the upper and lower quartiles, fold into one or more lines (see, for example, the GAL80_MBP_H9 and GAL80_MBP_M1 data in Figure 9A).

[0074] Figure 10 illustrates the switchability of different switch constructs containing MBP. Strains B, C, and D are syngenetic strains, and strains B and C contain specific switch constructs. B = pMAL32_v1>GAL80_MBPL8_v4d (a weak promoter driving the fusion of GAL80 with an MBP mutant exhibiting the best maltose-dependent difference for protein stability); C = pMAL32>GAL80_MBPL8_v4d (same as B, but with a stronger promoter); D = parental strain (a constitutive promoter driving mevalonate pathway gene expression in the absence of a maltose switch or a maltose-dependent degradation determinant).

[0075] Figure 11A illustrates the degradation of various MBP mutants fused with GFP in BSM sucrose medium. GFP expression was driven by the maltose-inducible promoter pMAL32. GFP protein was expressed alone or fused to the N-terminus of wild-type or mutant MBP (no MBP – x; GFP_MBP – ○; GFP_5A2 – □; GFP_5F3 – ▼; GFP_H8 – ◇; GFP_L8_v4d – ▲).

[0076] Figure 11B shows the half-life of GFP control and GFP fused with various MBP mutants in host cells cultured in BSM sucrose medium.

[0077] The results provided in Figures 12A and 12B demonstrate that host cells capable of generating the isoprene-like farnesene and containing the MEV pathway negatively regulated by a maltose-responsive promoter and a maltose-dependent degradation determinant exhibit improved farnesene generation stability over long fermentation periods when the construction phase of the fermentation is carried out in the presence of maltose (thus achieving a "closed" state), compared to constitutive farnesene-generating strains that generate farnesene throughout the construction phase.

[0078] Figure 13A shows a schematic diagram illustrating the construction of the pGMAL promoter.

[0079] Figure 13B shows the sequence alignment of MAL transcription activator binding sites from pMAL12 and pMAL32. The sequences of the binding sites were aligned using Clone Manager software with a scoring matrix: linear (Mismatch 2, OpenGap 4, ExtGap 1).

[0080] The results provided in Figures 14A and 14B show that the pGMAL promoter is maltose-inducible and is unaffected by the growth rate in the absence of maltose.

[0081] Figure 15 provides a schematic diagram of the mevalonic acid (“MEV”) pathway for the production of isopentenyl diphosphate (“IPP”).

[0082] Figure 16 provides a schematic diagram of the conversion of IPP and dimethylallyl pyrophosphate (“DMAPP”) to geraniyl pyrophosphate (“GPP”), farnesyl pyrophosphate (“FPP”) and geraniylgeraniyl pyrophosphate (“GGPP”).

[0083] Figure 17 illustrates a schematic diagram showing the use of a stabilized construct containing LYS9 operatively linked to the pGAL10v3 promoter to couple the expression of the conditionally essential gene LYS9 with the expression of genes involved in the biosynthetic pathway for the production of non-catabolite compounds. The embodiment shown in Figure 17 illustrates maltose, when present in a culture medium, binding to the Malx3 protein, which activates a maltose-responsive promoter, leading to the expression of the transcriptional repressor GAL80. Gal80p, encoded by the GAL80 gene, in turn represses the transcriptional activator Gal4p. Since both the LYS9 gene and the biosynthetic pathway genes for the production of non-catabolite compounds are co-regulated as GAL regulators, the expression of these genes is coupled via the transcriptional regulatory factors of the GAL regulator.

[0084] Figure 18 shows a schematic diagram illustrating the selection and screening of combinations of amino acid biosynthesis pathway genes and pGAL promoters operably linked thereto, wherein the pGAL promoters cause genetically modified host cells to express in a way that is dependent on their cell growth.

[0085] Figure 19 shows two stabilization constructs, each containing a lysine biosynthesis gene operatively linked to a pGAL promoter, which causes the genetically modified host cell to express the gene in a way that allows its cell growth.

[0086] The results shown in Figure 20 demonstrate that the maltose-switching yeast strains containing the stabilized constructs (strain F containing pGAL10_v3>LYS9; and strain G containing pGAL2_v3>LYS1) exhibit improved stability in the formation of isoprene-like farnesenes during long fermentation runs compared to their parental strain (strain E) which does not contain the stabilized constructs. Detailed Implementation

[0087] 6.1 Definition

[0088] The term "maltose-binding protein" or MBP used in this invention refers to a protein comprising a portion that specifically binds to and interacts with maltose. In one embodiment, MBP comprises a protein that is part of the maltose / maltodextrin system of *E. coli* and other bacteria, responsible for the uptake and efficient catabolism of maltodextrin. MBP exhibits binding affinity for both maltose and maltodextrin. Dextran linked to macromolecules α(1-4) also bind with high affinity. Ferenci & Klotz, FEBS Letters, vol. 94(2):213-217 (1978).

[0089] The term "wild-type" MBP as used in this invention includes MBPs that can be isolated from an organism. In one embodiment, the term "wild-type" MBP includes MBPs isolated from bacteria, such as MPBs isolated from *E. coli* containing the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 is a mature MBP (370 residues) during the N-terminal expansion of a precursor polypeptide encoded by the malE gene of *E. coli*. In some embodiments, the "wild-type" MPB contains a protein encoded by the nucleic acid sequence of SEQ ID NO: 1. In some embodiments, "wild-type" MBP refers to a reference sequence used as a background sequence to introduce mutations to generate MBP mutants. For example, "wild-type" MBP may further include a reference sequence containing SEQ ID NO: 2 and additional sequences, such as linker sequences. For example, "wild-type" MBP includes an MBP containing the amino acid sequence of SEQ ID NO: 28, which contains a linker sequence at the C-terminus of SEQ ID NO: 2. The “wild-type” MBP nucleic acid sequence also includes a nucleic acid sequence containing the nucleotide sequence of SEQ ID NO: 27, which contains a linker sequence at the C-terminus of SEQ ID NO: 1.

[0090] As used in this invention, the term "MBP mutant" refers to any variant of wild-type MBP. The MBP mutant may include, for example, a wild-type MBP having one or more added and / or substituted and / or deleted and / or inserted amino acids.

[0091] The term "degradation domain" or "degradation determinant" refers to a protein element that imparts instability to another protein with which it is fused.

[0092] The terms "maltose-dependent degradation determinant," "maltose-binding degradation determinant," or "maltose-binding protein degradation determinant" used in this invention refer to maltose-binding protein mutants that have become stable in dependence on binding to maltose. Maltose-dependent degradation determinants are more stable when in contact with or bound to maltose than when not in contact with or bound to maltose. When maltose-dependent degradation determinants are in contact with or bound to maltose, they also confer stability to another protein fused with them, compared to when they are not in contact with or bound to maltose.

[0093] The term "maltose-based inducer" includes maltose or any analogues and derivatives of maltose. The maltose-based inducer can bind to MBP, MBP mutants, or maltose-dependent degradation determinants. In one embodiment, when the maltose-based inducer binds to a maltose-dependent degradation determinant, it can stabilize the maltose-dependent degradation determinant. The maltose-based inducer can also induce activation of a maltose-responsive promoter. Although the term "maltose" is used throughout the specification for simplicity, any maltose-based inducer can be used instead of maltose in the compositions and methods provided by this invention, and throughout the invention, the term "maltose" can be replaced by "maltose-based inducer."

[0094] As used in this invention, the terms "sequence identity" or "percentage identity" in the context of two or more nucleic acid or protein sequences refer to two or more sequences or subsequences that are identical or have a specific percentage of identical amino acid residues or nucleotides. For example, when comparing and aligning over a comparison window or designated region to obtain maximum correspondence, as determined using sequence comparison algorithms or by manual alignment and visual inspection, the sequence may have a percentage identity of at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or higher, with a reference sequence. For example, the percentage of identity is determined by calculating the ratio of the number of identical nucleotides (or amino acid residues) in the sequence to the total length of the nucleotides (or amino acid residues) minus the length of any vacancies.

[0095] For convenience, computer programs and mathematical algorithms known in the art can be used to determine the degree of identity between two sequences. Such algorithms for calculating the percentage sequence identity typically take into account sequence gaps and mismatches in the comparison region. Procedures for comparing and aligning sequences, such as Clustal W (sequence alignment) (Thompson et al., (1994) Nucleic Acids Res., 22:4673-4680), ALIGN (Myers et al., (1988) CABIOS, 4:11-17), FASTA (Pearson et al., (1988) PNAS, 85:2444-2448; Pearson (1990), Methods Enzymol., 183:63-98), and BLAST for gaps (Altschul et al., (1997) Nucleic Acids Res., 25:3389-3402), can all be used for this purpose. The BLAST or BLAST 2.0 (Altschul et al., J.Mol.Biol.215:403-10, 1990) is available from multiple sources used for link sequence analysis programs BLASTP, BLASTN, BLASTX, TBLASTN, and TBLASTX, including the National Center for Biotechnology Information (NCBI) and the Internet. More information is available on the NCBI website.

[0096] In some implementations, sequence alignment and percentage identity calculations can be determined using the BLAST program with its standard default parameters. For nucleotide sequence alignment and sequence identity calculations, the BLASTN program can be used with its default parameters (vacancy open penalty = 5, vacancy extension penalty = 2, nuclear match = 1, nuclear mismatch = -3, expected value = 10.0, word size = 11). For peptide sequence alignment and sequence identity calculations, the BLASTP program can be used with its default parameters (vacancy open = 11, vacancy extension penalty = 2; nuclear match = 1; nuclear mismatch = -3, expected value = 10.0; word size = 11; matrix Blosum62). Alternatively, the following program and parameters can be used: Clone Manager Suite version 5 (Sci-Ed software) Align Plus software; DNA comparison: Global comparison, Standard Linear Scoring matrix, mismatch penalty = 2, open vacancy penalty = 4, extension vacancy penalty = 1. Amino acid comparison: Global comparison, BLOSUM62 score matrix.

[0097] The term "homology" as used in this invention refers to the similarity between two or more nucleic acid sequences or two or more amino acid sequences. Sequence identity can be measured as a percentage of identity (or similarity or homology); the higher the percentage, the closer the sequences are to being identical. When aligned using standard methods, homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity. For example, "homologies" of a reference protein or nucleic acid include those with at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% sequence identity with the reference protein or nucleic acid. As mentioned above, various procedures for sequence alignment and analysis are well known and can be used to determine whether two sequences are homologs of each other.

[0098] The phrase "strict hybridization conditions" refers to conditions under which a probe typically hybridizes with its target sequence in a complex mixture of nucleic acids, but not with other sequences. Strict conditions are sequence-dependent and vary depending on the specific conditions. Longer sequences hybridize specifically at higher temperatures. For extensive guidelines on nucleic acid hybridization, see Tijssen, Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Typically, strict conditions are chosen as the melting point (T0) of the specific sequence at a pH value higher than a defined ionic strength. m The temperature is approximately 5-10°C lower. The T... m It is at equilibrium (because the target sequence is in excess, at T) m The stringent conditions are determined by the temperature at which 50% of the probe is used in equilibrium and the 50% of the probe complementary to the target sequence hybridizes with the target sequence (under defined ionic strength, pH, and nucleic acid concentration). Strict conditions can also be achieved by adding a destabilizing agent such as formamide. For selective or specific hybridization, the positive signal must be at least twice the background signal, preferably 10 times the background signal. Exemplary stringent hybridization conditions can be as follows: incubation at 42°C with 50% formamide, 5×SSC, and 1% SDS, or incubation at 65°C with 5×SSC and 1% SDS, followed by washing at 65°C with 0.2×SSC and 0.1% SDS.

[0099] If the peptides they encode are substantially the same, then nucleic acids that do not hybridize under stringent conditions are still substantially the same. This occurs, for example, when copies of nucleic acids are generated using the maximum codon degeneracy allowed by the genetic code. In this case, nucleic acids are typically hybridized under moderately stringent hybridization conditions. An exemplary “moderately stringent hybridization condition” involves hybridization at 37°C in a buffer of 40% formamide, 1M NaCl, and 1% SDS, followed by washing at 45°C in 1×SSC. Positive hybridization is at least twice the background. Those skilled in the art will readily recognize that other hybridization and washing conditions can be used to provide similar stringent conditions. Additional guidelines for determining hybridization parameters are provided in numerous references, such as Current Protocols in Molecular Biology, ed. Ausubel et al.

[0100] "Conservative amino acid substitution" is a situation where an amino acid residue is replaced by another amino acid residue in a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitution does not substantially alter the functional properties of a protein. In cases where two or more amino acid sequences differ from each other due to conservative substitution, the percentage sequence identity or homology can be adjusted upwards to correct for the conservatism of the substitution. Methods for making such adjustments are well known to those skilled in the art (see, for example, Pearson WR, 1994, Methods in Mol. Biol 25:365-89).

[0101] The following six groups each contain amino acids that are conserved and substituted for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), alanine (A), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0102] As used in this invention, the term "variant amino acid residue" refers to an amino acid change or substitution in a variant form of the reference protein. For example, the variant amino acid residue "I10T" means that position 10 of the reference protein, which normally contains isoleucine (I) in the variant protein, is replaced by the amino acid residue threonine (T). In another embodiment, the variant amino acid residue "A216V" means that position 216 of the reference protein, which normally contains the amino acid residue alanine (A) in the variant protein, is replaced by the amino acid residue valine (V).

[0103] An example of an algorithm suitable for determining the identity of percentage sequences is the one used in the Basic Local Alignment Search tool (hereinafter referred to as "BLAST"), see, for example, Altschul et al., J.Mol.Biol.215:403-410 (1990) and Altschul et al., Nucleic Acids Res.,15:3389-3402 (1997), which are publicly available through the National Center for Biotechnology Information (hereinafter referred to as "NCBI").

[0104] As used in this invention, the term "target protein" refers to any protein or polypeptide whose formation is desired. In some embodiments, the term "target protein" refers to the protein that is required to be expressed within the fusion protein.

[0105] As used in this invention, the term "fusion protein" refers to a protein formed by the linkage of two or more proteins through a peptide bond between the amino terminus of one protein and the carboxyl terminus of another. Translation of the "fusion gene" produces a single fusion protein possessing functional properties derived from each of the original proteins.

[0106] The term "protein stability" as used in this invention, when applied in a structural context, relates to the structural integrity of the protein. In one embodiment, protein stability refers to a net balance of forces that determines whether the protein is in its native folded conformation, which is protected from degradation or is in a denatured state that is prone to degradation. In one embodiment, protein stability can be determined in a functional context, i.e., in relation to the state of the protein or its fusion protein to confer function and / or activity over time.

[0107] The term "stability" in relation to proteins can be used as a relative term to describe the relative state in which a protein is protected from protein degradation. For example, a maltose-dependent degradation determinant is considered more stable in the presence or absence of maltose compared to the absence of maltose. In one embodiment, the stability of the maltose-dependent degradation determinant in the presence or absence of maltose is determined by comparing the activity of a reporter gene (e.g., green fluorescent protein) fused to it.

[0108] The term "half-life" of a protein typically refers to the time required for the protein concentration in a host cell or cell extract to decrease by half.

[0109] The maltose-dependent degradation determinant or fusion protein that is "in contact with maltose" or "bound to maltose" as used in this invention refers to a maltose-dependent degradation determinant that has a physical dependence on or close correlation with maltose. For example, the binding can be formed by hydrogen bonds, hydrophobic forces, van der Waals forces, covalent bonds, or ionic bonds.

[0110] As used in this invention, the term "biomolecule" refers to any endogenous or heterologous molecule that can exist in cells. The term "biomolecule" can refer to, for example, nucleic acids, proteins, peptides, amino acids, lipids, carbohydrates, metabolites, and metabolic products.

[0111] As used in this invention, the term "target molecule" refers to a molecule whose quantity or expression level is directly or indirectly affected by the activity of a fusion protein, said fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame. The term "target molecule" can refer to, for example, enzymes, other proteins, peptides, amino acids, nucleic acids, lipids, carbohydrates, metabolites, metabolites, and non-catabolizable compounds.

[0112] The GAL80 gene refers to the gene encoding the transcriptional regulator Gal80p, which is involved in repressing the expression of GAL regulator genes. Transcriptional regulation in the yeast galactosidase regulator is determined by the interaction between the positive regulator Gal4p and the negative regulator Gal80p. The Gal80p used in this invention can refer to wild-type Gal80p, any variant, or any modified form. For example, the GAL80 gene used in this invention can encode wild-type Gal80p or Gal80p modified with a constitutive degradation determinant fused to its N-terminus to increase protein turnover.

[0113] As used in this invention, phrases such as "functionally disrupted" or "functionally disrupted" of a selected gene refer to alterations to the selected gene in such a manner that the activity of the protein encoded by the selected gene in the host cell is reduced. Similarly, "functionally disrupted" or "functionally disrupted" of a selected protein refers to alterations to the protein in such a manner that the activity of the protein in the host cell is reduced. In some embodiments, the activity of the selected protein encoded by the selected gene is eliminated in the host cell. Functional disruption of the selected gene can be achieved by deleting all or part of the gene, thereby eliminating or reducing gene expression, or eliminating or reducing the activity of the gene product. Functional disruption of the selected gene can also be achieved by mutating the regulatory elements of the gene, such as mutating the promoter of the gene, thereby eliminating or reducing expression, or by mutating the coding sequence of the gene, thereby eliminating or reducing the activity of the gene product. In some embodiments, functional disruption of the selected gene results in the removal of the entire open reading frame of the selected gene.

[0114] The terms “natural” or “endogenous” as used in this invention refer to substances or processes / methods that can be naturally present in host cells.

[0115] The term "genetically modified" as used in this invention refers to a host cell containing a heterologous nucleotide sequence.

[0116] The term "heterologous" as used in this invention refers to something that is not normally found in nature. For example, when used in connection with nucleic acids (DNA or RNA) or proteins, "heterologous" refers to nucleic acids or proteins that are not naturally occurring as part of an organism, cell, genome, or DNA or RNA sequence present therein, or that are present on cells or sites within a genome or DNA or RNA sequence and are distinct from naturally occurring ones. When used in connection with nucleic acids (DNA), the term "heterologous" can also refer to nucleic acids that are operatively linked to promoters other than endogenous promoters. The term "heterologous compound" refers to a compound generated by cells that do not normally produce the compound, or a compound generated at levels not normally produced by cells.

[0117] The term "heterogeneous enzyme" as used in this invention refers to an enzyme that is not normally present in a given cell in nature. The term includes enzymes that are: (a) exogenous to a given cell (i.e., encoded by nucleotide sequences that are not naturally present in the host cell or are not naturally present in the given context of the host cell); and (b) naturally present in the host cell (e.g., the enzyme is encoded by nucleotide sequences that are endogenous to the cell), but are produced in the host cell in a non-natural amount (e.g., more or less than naturally present).

[0118] The term "naturally occurring" as used in this invention refers to something found in nature. For example, maltose-binding proteins present in organisms can be isolated from natural sources and are not intentionally modified in the laboratory; these are naturally occurring maltose-binding proteins (e.g., maltose-binding protein sequences in GenBank). Conversely, the term "not naturally occurring" as used in this invention refers to something not found in nature but which can be generated through human intervention.

[0119] The terms “amino acid sequence,” “peptide,” “oligopeptide,” “polypeptide,” and “protein” are used interchangeably in this invention and refer to a polymeric form of amino acids of any length that may or may not be chemically or biochemically modified.

[0120] The terms “polynucleotide” and “nucleic acid” are used interchangeably in this invention and refer to polymeric forms of any length, both ribonucleotides and deoxyribonucleotides.

[0121] The term "isolated nucleic acid," when applied to DNA, refers to a DNA molecule that has been isolated from the sequence immediately adjacent to the naturally occurring genome of the organism of its origin. "Isolated nucleic acid" also includes non-genomic nucleic acids, such as cDNA or other non-naturally occurring nucleic acid molecules.

[0122] The term "cDNA" is defined in this invention as a DNA molecule that can be produced by reverse transcription from a mature, spliced ​​mRNA molecule obtained from a cell. cDNA lacks the intron sequences that are normally present in the corresponding genomic DNA.

[0123] The phrase "operably linked" as used in this invention refers to a functional link between nucleic acid sequences such that the promoters and / or regulatory regions of the link functionally control the expression of the coding sequence.

[0124] The term "production amount" as used in this invention generally refers to the amount of non-catabolite compound produced by the genetically modified host cell provided by this invention. In some embodiments, production amount is expressed as the yield of the non-catabolite compound produced by the host cell. In other embodiments, production amount is expressed as the productivity of the host cell in producing the non-catabolite compound.

[0125] The term "yield" refers to the amount of non-catabolite compound produced by the host cell, expressed as the amount of non-catabolite compound produced per unit of carbon source consumed by the host cell, by weight. In some embodiments, the term "yield" refers to the amount of non-catabolite compound produced per unit of total reducing sugar added to the fermentation vessel or flask (i.e., grams of non-catabolite product divided by grams of total reducing sugar added, expressed as a percentage). The total reducing sugar is a unit of measurement for sugars in grams. Reducing sugars are any sugars that can be used as reducing agents because they have a free aldehyde group or a free ketone group. All monosaccharides, such as galactose, glucose, and fructose, are reducing sugars. For example, if 10 grams of non-catabolite compound are produced by adding 100 grams of glucose (i.e., 100 grams of reducing sugar) to the host cell, the yield per reducing sugar product is 10%.

[0126] The term “productivity” as used in this invention refers to the amount of non-catabolite compounds produced by host cells, expressed as the amount of non-catabolite compounds produced per unit volume of fermentation broth in which the host cells are cultured over time (per hour) (by volume).

[0127] The term "fermentation" is used to refer to the cultivation of host cells that use a carbon source (such as sugar) as an energy source to produce desired products.

[0128] The term "culture medium" refers to a medium in which cell biomass grows and metabolites are produced by the host cells. It contains a carbon source and may further contain nitrogen, phosphorus, vitamins, minerals, etc.

[0129] The term "fermentation medium" as used in this invention may be used synonymously with "culture medium." Generally, the term "fermentation medium" can be used to refer to a culture medium suitable for long-term culture of host cells to produce desired compounds.

[0130] The term "culture medium / medium" refers to a culture medium and / or a fermentation medium. The "culture medium / medium" can be liquid or semi-solid. A given culture medium / medium can be both a culture medium and a fermentation medium.

[0131] The term "whole cell broth" refers to the entire contents of a container (e.g., flask, plate, fermenter, etc.), including cells, the aqueous phase, and compounds generated in the hydrocarbon phase and / or emulsion. Therefore, whole cell broth comprises a mixture of culture media containing water, carbon sources (e.g., sugars), minerals, vitamins, other dissolved or suspended substances, microorganisms, metabolites, and compounds generated by the host cells, as well as all other components of the material held in the container from which non-catabolite compounds are prepared by the host cells.

[0132] The term "fermentation composition" may be used interchangeably with "whole-cell fluid". If the fermentation composition is added to the fermentation vessel during fermentation, it may also include a covering.

[0133] The term "biosynthetic pathway" refers to a pathway with a set of anabolic or catabolic biochemical reactions that convert one chemical substance into another to generate a molecule. Gene products belong to the same "biosynthetic pathway" if they act in parallel or in series with the same substrate to produce the same product, or act on or produce metabolic intermediates (e.g., metabolites) between the same substrate and the final metabolite product.

[0134] As used in this invention, the term "promoter" refers to a synthetic or naturally derived nucleic acid capable of conferring, activating, or enhancing the expression of a DNA-coding sequence. A promoter may contain one or more specific transcriptional regulatory sequences to further enhance expression and / or alter the spatial and / or temporal expression of the coding sequence. The promoter may be located at the 5' position (upstream) of the coding sequence it controls. The distance between the promoter and the coding sequence to be expressed may be approximately the same as the distance between the promoter and the natural nucleic acid sequence it controls. As is known in the art, variations in this distance can be accommodated without loss of promoter function. In some embodiments, the regulatory promoters used in this invention typically enable transcription of nucleic acid sequences encoding transcriptional regulatory factors (e.g., activators such as Gal4p, or repressors such as Gal80p) in a permissive environment (e.g., in the presence of maltose), but halt transcription of the nucleic acid sequences encoding transcriptional regulatory factors in a non-permissive environment (e.g., in the absence of maltose).

[0135] The term "maltose-responsive promoter" or "pMAL" promoter refers to a promoter sequence that is bound to and regulated by a maltose-regulated transcriptional activator. For example, maltose-inducible promoters are regulated by MAL operon activators (e.g., MAL transcriptional activators) or their functional homologs.

[0136] The term "MAL operon activator" or "MAL transcription activator" refers to the DNA-binding, maltose-dependent transcription activator of the maltose operon or maltose-responsive promoter.

[0137] The term "galactose-inducible promoter" or "pGAL" promoter refers to a promoter sequence that is bound to and regulated by a galactose-regulated transcriptional activator. For example, the galactose-inducible promoter is regulated by Gal4p or its functional homologs.

[0138] The term "pGMAL" promoter refers to a synthetic promoter having a pGAL promoter sequence in which the GAL transcription activator (e.g., GAL4p) binding site is replaced by one or more binding sites of a MAL transcription activator. Therefore, the pGMAL promoter is activated by a MAL transcription activator, not a GAL transcription activator.

[0139] The term "synthetic promoter" refers to a nucleotide sequence that has promoter activity and is unknown in nature. In one embodiment, a synthetic promoter is assembled from multiple heterologous elements.

[0140] The phrase "strain stability" generally refers to the stability of genetically modified host cells, as described in this invention, in producing heterologous compounds during prolonged fermentation. Specifically, stability refers to the ability of microorganisms to maintain favorable characteristics for the production of non-catabolite fermentation products (i.e., high yield (grams of compound per gram of substrate) and / or productivity (grams per liter of fermentation broth per hour)) over extended culture periods, such as approximately 3 to 20 days. Genetic stability refers to the tendency of the generating microbial community to maintain relatively stable allele frequencies of genes associated with product production over time, which plays a major role in the sustained production of the product.

[0141] Unless otherwise stated, the concentration of maltose or other components in the culture medium or culture solution is expressed as a weight / volume percentage. It is defined as: solute concentration (w / v%) = (solute weight (g) / solution volume (mL)) × 100.

[0142] The term "transcriptional regulatory factor" refers to proteins that control gene expression.

[0143] The term "transcription activator" refers to transcriptional regulatory factors that activate or positively regulate gene expression.

[0144] The term "transcriptional repressor" refers to transcriptional regulatory factors that inhibit or negatively regulate gene expression.

[0145] The terms "genes that affect cell growth" or "nucleic acids that encode proteins that affect cell growth" refer to nucleic acids that encode proteins that affect cell growth (e.g., growth rate or cell biomass).

[0146] The term "essential genes" refers to genes that are absolutely necessary to maintain life under optimal conditions where all nutrients are available.

[0147] The term "conditionally essential gene" refers to a gene that is required only under certain conditions or growth conditions.

[0148] The term "regulator" refers to a genome or nucleotide sequence regulated by the same regulatory protein (e.g., a transcription regulator). The gene containing the regulator has a regulatory binding site or a promoter regulated by a common transcription regulator. The genome or nucleotide sequence containing the regulator may be located contiguously or discontinuously within the host cell's genome.

[0149] The term "inducible promoter" refers to a promoter that is activated by an inducer to induce transcription of the gene it controls.

[0150] The phrase "constitutive promoter" refers to a promoter that does not require an inducer to induce the transcription of the gene it controls.

[0151] Unless otherwise stated, the term "expression" refers to the generation of mRNA through the transcription of a relevant gene and / or the production of a protein through gene transcription, followed by mRNA translation.

[0152] The term "catabolism" used in this invention refers to the process / method of breaking down molecules or degrading macromolecules into smaller molecules.

[0153] The term "non-catabolism" refers to processes / methods for constructing molecules from smaller units, and these reactions typically require energy. The term "non-catabolistic compound" refers to a compound produced through non-catabolistic processes / methods.

[0154] Unless the context clearly indicates otherwise, the terms “a,” “an,” and “the” mean “at least one.”

[0155] 6.2 Maltose-dependent degradation determinants

[0156] 6.2.1. Determination of maltose-dependent degradation determinants and maltose-dependent stability of MBP mutants

[0157] The maltose-dependent degradation determinants useful in this invention rely on binding to maltose to maintain their stability and that of their fusion proteins. In the compositions and methods provided by this invention, the maltose-dependent stability of the maltose-dependent degradation determinants and their fusion proteins is, in a sense, conditional on the fact that the maltose-dependent degradation determinants are stable when in contact with maltose, and unstable when maltose is removed from contact with the maltose-dependent degradation determinants. For example, when host cells expressing maltose-dependent degradation determinants and / or their fusion proteins are cultured in a medium containing maltose, they are more stable than when cultured in a medium without maltose. In some embodiments, when maltose is removed from the culture medium, the half-life of the fusion protein is reduced compared to a control (e.g., a fusion protein comprising a target protein fused with a wild-type maltose-binding protein or a target protein alone).

[0158] Any suitable amount of maltose can be contacted with the maltose-dependent degradation determinant and / or its fusion protein to maintain its stability. For example, genetically modified host cells expressing the maltose-dependent degradation determinant and / or its fusion protein can be cultured in a medium containing maltose at concentrations (w / v) of at least about 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2.0%, 2.25%, 2.5%, 2.75%, 3.0%, 3.25%, 3.75%, 4.0%, 4.25%, 4.5%, 4.75%, 5.0% or more. When it is desired to destabilize or degrade maltose-dependent degradation determinants and / or their fusion proteins at a faster rate in host cells, the host cells may be cultured in a new medium that is maltose-free or contains a sufficiently low amount of maltose (e.g., less than about 0.25%, less than about 0.1%, 0%, etc.). The appropriate amount of maltose for maintaining the stability of maltose-dependent degradation determinants and / or their fusion proteins is described in further detail in Section 6.4 below.

[0159] In some implementations, the maltose-dependent degradation determinant can be derived from a maltose-binding protein (MBP) by introducing a destabilizing mutation into a native maltose-bound protein. Without any theoretical limitations, the mutation in the MBP causes the mutant protein to fold properly in dependence of maltose binding energy, and in the absence of maltose, the mutant protein may fail to fold properly and degrade at a faster rate. When fused with another protein, the entire fused protein becomes a target for degradation in the absence of maltose.

[0160] In some embodiments, MBP mutants can be obtained by mutagenesis of the nucleic acid encoding wild-type MBP (e.g., the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of SEQ ID NO: 27, which, in addition to the nucleotide sequence of SEQ ID NO: 1, also contains a linker sequence). In some embodiments, the nucleic acid encoding wild-type MBP can be randomly mutagenesised. In other embodiments, the nucleic acid encoding wild-type MBP can be rationally mutagenesised based on its known structure and / or function. Furthermore, mutations in MBP mutants obtained by random or rational mutagenesis can be combined to generate additional MBP mutants. MBP mutants that can be used as maltose-dependent degradation determinants have been identified and described in Section 6.2.2 and the Examples section below by introducing one or more mutations that disrupt the protein conformation in the absence of binding ligands.

[0161] MBP mutants that can be used as maltose-dependent degradation determinants exhibit one or more of the following characteristics. One characteristic of useful maltose-dependent degradation determinants includes increased stability of the fusion protein in the presence of maltose compared to its absence or a sufficiently low amount. Without being bound by any theory, if the binding of maltose to a maltose-dependent degradation determinant results in a more stable conformation of the fusion protein, then the fusion protein can be degraded at a slower rate by the host cell's degradation mechanisms. Another characteristic of useful maltose-dependent degradation determinants includes their conditional stability in regulating the expression levels or amounts of downstream target molecules by manipulating the maltose content in the culture medium. These and other characteristics of the maltose-dependent stability of maltose-binding proteins and / or their fusion proteins can be determined using any suitable assay method described below.

[0162] In some embodiments, suitable assays for screening MBP mutants (and determining the maltose-dependent characteristics of maltose-dependent degradation determinants) may include measuring the maltose-dependent stability of the fusion protein containing the reporter protein. For example, a reporter protein fused to an MBP mutant frame can be used to measure reporter gene activity in the presence or absence of maltose. As described in Example 7.8 in the Examples section, a reporter gene, such as a fluorescently labeled protein (e.g., GFP), can be used as a fusion partner. In these embodiments, a genetically modified host cell population containing the reporter fusion protein may be pre-cultured to express the fusion protein in a maltose-containing medium. The genetically modified host cell population or subpopulation is then divided into two groups and cultured in parallel, one group of cells in a maltose-containing medium and the other group of cells in a maltose-free medium. After an appropriate time period (e.g., after 48 hours of culture or when the control GFP reporter gene is maximally expressed in the host cells), the fluorescence levels of the host cells cultured in the presence and absence of maltose are compared. If the relative GFP intensity of the GFP fusion protein is higher in the presence of maltose than in the absence of maltose (or in the presence of low amounts of maltose), then the MBP mutant is considered a maltose-dependent degradation determinant.

[0163] For these comparative experiments, genetically modified host cells are typically cultured in a medium containing the same amount (e.g., molar or weight) of carbon source. For example, a medium containing maltose may contain about 2.3% (w / v) sucrose and about 1.7% (w / v) maltose, while a medium without maltose may contain about 4% sucrose.

[0164] In some implementations, if the relative GFP intensity of the fusion protein is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or more higher in the presence of maltose than in the absence of maltose, then the MBP mutant is considered usable as a maltose-dependent degradation determinant. For example, compared to the control (i.e., the GFP intensity of GFP alone in the presence of maltose), host cells expressing the MBP mutant L8_v4d, as shown in Figure 8B, exhibited approximately 95% of the relative GFP intensity in the presence of maltose, compared to approximately 30% in the absence of maltose. Since the reporter gene activity (i.e., fluorescence intensity) of the MBP mutant L8_v4d fusion protein is approximately 316% higher in the presence of maltose than in the absence of maltose, the MBP mutant L8_v4d is useful and is considered a maltose-dependent degradation determinant.

[0165] In Figure 8B, the relative GFP intensity of the fusion protein is normalized to 100% relative to the GFP intensity of the unfused GFP. In some embodiments, the relative GFP intensity of the fusion protein can be normalized relative to the GFP intensity of a GFP fused with wild-type MBP expressed in the presence or absence of maltose. For example, the GFP intensity of a GFP fused with wild-type MBP in the presence of maltose can be calibrated to have 100% intensity, and the GFP intensity of other fusion proteins can be calibrated relative to the wild-type MBP fusion protein. In another embodiment, as shown in Figure 8C, the ratio of GFP fluorescence of each GFP fused to a control (e.g., GFP fused with wild-type MBP) and fused to various MBP mutants in the presence and absence of maltose can be calculated. The calculated ratio for each MBP mutant can be compared to the calculated ratio for the control. If the calculated GFP fluorescence ratio of the MBP mutant is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or more higher than that of the control, then the MBP mutant is considered to be usable as a maltose-dependent degradation determinant.

[0166] Although the use of GFP as a reporter gene has been described above, the determination of fusion protein stability is not limited to the use of GFP or relative GFP intensity assays. Other suitable reporter genes or assays that a person skilled in the art would consider appropriate may be used to compare the stability of fusion proteins in the presence or absence (or in a sufficiently low amount) of maltose to determine the maltose dependence of MBP mutants on their stability.

[0167] In another embodiment, a suitable assay for screening MBP mutants (or determining the maltose-dependent signature of a maltose-dependent degradation determinant) may include measuring the expression level or amount of a target molecule that is directly or indirectly affected by the stability of the fusion protein containing the MBP mutant. Such a screening assay using a target molecule can be illustrated using the exemplary embodiments shown in Figures 1A and 1B. As shown in the figures, the fusion protein comprises Gal80p fused to the maltose-dependent degradation determinant (or MBP mutant) frame. Gal80p is a repressive transcriptional regulator that binds to Gal4p, the major transcriptional activator of the Gal promoter. In the embodiments shown in Figures 1A and 1B, one or more pGal promoters are operatively linked to a biosynthetic pathway gene. If the Gal80p fusion protein in the host cell is stable with maltose bound to the portion of the maltose-dependent degradation determinant (or MBP mutant), it will repress the Gal4p transcriptional activator, resulting in minimal or no expression of the pathway enzyme. This, in turn, will eliminate or reduce the amount of any heterologous compounds generated by said pathway enzyme. If the Gal80p fusion protein is unstable due to the maltose-dependent degradation determinant (or MBP mutant) not contacting maltose, as shown in Figure 1B, then the fusion protein is also unstable or inactivated, thereby relieving Gal80p's inhibition of Gal4p. This will lead to higher expression levels of the pathway enzyme and an increased amount of heterologous compound generated by the pathway enzyme. In this exemplary embodiment, the amount of maltose-dependent heterologous compound generated can be used as a downstream target molecule to screen whether the MBP mutant fused with Gal80p is suitable as a maltose-dependent degradation determinant.

[0168] In some embodiments, isoprene-like farnesene can be used as a downstream target molecule. In this embodiment, the pathway enzyme genes shown in Figures 1A and 1B may include genes encoding mevalonate pathway enzymes and farnesene synthase, and the host cells will generate isoprene-like farnesene as one of the downstream target molecules. When stable Gal80p inhibits the expression of biosynthetic pathway genes, genetically modified host cells will not generate or generate small amounts of farnesene in a medium containing maltose (see Figure 1A). When these host cells are cultured in the absence of maltose, as shown in Figure 1B, the host cells will generate farnesene (when unstable or inactivated Gal80p no longer inhibits the expression of biosynthetic pathway genes). Therefore, in this exemplary embodiment, the amount of farnesene generated by the host cells in the absence or presence of maltose can be used to screen whether MBP mutants fused with Gal80p exhibit maltose-dependent degradation determinants, which depend on maltose binding for stability.

[0169] In the exemplary embodiments shown in Figures 1A and 1B, the fusion protein acts as a negative regulator of the target molecule. If the maltose-dependent stability of the MBP mutant leads to the generation of a greater amount of the target molecule (e.g., farnesene) in the absence of maltose than in the presence of maltose, the MBP mutant can be considered a useful maltose-dependent degradation determinant. The amount of the target molecule (e.g., farnesene) generated in the host cell can be determined using any technique known in the art. For example, the farnesene potency determination method described in Examples 7.2, 7.3, or 7.4 in the Examples section can be used. In the exemplary embodiments shown in Figures 1A and 1B, the MBP mutant is considered a useful maltose-dependent degradation determinant if the amount of target molecules (e.g., farnesene) generated from host cells cultured without maltose is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, or more higher than the amount of target molecules generated from host cells cultured with maltose.

[0170] In some embodiments, the relative percentage increase in target molecule production discussed above can be calculated by dividing the original measurement of target molecule production in the absence of maltose (e.g., the UV farnesene potency assay described in Example 7.2) by the original measurement of target molecule production in the presence of maltose. In some embodiments, the amount of target molecules produced by the host cells can be normalized relative to a control before allocating the two values. For example, the control may include the amount of target molecules produced by host cells constitutively producing target molecules in the presence of maltose. The control amount can be calibrated to 100, and all other target molecule production values ​​obtained from host cells containing the MBP mutant can be normalized relative to this value.

[0171] In another embodiment, suitable assays for screening MBP mutants include using auxotrophic strains and appropriate positive and negative selection protocols to screen for MBP mutants exhibiting maltose-dependent stability. For example, genetic strategies can be designed to screen for MBP mutants that, upon fusion with constitutively transcribed Gal80p, cause Gal80p to transition from a functional (e.g., stable) state in the presence of maltose to a non-functional (e.g., unstable) state in the absence of maltose.

[0172] An exemplary genetic strategy for screening assays is illustrated in Figure 4 and described in Example 7.6 of the Examples section. As shown in Figure 4, the GAL80 gene is fused within the nucleic acid frame encoding the MBP mutant. In this exemplary embodiment, a lysine auxotrophic yeast strain containing the LYS2 gene operatively linked to pGAL10 can be used as a reporter strain to screen for MBP mutants exhibiting a maltose-dependent degradation determinant. If the MBP mutant fused to Gal80p depends on maltose binding to maintain its stability, then it confers maltose-dependent stability to the Gal80p fusion protein. Therefore, in the reporter strain, the function of Gal80p and its maltose-dependent stability can be reported by a phenotype attributable to the expression or repression of the LYS2 gene from a promoter regulated by Gal80 (e.g., pGAL10).

[0173] In this exemplary screening assay, the LYS2 gene is operatively linked to the pGAL10 promoter, as shown in Figure 4. The LYS2 gene encodes aminoadipic acid reductase, an enzyme required for lysine biosynthesis. When the MBP mutant possesses the properties of a maltose-dependent degradation determinant, the Gal80p fused to the MBP mutant will be unstable in the absence of maltose and will not inhibit the expression of LYS2 operatively linked to pGAL10. As a result, aminoadipic acid reductase will be expressed from the LYS2 gene, allowing the reporter yeast to grow on a lysine-deficient medium. To exclude normally destabilized MBP mutants exhibiting maltose-dependent stability, negative or reverse selection can be performed. In reverse selection, the reporter strain is cultured on a medium containing α-aminoadipic acid as the sole nitrogen source. Reporter strains expressing LYS2 will not grow on this medium. If the MBP mutant relies on maltose for stability, then the Gal80p fused to it will be stable and functional in the presence of maltose, thereby inhibiting pGAL10 and preventing LYS2 expression. Therefore, during the reverse selection process, reporter strains containing MBP mutants that are stable due to maltose binding can be selected. The positive and negative selection protocols for screening MBP mutants shown in Figure 4 are merely exemplary. Other suitable auxotrophic reporter strains (e.g., URA3 or TRP1 auxotrophs) with positive and reverse selection protocols can be used to screen for MBP mutants.

[0174] The methods used to determine the maltose-dependent stability of the MBP mutants, maltose-dependent degradation determinants, and their fusion proteins described in this invention are merely exemplary. Those skilled in the art can readily determine other maltose-dependent stability determination methods to screen for MBP mutants that can be used as maltose-dependent degradation determinants in the compositions and methods provided in this invention. For example, the several rounds of competitive selection / anti-selection growth schemes described in Example 7.7 can also be used.

[0175] 6.2.2. Maltose-dependent degradation determinant sequence

[0176] 6.2.2.1 Maltose-dependent degradation determinant amino acid sequence

[0177] On one hand, the present invention provides an amino acid sequence of a maltose-dependent degradation determinant exhibiting maltose-dependent stability. In some embodiments, the maltose-dependent degradation determinant is a mutant derived from any suitable wild-type maltose-binding protein capable of binding maltose. In some embodiments, the maltose-dependent degradation determinant contains one or more destabilizing mutations (e.g., one or more amino acid additions, substitutions, deletions, or insertions) compared to their wild-type counterparts. The maltose-dependent degradation determinant is less stable when not bound to maltose compared to when bound to maltose.

[0178] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having a degree of sequence identity with the wild-type maltose-binding protein having SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 2, and includes at least one variant amino acid residue compared to SEQ ID NO: 2.

[0179] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having a degree of sequence identity with the wild-type maltose-binding protein having SEQ ID NO: 28, wherein SEQ ID NO: 28 has a linker sequence at the C-terminus of SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with SEQ ID NO: 28, and includes at least one variant amino acid residue compared to SEQ ID NO: 28.

[0180] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence containing at least one or more variant amino acid residues located at one or more positions selected from the following: 7, 10, 11, 21, 24, 28, 42, 43, 64, 68, 83, 88, 92, 95, 98, 101, 110, 117, 134, 135, 136, 149, 168, 177, 186, 187, 193, 198, 210, 216, 217, 229, 236, 237, 242, 263, 291, 304, 321, 322, 339, 351, 357, 367, 370, and 374, wherein the positions of these variant amino acid residues correspond to the amino acid positions of SEQ ID NO: 2 or SEQ ID NO: 28.

[0181] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence containing one or more variant amino acid residues, wherein the one or more variant amino acid residues are selected from K7R, I10T, W11G, L21S, V24A, F28Y, D42V, K43E, A64T, F68S, D83G, D88N, P92T, W95R, V98I, N101I, A110T, I117V, P134S, A135T, The group consisting of L136M, M149I, Y168C, Y168N, Y177H, N186S, A187P, L193S, D198V, D210E, A216V, A217D, G229C, I236N, D237N, N242D, L263M, L291V, A304S, T321N, M322L, A339T, A351T, T357S, T367S, S370P, and N374S. The positions of the amino acid residues in these variants correspond to the amino acid positions in SEQ ID NO: 2 or SEQ ID NO: 28.

[0182] In some embodiments, the maltose-dependent degradation determinant may comprise a truncated amino acid sequence compared to the wild-type MBP having SEQ ID NO: 2. For example, the maltose-dependent degradation determinant may comprise an amino acid sequence truncated after amino acid position 365 of SEQ ID NO: 2. Therefore, in some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of positions 1 to 365 of SEQ ID NO: 2, and contains one or more variant amino acid residues at positions selected from the group consisting of: 7, 10, 11, 21, 24, 28, 42, 43, 64, 68, 83, 88, 92, 95, 98, 101, 110, 117, 134, 135, 136, 149, 168, 177, 186, 187, 193, 198, 210, 216, 217, 229, 236, 237, 242, 263, 291, 304, 321, 322, 339, 351, and 357, wherein the positions of these variant amino acid residues correspond to the amino acid positions of SEQ ID NO: 2.

[0183] In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence at positions 1 to 365 of SEQ ID NO: 2, and includes one or more of the following variant amino acid residues: K7R, I10T, W11G, L21S, V24A, F28Y, D42V, K43E, A64T, F68S, D83G, D88N, P92T, W95R, V98I, N101I, A110T, I117V, P134S, A135T, L136M, M149I, Y168C, Y168N, Y177H, N186S, A187P, L193S, D198V, D210E, A216V, A217D, G229C, I236N, D237N, N242D, L263M, L291V, A304S, T321N, M322L, A339T, A351T, and T357S, wherein the positions of these variant amino acid residues correspond to the amino acid positions of SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant may comprise an amino acid sequence shorter than 365 amino acid residues and include one or more variant amino acid residues as described in this invention.

[0184] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having four variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having five variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having six variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having seven variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having eight variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having nine variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having ten variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having eleven variant amino acid residues compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having fewer than four variant amino acid residues (e.g., 1, 2, or 3) compared to SEQ ID NO: 2 or SEQ ID NO: 28. In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence having more than eleven variant amino acid residues (e.g., 12, 13, 14, 15, or more) compared to SEQ ID NO: 2 or SEQ ID NO: 28.

[0185] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence containing at least five variant amino acid residues at positions 10, 24, 42, 149, and 216, wherein the positions of these variant amino acid residues correspond to the positions in SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant comprises at least five variant amino acid residues comprising I10T, V24A, D42V, M149I, and A216V, wherein the positions of these variant amino acid residues correspond to the positions in SEQ ID NO: 2. In some embodiments, the maltose-dependent degradation determinant comprising the amino acid sequence containing these variant amino acid residues is truncated at amino acid position 365 or less.

[0186] In some embodiments, the maltose-dependent degradation determinant comprises an amino acid sequence containing at least one set of variant amino acid residues selected from the group consisting of variant amino acid residues, wherein the position of the variant amino acid residue corresponds to the amino acid position of SEQ ID NO: 2 or SEQ ID NO: 28:

[0187] (a) I10T, V24A, D42V, K43E, D83G, P92T, M149I, Y168N, N186S, A216V, and T357S;

[0188] (b) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and D237N;

[0189] (c)I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and A339T;

[0190] (d)I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and N242D;

[0191] (e)I10T, V24A, D42V, A110T, M149I, and A216V;

[0192] (f) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, and A216V;

[0193] (g)L21S, A64T, L136M, Y177H, A187P, A304S, T321N, and A351T;

[0194] (h)K7R, D83G, V98I, L193S, I236N, and N374S;

[0195] (i)W11G, D88N, P134S, A135T, D210E, and M322L;

[0196] (j)I117V, Y168N, G229C, L263M, T367S, and S370P;

[0197] (k)F68S, W95R, N186S, and D198V; and

[0198] (l)F28Y, K43E, N101I, Y168C, A217D, and L291V.

[0199] In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 4 (MBP mutant 3A6). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 6 (MBP mutant 4D3). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 8 (MBP mutant 5A2). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 10 (MBP mutant 5F3). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 12 (MBP mutant L8). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 14 (MBP mutant L8_v4d). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 16 (MBP mutant H8). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 18 (MBP mutant H9). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 20 (MBP mutant H10). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 22 (MBP mutant M1). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 24 (MBP mutant M5). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence of SEQ ID NO: 26 (MBP mutant M13).

[0200] Many of the aforementioned MBP mutants (e.g., 5A2, 5F3, L8, etc.) are truncated at 365 amino acids and function as maltose-dependent degradation determinants. Therefore, in some embodiments, longer MBP mutants can be used as maltose-dependent degradation determinants in a truncated form. For example, in some embodiments, a useful maltose-dependent degradation determinant comprises the amino acid sequence from position 1 to 365 of SEQ ID NO: 16 (MBP mutant H8). In some embodiments, a maltose-dependent degradation determinant comprises the amino acid sequence from position 1 to 365 of SEQ ID NO: 18 (MBP mutant H9). In some embodiments, a maltose-dependent degradation determinant comprises the amino acid sequence from position 1 to 365 of SEQ ID NO: 20 (MBP mutant H10). In some embodiments, a maltose-dependent degradation determinant comprises the amino acid sequence from position 1 to 365 of SEQ ID NO: 22 (MBP mutant M1). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence from positions 1 to 365 of SEQ ID NO: 24 (MBP mutant M5). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence from positions 1 to 365 of SEQ ID NO: 26 (MBP mutant M13).

[0201] Compared to the wild-type MBP having SEQ ID NO: 2, some MBP mutants contain an additional linker sequence at the C-terminus. Since the linker sequence is not necessarily essential for the function of the maltose-dependent degradation determinant, useful maltose-dependent degradation determinants contain an amino acid sequence of the same length as the wild-type MBP. For example, in some embodiments, useful maltose-dependent degradation determinants contain the amino acid sequence from positions 1 to 370 of SEQ ID NO: 16 (MBP mutant H8). In some embodiments, the maltose-dependent degradation determinant contains the amino acid sequence from positions 1 to 370 of SEQ ID NO: 18 (MBP mutant H9). In some embodiments, the maltose-dependent degradation determinant contains the amino acid sequence from positions 1 to 370 of SEQ ID NO: 20 (MBP mutant H10). In some embodiments, the maltose-dependent degradation determinant contains the amino acid sequence from positions 1 to 370 of SEQ ID NO: 22 (MBP mutant M1). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence from positions 1 to 370 of SEQ ID NO: 24 (MBP mutant M5). In some embodiments, the maltose-dependent degradation determinant comprises the amino acid sequence from positions 1 to 370 of SEQ ID NO: 26 (MBP mutant M13).

[0202] In some embodiments, the maltose-dependent degradation determinant comprises any suitable amino acid sequence described in this invention, with substitutions, deletions, or insertions. Typically, the amino acid changes may be minor, allowing the maltose-dependent degradation determinant to retain its maltose-dependent conditional stability. For example, the substitutions, deletions, or insertions may comprise 1 to about 30 amino acids, and may include, for example, smaller peptide linkers having about 30 amino acid residues or less at the carboxyl terminus or amino terminus.

[0203] In some embodiments, the maltose-dependent degradation determinant comprises any suitable amino acid described in this invention with conserved amino acid substitutions. The following six groups each contain amino acids that are conservedly substituted for each other: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), alanine (A), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). Amino acid substitutions that do not typically alter a particular activity are known in the art, for example, as described in H. Neurath and R.L. Hill, 1979, in *The Proteins*, Academic Press, New York. The most common substitutions are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0204] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaleine, and α-methylserine) can replace amino acid residues in the maltose-dependent degradation determinant described in this invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace amino acid residues. Non-natural amino acids can be modified post-protein synthesis and / or have a chemical structure in their side chains that differs from that of standard amino acids.

[0205] Although this invention describes specific amino acid sequences and specific variant amino acid residues of maltose-dependent degradation determinants, the amino acid sequences of maltose-dependent degradation determinants applicable to the compositions and methods of this invention are not limited to these specific amino acid sequences or variants. For example, Figure 8B shows many other maltose-dependent degradation determinants (e.g., 1-B9, 4-E12, 4-G10, 4-F11, 4-F4, 2-F10, 2-E8, 2-G8, 1-F7, 4-H4, and 2-A4) that exhibit maltose-dependent stability.

[0206] Furthermore, the destabilizing mutations described in this invention can be introduced into any homolog of the MBP containing SEQ ID NO: 2 or SEQ ID NO: 28. For example, other homologs at the corresponding positions of the destabilizing mutations can be readily identified using sequence alignment algorithms known in the art. The amino acid substitutions described in this invention with reference to SEQ ID NO: 2 or SEQ ID NO: 28 can be applied to homologs derived from different species or organisms. For example, the amino acid substitutions described with reference to the positions in SEQ ID NO: 2 or SEQ ID NO: 28 can be derived from homologs of Escherichia coli MBP (e.g., MBPs derived from Yersiniapestis, Vibrio cholerae, Thermotoga maritima, Thermococcus litoralis, Pyrococcus furiosus, etc.). In some embodiments, some of these homologs may have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 28. In other embodiments, the maltose-dependent degradation determinant may be derived from MBP that is not a homolog of *E. coli* MBP, but is stable due to binding to maltose. Therefore, other maltose-dependent degradation determinants (e.g., MBP mutants screened using the assay methods described in this invention) are within the scope of this invention and may be used in the compositions and methods provided by this invention.

[0207] 6.2.2.2 Maltose-dependent degradation determinant nucleic acid sequence

[0208] On the other hand, the present invention provides isolated nucleic acid molecules encoding maltose-dependent degradation determinants. The term "nucleic acid molecule" refers to DNA, RNA, or a combination of both, or any modification thereof, as known in the prior art. Such nucleic acid molecules are single-stranded or double-stranded, straight-chain or circular, and there are no size limitations. The nucleic acid molecules of the present invention can be obtained by recombinant techniques such as PCR, or can be synthesized. In a specific embodiment, the nucleic acid molecule of the present invention is a DNA molecule, such as cDNA.

[0209] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding any of the maltose-dependent degradation determinants described in this invention (e.g., as described in section 6.2.2.1). For example, the isolated nucleic acid molecule comprises a nucleic acid sequence encoding a maltose-dependent degradation determinant having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 90% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 28, and contains at least one variant amino acid residue compared to SEQ ID NO: 2 or SEQ ID NO: 28.

[0210] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a maltose-dependent degradation determinant, the maltose-dependent degradation determinant comprising one or more variant amino acid residues located at one or more positions selected from the group consisting of: 7, 10, 11, 21, 24, 28, 42, 43, 64, 68, 83, 88, 92, 95, 98, 101, 110, 117, 134, 135, 136, 149, 168, 177, 186, 187, 193, 198, 210, 216, 217, 229, 236, 237, 242, 263, 291, 304, 321, 322, 339, 351, 357, 367, 370, and 374, wherein the positions of these variant amino acid residues correspond to SEQ ID NO: 2 or SEQ ID NO: 370. The amino acid position of wild-type MBP at NO:28.

[0211] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a maltose-dependent degradation determinant, wherein, compared to SEQ ID NO: 2 or SEQ ID NO: 28, the maltose-dependent degradation determinant comprises one or more variant amino acid residues, and wherein said one or more variant amino acid residues are selected from K7R, I10T, W11G, L21S, V24A, F28Y, D42V, K43E, A64T, F68S, D83G, D88N, P92T, W95R, V98I, N101I, A110T, I117V, P134S, A135T, L136M, M149I, Y168C, Y168N, Y177H, N186S, A187P, L193S, D198V, D210E, A216V, The group consists of A217D, G229C, I236N, D237N, N242D, L263M, L291V, A304S, T321N, M322L, A339T, A351T, T357S, T367S, S370P, and N374S.

[0212] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a maltose-dependent degradation determinant that is truncated compared to wild-type MBP having SEQ ID NO: 2. For example, the nucleotide sequence may encode a maltose-dependent degradation determinant comprising amino acid residues 1 to 365 of SEQ ID NO: 2, and may also include one or more variant amino acid residues as described in this invention. In some embodiments, the isolated nucleic acid sequence comprises a nucleotide sequence encoding a maltose-dependent degradation determinant that is truncated at a position shorter than 365 amino acid residues.

[0213] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a maltose-dependent degradation determinant, the maltose-dependent degradation determinant comprising an amino acid sequence having at least one set of variant amino acid residues selected from the group consisting of variant amino acid residues, wherein the positions of the variant amino acid residues correspond to the amino acid positions of SEQ ID NO: 2 or SEQ ID NO: 28:

[0214] (a) I10T, V24A, D42V, K43E, D83G, P92T, M149I, Y168N, N186S, A216V, and T357S;

[0215] (b) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and D237N;

[0216] (c)I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and A339T;

[0217] (d)I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, A216V, and N242D;

[0218] (e)I10T, V24A, D42V, A110T, M149I, and A216V;

[0219] (f) I10T, V24A, D42V, K43E, D83G, M149I, Y168N, N186S, and A216V;

[0220] (g)L21S, A64T, L136M, Y177H, A187P, A304S, T321N, and A351T;

[0221] (h)K7R, D83G, V98I, L193S, I236N, and N374S;

[0222] (i)W11G, D88N, P134S, A135T, D210E, and M322L;

[0223] (j)I117V, Y168N, G229C, L263M, T367S, and S370P;

[0224] (k)F68S, W95R, N186S, and D198V; and

[0225] (l)F28Y, K43E, N101I, Y168C, A217D, and L291V.

[0226] In some embodiments, the isolated nucleic acid molecule comprises a nucleic acid molecule that hybridizes under stringent conditions to a nucleotide sequence complementary to SEQ ID NO: 1 or SEQ ID NO: 27 and encodes a maltose-dependent degradation determinant, said maltose-dependent degradation determinant containing at least one of the aforementioned specific mutations and preserving the stability of the maltose-dependent protein. In some embodiments, the stringent conditions are one, two, or three washes at 65°C with salt concentrations corresponding to 0.1 × SSC and 0.1% SDS.

[0227] In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3 (MBP mutant 3A6). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 5 (MBP mutant 4D3). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 7 (MBP mutant 5A2). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 9 (MBP mutant 5F3). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 11 (MBP mutant L8). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 13 (L8_v4d). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 15 (MBP mutant H8). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 17 (MBP mutant H9). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the sequence of SEQ ID NO: 19 (MBP mutant H10). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 21 (MBP mutant M1). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 23 (MBP mutant M5). In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 25 (MBP mutant M13).

[0228] Many of the aforementioned MBP mutant nucleic acids (e.g., 5A2, 5F3, L8, etc.) are truncated at 1098 nucleotides and, when translated into proteins, act as maltose-dependent degradation determinants. Therefore, in some embodiments, maltose-dependent degradation determinants having sequences longer than 1098 nucleotides can be used in a truncated form. For example, in some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1098 of SEQ ID NO: 11. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1098 of SEQ ID NO: 15. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1098 of SEQ ID NO: 17. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the sequence at positions 1 to 1098 of SEQ ID NO: 19. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises nucleotide sequences from positions 1 to 1098 of SEQ ID NO: 21. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises nucleotide sequences from positions 1 to 1098 of SEQ ID NO: 23. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 25.

[0229] Compared to the wild-type MBP nucleic acid having SEQ ID NO: 1, some MBP mutant nucleic acids contain an additional linker sequence at their C-terminus. Therefore, in some embodiments, these MBP mutant nucleic acids may be used without the additional linker sequence at their C-terminus. For example, in some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1113 of SEQ ID NO: 11. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1113 of SEQ ID NO: 15. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1113 of SEQ ID NO: 17. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the sequence at positions 1 to 1113 of SEQ ID NO: 19. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1113 of SEQ ID NO: 21. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence at positions 1 to 1113 of SEQ ID NO: 23. In some embodiments, the maltose-dependent degradation determinant nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 25.

[0230] Mutations can be introduced into any sequence disclosed in this invention (e.g., wild-type MBP having SEQ ID NO: 1) by conventional methods such as polymerase chain reaction (PCR, see Sambrook J et al., Molecular cloning: a laboratory manual, Cold Spring Harbor Press, New York (2001), Ausubel FM et al., Current protocols in molecular biology, John Wiley and sons, New York (1999), Adams A et al., Methods in yeast genetics, Cold Spring Harbor Press, New York (1997)), or by random mutagenesis techniques, such as using mutagens (ultraviolet light or chemicals such as nitrosoguanidine (NTG) or ethyl methanesulfonate (EMS)), or by using PCR techniques (DNA shuffling or error-prone PCR). In some embodiments, any nucleic acid sequence encoding the maltose-dependent degradation determinant can be optimized by genetic / protein engineering techniques known to those skilled in the art, such as directed evolution or rational mutagenesis. Mutants generated using these methods can be screened for maltose-binding stability using any of the assays described in this invention or other assays deemed appropriate by those skilled in the art. Therefore, the scope of the maltose-dependent degradation determinant nucleic acid is not limited to the specific sequences disclosed in this invention, but also includes any MBP mutant nucleic acid that exhibits maltose-dependent stability when encoded as a protein.

[0231] Due to the inherent degeneracy of the genetic code, other polynucleotides encoding essentially the same or functionally equivalent polypeptides described in Section 6.2.2.1 can also be used in the compositions and methods provided by this invention.

[0232] As those skilled in the art will understand, modifying coding sequences to enhance their expression in a particular host can be advantageous. The genetic code is redundant, with 64 possible codons, but most organisms typically use a subset of these codons. The most frequently used codons in a species are called optimal codons, while those that are less frequently used are categorized as rare or low-use codons. In a process sometimes referred to as “codon optimization” or “controlling species codon bias,” codons can be substituted to reflect the host’s preferred codon usage.

[0233] Optimized coding sequences containing codons preferred by specific prokaryotic or eukaryotic hosts (Murray et al., 1989, Nucl Acids Res. 17:477-508) can be prepared, for example, to improve translation rate or generate recombinant RNA transcripts with desired properties. Translation stop codons can also be modified to reflect host preferences. For example, typical stop codons for Saccharomyces cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledons is UGA, while insects and Escherichia coli typically use UAA as their stop codon (Dalphin et al., 1996, Nucl Acids Res. 24:216-8).

[0234] Although the degeneracy of the genetic code and the optimization of coding sequences are discussed in the context of maltose-dependent degradation determinants, this principle can be applied to any coding sequence described in this invention.

[0235] 6.2.3. Fusion Protein

[0236] On the other hand, the present invention provides a maltose-dependent degradation determinant that can fuse with any target protein to control its stability by manipulating the maltose content in the culture medium. For example, the target protein may include transcription factors, enzymes, signal transduction proteins, transport proteins, etc. In some embodiments, the target protein may include proteins that directly affect cellular functions such as cell growth rate. In other embodiments, the target protein is selected such that its maltose-dependent stability can be temporarily altered by manipulating the maltose content to change the level of the desired target molecule.

[0237] In some implementations, transcriptional regulators can be selected as target proteins to fuse with maltose-dependent degradation determinants. Transcriptional regulators fused within the maltose-dependent degradation determinant frame can comprehensively influence the expression of many different downstream target molecules. Examples of transcriptional regulators that can fuse with maltose-dependent degradation determinants include Gal80p or Gal4p. The maltose-dependent stability of the transcriptional regulator fused within the maltose-dependent degradation determinant frame can further modulate the expression level or amount of downstream target molecules. For example, the target molecules may include enzymes, metabolites, or heterologous compounds encoded by biosynthetic genes in a biosynthetic pathway, or generated through enzymatic catalytic reactions.

[0238] Methods for generating fusion proteins and fusion DNA constructs are well known in the art. In short, the method involves ligating DNA encoding a target gene or a portion thereof into DNA encoding a maltose-dependent degradation determinant within the same reading frame. The encoded target protein may be in-frame ligated to the amino or carboxyl terminus of the maltose-dependent degradation determinant. In some embodiments, the coding sequence of the maltose-dependent degradation determinant may be directly ligated to the coding sequence of the target protein. In other embodiments, the fusion protein may comprise a linker (e.g., a peptide linker) ligating the maltose-dependent degradation determinant to the target protein.

[0239] 6.2.4. Nucleic acid constructs and expression vectors

[0240] On the other hand, the present invention provides nucleic acid constructs comprising nucleic acids encoding fusion proteins, said fusion proteins comprising a maltose-dependent degradation determinant fused within a target protein frame. In some embodiments, the nucleic acid encoding the fusion protein may be operatively linked to one or more control sequences that direct the expression of the fusion protein in a host cell under conditions suitable for said control sequences. The control sequences may include any suitable promoter sequences, transcriptional terminal sequences, polyadenylated sequences, etc. These control sequences may be any nucleotide sequences that regulate transcriptional activity in selected host cells and may be obtained from genes encoding target proteins homologous or heterologous to those of the host cells. In some embodiments, nucleic acid constructs for genetically modified host cells comprise one or more optional markers for selecting transformed host cells and applying selection pressure to the host cells to maintain exogenous DNA. Any suitable control or marker sequences known in the art can be used to generate the nucleic acid constructs.

[0241] In some implementations, the promoter sequence operatively linked to the fusion nucleic acid can be an endogenous promoter sequence of the nucleic acid encoding the target protein. In some cases, it may be necessary to match the time response of the fusion protein expression level with the time response of the endogenous expression level of the target protein. For example, if Gal80p is selected as the target protein, its endogenous promoter can be used to drive the expression of the fusion Gal80p protein.

[0242] In some implementations, the promoter sequence operatively linked to the fusion nucleic acid can be a maltose-responsive promoter. By combining a maltose-responsive promoter with a nucleic acid construct encoding a fusion protein containing a maltose-dependent degradation determinant, the composition and method can use a single ligand (e.g., maltose) to control the transcription of the fusion nucleic acid construct and the post-translational stability of the fusion protein encoded therefrom. Examples of suitable maltose-responsive promoters will be described in further detail in Section 6.3 below.

[0243] On the other hand, the present invention provides an expression vector or chromosome integration construct comprising nucleic acid encoding a fusion protein, said fusion protein comprising a target protein and a maltose-dependent degradation determinant. The recombinant expression vector can be any vector suitable for expressing the fusion protein (e.g., plasmid, viral vector, granule). The choice of vector will depend on the compatibility of the vector with the host cell to which the vector is to be introduced and the final application of said host cell. In some embodiments, the vector may further include elements that allow the vector to integrate into the genome of the host cell. In other embodiments, the vector may be an extrachromosomal, autonomously replicating vector present in the host cell.

[0244] 6.2.5. Use of maltose-dependent degradation determinants in methods for regulating protein stability and generating non-catabolizable compounds

[0245] On the other hand, the present invention provides a method for regulating protein stability using maltose-dependent degradation determinants and maltose. The method includes providing a fusion protein comprising a target protein fused within any suitable maltose-dependent degradation determinant frame. In some embodiments, the method includes contacting the fusion protein with maltose such that the fusion protein is more stable when the maltose-dependent degradation determinants are in contact with (e.g., bound to) maltose compared to when the maltose-dependent degradation determinants are not in contact with maltose.

[0246] In some embodiments, the method can be performed in a cell-free system (e.g., a cell extract). For example, a cell extract obtained from a host cell genetically modified to express a fusion protein can be used in the method provided by this invention. The fusion protein in a maltose-free cell extract may be unstable and degrade at a faster rate than desired. When it is desired to increase the stability of the fusion protein and reduce the degradation rate, maltose can be added to the cell extract in the method provided by this invention. In a cell-free system, any suitable technique can be used to determine the maltose-dependent conditional stability of the fusion protein. For example, if the fusion protein contains a fluorescent label as a fusion partner of a maltose-dependent degradation determinant, suitable imaging techniques and / or quantitative kits can be used to determine the fluorescence level of the cell extract.

[0247] In some embodiments, methods for regulating protein stability can be performed using genetically modified host cells expressing the fusion protein of the embodiments of the present invention. For example, host cells genetically modified with nucleic acids encoding the fusion protein can be cultured in a medium containing maltose to maintain the stability of the fusion protein. When it is desired to eliminate or reduce the stability of the fusion protein in the host cells, the culture medium can be modified so that maltose is absent or present in a sufficiently low amount. For example, the host cells can be transferred to a new fermenter containing a maltose-free culture medium. Any residual maltose transferred with the host cells can be consumed by the host cells in the new fermenter, and a carbon source other than maltose (e.g., sucrose) can be added to maintain the host cells.

[0248] The appropriate amount of maltose for maintaining the stability of maltose-dependent degradation determinants (and fusion proteins) can be determined empirically. For example, the saturation or optimal amount of maltose for maintaining the stability of maltose-dependent degradation determinants and their fusion proteins can be determined by plotting protein stability (maltose-dependent degradation determinants and / or fusion proteins) curves with increasing amounts of maltose in the culture medium, i.e., maltose titration. For example, a genetically modified host cell population expressing maltose-dependent degradation determinants (or their fusion proteins) can be divided into multiple subpopulations and cultured in parallel, each subpopulation being grown in a medium containing different amounts, such as increased amounts of maltose (including no maltose), and the expression of maltose-dependent degradation determinants or fusion proteins can be measured after a defined time period. The maltose titration curves will show at least two concentrations of maltose where the stability level of the maltose-dependent degradation determinants or fusion proteins in the host cells is at its lowest level, and therefore their stability level in the host cells reaches its highest level. The saturated or optimal amount of maltose is the quantity or concentration of maltose in the culture medium at which the stability of maltose-dependent degradation determinants or fusion proteins in host cells reaches its highest level. The appropriate amount of maltose for stabilizing maltose-dependent degradation determinants and their fusion proteins will be described in further detail in Section 6.4.

[0249] In some embodiments, the host cell is genetically modified to include a maltose-responsive promoter operatively linked to the nucleic acid encoding the fusion protein. These embodiments will be described in further detail in Sections 6.2.6 and 6.8. In these embodiments, the saturated or optimal amount of maltose includes an amount capable of activating the maltose-responsive promoter to drive the fusion gene to express at the highest level and maintaining maximum post-translational stability of the fusion protein encoded therein. Typically, to increase the activity of the maltose-responsive promoter and the stability of the fusion protein, maltose is present in the culture medium at a level of at least about 5 g / L, typically at least about 10 g / L, and more typically at least about 20 g / L. Typically, maltose is present in the culture medium at a level of less than about 100 g / L, typically less than about 60 g / L, and more typically less than about 50 g / L.

[0250] In some embodiments, when the maltose-dependent degradation determinant in the fusion protein comes into contact with maltose, the fusion protein of the present invention is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300% or more stable than when not in contact with maltose.

[0251] For a given fusion protein, its maltose-dependent stability can be modulated in many different ways. In one embodiment, the concentration of maltose can be adjusted in the culture medium. For example, if the fusion protein requires maximum stability, the optimal amount of maltose determined by the titration profile can be added to the culture medium. In some embodiments, maltose can be used at a higher concentration than the optimal amount determined by the maltose titration profile to ensure that maximum stability of the fusion protein is achieved. In another embodiment, if the fusion protein requires a moderate level of stability, a suboptimal amount of maltose (e.g., half the optimal amount) can be added to the culture medium.

[0252] In another embodiment, the maltose-dependent stability of the fusion protein can be modulated by selecting a suitable maltose-dependent degradation determinant as the fusion partner of the target protein. Different maltose-dependent degradation determinants can confer different levels of stability (e.g., based on fusion protein activity assays) when fused with the target protein, whether in the presence or absence of maltose. For example, as shown in Figure 8B, the stability level of a fusion protein containing GFP fused with the maltose-dependent degradation determinant 4-H10 (e.g., H10) is lower than that of a fusion protein containing GFP and the maltose-dependent degradation determinant L8_v4d, regardless of whether maltose is present or absent. In embodiments where a higher level of stability is required for the fusion protein, a maltose-dependent degradation determinant such as L8_v4d can be used as the fusion partner. In another embodiment where a lower level of stability is required for the fusion protein, the maltose-dependent degradation determinant 4-H10 can be used as the fusion partner. In some implementations, it may be necessary to match the stability level of the fusion protein (when contacted with maltose) to the endogenous stability level of the target protein in the host cell. In such implementations, a suitable maltose-dependent degradation determinant that confers an appropriate level of maltose-dependent stability can be selected as the fusion partner of the target protein.

[0253] In some implementations, maltose-dependent degradation determinants can be selected as fusion partners based on their degradation rate distribution. As shown in Figures 11A and 11B, some maltose-dependent degradation determinants exhibit faster degradation rates in maltose-free cells than other maltose-dependent degradation determinants. For example, when fused with GFP, maltose-dependent degradation determinant 5F3 degrades the fusion protein at a faster rate than maltose-dependent degradation determinant H8. If a slower degradation rate is required for the target protein after maltose removal from the culture medium, a maltose-dependent degradation determinant such as H8 with a slower degradation rate can be used as a fusion partner. On the other hand, if a faster degradation rate is required for the target protein after maltose removal, a maltose-dependent degradation determinant such as 5F3 with a faster degradation rate can be used as a fusion partner for the target protein.

[0254] In some implementations, it may be necessary to use maltose-dependent degradation determinants in genetically modified host cells to control the stability of multiple proteins. For example, maltose may be used to control the stability of two or more enzymes in a biosynthetic pathway. In these implementations, the same maltose-dependent degradation determinant can be used as a fusion partner for all target proteins. Alternatively, each target protein can be fused with a different maltose-dependent degradation determinant, depending on the stability required for each target protein.

[0255] In some embodiments, the target protein selected as the fusion partner for maltose-dependent degradation determinant can be endogenously expressed in the host cell. Since the expression of the endogenous target protein can obscure the maltose-dependent stability of the fusion protein, in some embodiments, the endogenous gene encoding the target protein (e.g., Gal80p) can be functionally disrupted. For example, the endogenous gene can be deleted from the host genome. In another embodiment, a nucleic acid construct containing the nucleic acid encoding the fusion protein can be integrated into the host genome at the site of the endogenous gene encoding the target protein. Any suitable method known in the art can be used to integrate the nucleic acid encoding the fusion protein into the desired target site in the host genome. For example, the heterologous nucleic acid encoding the fusion protein can be integrated into the selected gene after enzymatic cleavage by a nuclease, such as a zinc finger nuclease, a TAL effector domain nuclease, and / or a CRISPR / Cas nuclease system. These techniques are known and described, for example, in U.S. Patent No. 8,685,737; and Horwitz et al. (2015), Cell Systems 1, 1-9, which are incorporated herein by reference in their entirety.

[0256] Any suitable target protein can be selected as the fusion partner of the maltose-dependent degradation determinant described in the embodiments of the present invention. In some embodiments, the target protein is selected such that when fused with the maltose-dependent degradation determinant, it can directly affect cell function by manipulating maltose concentration. For example, the target protein may include a gene product involved in fatty acid synthesis, which can improve cell growth. When cell growth is required, this gene product fused with the maltose-dependent degradation determinant is activated in the culture medium using maltose during the cell biomass building phase of fermentation. When reduced cell growth is required, it can be deactivated by removing maltose during the generation phase of fermentation, thereby diverting cellular resources to the preparation of desired heterologous compounds from host cells. By adjusting the maltose concentration in the culture medium, the stability of the fusion protein containing such a target protein can be directly and temporarily regulated as needed at different stages of the fermentation process.

[0257] In other embodiments, the target protein is selected as a fusion partner of a maltose-dependent degradation determinant, such that the maltose-dependent stability of the target protein can be used to cascade its effects to one or more downstream target molecules by manipulating maltose content. In these embodiments, the fusion protein containing the maltose-dependent degradation determinant can interact with one or more biomolecules (e.g., other proteins or nucleic acids) in the host cell to indirectly regulate the expression level or amount of one or more target molecules. For example, indirect regulation of one or more enzymes in a biosynthetic pathway can be achieved by fusing a maltose-dependent degradation determinant with a single heterologous transcription factor, the expression of which in turn regulates the expression of one or more enzymes (e.g., all enzymes) in the biosynthetic pathway. Exemplary embodiments of indirect regulation are illustrated in the schematic diagrams of Figures 1A to 2B, which show the regulation of genes in various biosynthetic pathways as GAL regulators.

[0258] The GAL regulator in yeast provides an exemplary regulatory network of activators, repressors, and promoters that can be used in combination with the maltose-dependent degradation determinants described in this invention. Yeast utilizes galactose as a carbon source to access and metabolize galactose within the cell through the expression of GAL genes. The GAL genes include structural genes GAL1, GAL2, GAL7, and GAL10 encoding galactokinase, galactose permease, α-D-galactose-1-phosphate uridineyltransferase, and uridine diphosphate galactose-4-epimerase, respectively, and regulatory genes GAL4, GAL80, and GAL3. The GAL4 gene product is a positive regulator (i.e., an activator), while the GAL80 gene product is a negative regulator (i.e., a repressor / repressor) of the expression of GAL1, GAL2, GAL7, and GAL10 genes. Gal4p activates transcription within the promoters of pGAL1, pGAL7, and pGAL10 by binding to upstream activating sequences (UAS), such as those of the GAL structural genes. In the absence of galactose, the expression of structural proteins (Gal1p, Gal2p, Gal7p, and Gal10p) is typically detected in very small amounts because Gal80p interacts with Gal4p and inhibits Gal4p transcriptional activity. However, in the presence of galactose, Gal3p interacts with Gal80p, thereby relieving Gal80p's inhibition of Gal4p. This allows downstream genes of Gal4p binding sequences such as GAL1, GAL2, GAL7, and GAL10 gene products to be expressed.

[0259] Figures 1A and 1B illustrate exemplary embodiments in which a fusion protein that indirectly and negatively interacts with one or more biomolecules uses the GAL regulator to regulate the expression level or amount of one or more target molecules. In the embodiments shown in Figures 1A and 1B, the transcriptional regulator Gal80p is a target protein fused within a maltose-dependent degradation determinant frame. Gal80p is a repressive cofactor (i.e., a transcriptional repressor) of the transcriptional activator Gal4p (e.g., a biomolecule) that binds to a pGal promoter such as pGal1, pGal2, pGal7, or pGAL10 (e.g., a biomolecule). As shown in Figure 1A, the pGal promoter is operatively linked to one or more genes (e.g., target molecules) encoding enzymes in a biosynthetic pathway. In the presence of maltose in the culture medium, as shown in Figure 1A, maltose binds to the maltose-dependent degradation determinant, and the fusion protein is stabilized and expressed at a relatively high level. The Gal80p portion of the fusion protein binds to and inhibits the transcriptional activator Gal4p, thereby activating the pGal promoter. This action, in turn, inhibits the transcription of biosynthetic pathway genes from the pGal promoter, reducing the expression levels of enzymes and other downstream target molecules generated by the catalytic reactions of enzymes (e.g., non-catabolite compounds). Therefore, in the embodiment shown in Figure 1A, when maltose comes into contact with the maltose-dependent degradation determinant, the fusion protein negatively regulates the expression levels or amounts of target molecules (e.g., enzymes, non-catabolite compounds, etc.).

[0260] In Figure 1B, when maltose is removed from the culture medium, the fusion protein containing Gal80p in the host cells becomes unstable and degrades at a faster rate, resulting in a lower Gal80p expression level in the host cells compared to the stage shown in Figure 1A. This relieves the inhibition of the transcription activator Gal4p, which can then bind to the pGal promoter and drive the expression of genes involved in biosynthetic pathways. Therefore, when maltose is present in the culture medium, the levels of target molecules catalyzed by the enzymatic reaction, such as enzymes, metabolites, and heterologous compounds, increase compared to the stage shown in Figure 1A.

[0261] Figures 2A and 2B illustrate fusion proteins that interact with one or more biomolecules to indirectly and positively regulate the expression levels or amounts of one or more target molecules. As shown in Figure 2A, the host cell contains the transcriptional activator Gal4p as a target protein fused with a maltose-dependent degradation determinant. When maltose is added to the culture medium, as shown in Figure 2A, the fusion protein is stabilized by contact between maltose and the maltose-dependent degradation determinant. This increases the overall stability of the fusion protein containing Gal4p, which in turn binds to the pGal promoter (e.g., a biomolecule) to drive the expression of biosynthetic pathway genes (e.g., target molecules). The expression of biosynthetic pathway genes, in turn, increases the production of heterologous compounds from the catalytic reaction products of downstream target molecules, such as these enzymes. In this embodiment, when it is necessary to reduce or eliminate the expression levels or amounts of one or more target molecules produced in the host cell, the maltose content can be reduced or maltose can be removed, as shown in Figure 2B. In this exemplary embodiment, the expression of the endogenous GAL80 gene can be functionally disrupted, so that the endogenous Gal80p, as a repressor of Gal4p, does not negatively regulate Gal4p activity.

[0262] In some embodiments, different modes of regulation by the fusion protein may coexist in a single host cell. For example, the host cell may contain a fusion protein that directly affects cell function (e.g., cell growth) and another fusion protein that positively regulates the expression levels or amounts of one or more target molecules. In another embodiment, the host cell may contain a fusion protein that directly affects cell function and another fusion protein that negatively regulates the expression levels or amounts of one or more target molecules. In yet another embodiment, the host cell may contain a fusion protein that negatively regulates the expression levels or amounts of one or more target molecules and a fusion protein that positively regulates the expression levels or amounts of one or more target molecules.

[0263] The embodiments shown in Figures 1A to 2B are merely exemplary. Any suitable combination of target proteins can be used as a fusion partner of the maltose-dependent degradation determinant, and any combination of suitable regulatory modes can be used in the compositions and methods provided in this invention.

[0264] 6.2.6. Dual transcriptional and post-translational control by maltose-responsive promoters and maltose-dependent degradation determinants

[0265] On the other hand, the present invention provides a method for providing transcriptional control of gene expression and post-translational stability control of gene products by manipulating maltose content. By combining genetic elements that confer maltose-dependent post-translational stability with any gene fused within a maltose-induced promoter frame, the compositions and methods provided by the present invention can impose very robust and stringent control over the timing of expression and stability of the target protein (and any downstream target molecule).

[0266] Therefore, the present invention provides a method for controlling the expression and stability of a target protein in a genetically modified host cell using a maltose-dependent degradation determinant and a maltose-responsive promoter. In some embodiments, the host cell contains a maltose-responsive promoter operably linked to a heterologous nucleic acid encoding a fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame. In the presence of sufficient maltose, transcription of the heterologous nucleic acid is activated (or increased), and the fusion protein encoded therefrom is stabilized. At appropriate time points, the host cell can be cultured in a medium in the absence of maltose or in a sufficiently low amount, such that the activity of the maltose-responsive promoter and the stability of the fusion protein are reduced compared to when the medium contains sufficient maltose. As a result, the expression of the heterologous nucleic acid can be negatively regulated in the absence (or in a sufficiently low amount) of maltose. Therefore, in these embodiments, the same effector molecule (e.g., maltose) can be used to provide simultaneous transcriptional control of the target gene and post-translational control of the gene product.

[0267] Figures 3A and 3B illustrate schematic diagrams demonstrating dual transcriptional control and post-translational control of the gene product using an exemplary gene expression method employing maltose and a maltose-dependent degradation determinant. As shown in Figures 3A and 3B, the nucleic acid encoding a fusion protein containing Gal80p and the maltose-dependent degradation determinant is operatively linked to a maltose-responsive promoter (e.g., the natural promoter pMAL or a synthetic maltose-responsive promoter such as the pGMAL promoter). In the presence of maltose, as shown in Figure 3A, the pMAL or pGMAL promoter is activated (or its activity is increased), and the fusion DNA construct is transcribed to encode a fusion protein containing Gal80p fused within the maltose-dependent degradation determinant frame. In the presence of maltose, the fusion protein is stable with maltose bound to the maltose-dependent degradation determinant, thereby providing high levels of fusion protein expression and stability. As shown in Figure 3B, when maltose is removed from the culture medium, the maltose-responsive promoter is inactivated or its activity is reduced, and any fusion protein encoded by the fusion DNA construct becomes unstable and degrades at a faster rate.

[0268] The embodiments shown in Figures 3A and 3B are merely exemplary. Dual transcriptional and post-translational control using a maltose-responsive promoter and a maltose-dependent degradation determinant can be applied to any situation where tight control over the expression and stability of the fusion protein and / or downstream target molecules is required.

[0269] 6.2.6.1 Using a combination of a maltose-responsive promoter, a maltose-dependent degradation determinant, and maltose content manipulation as a switch to generate non-catabolite compounds.

[0270] In specific embodiments, the methods and compositions provided by the present invention utilize maltose-responsive promoters and maltose-dependent degradation determinants to manipulate maltose content in fermentation media to directly or indirectly regulate the expression and / or stability of heterologous enzymes capable of influencing the production of non-catabolite compounds in genetically modified host cells. In these embodiments, the nucleic acid encoding one or more target molecules shown in Figures 3A and 3B may include a heterologous nucleic acid encoding a biosynthetic pathway gene that encodes an enzyme (e.g., a mevalonate pathway enzyme) capable of producing non-catabolite compounds (e.g., isoprene-like compounds).

[0271] Maltose (or its analogues or derivatives) is inexpensive, non-toxic, and stable. It is an attractive molecule for controlling the timing of gene expression and protein stability, particularly for large-scale production processes. In some embodiments, naturally occurring maltose-responsive promoters do not always provide the tight transcriptional control required for extended production processes when operatively linked to a target gene. Therefore, in some embodiments, maltose-dependent degradation determinants can be used in combination with maltose-responsive promoters to simultaneously control the timing of gene expression and / or protein stability (e.g., enzymes in biosynthetic pathways that generate non-catabolite compounds in genetically modified host cells during fermentation). In other embodiments, synthetic maltose-responsive promoters can also be used in combination with maltose-dependent degradation determinants to simultaneously control the timing of gene expression and / or protein stability in the production of non-catabolite compounds.

[0272] In one embodiment, when host cell fermentation is carried out in the presence of maltose (e.g., at least about 0.1% maltose), the production of non-catabolite compounds is substantially reduced or shut off. When the amount of maltose in the fermentation medium is reduced or eliminated, the production of non-catabolite compounds is turned on or increased. Therefore, in some embodiments, the genetically modified cells of the present invention contain heterologous biosynthetic pathway genes regulated by a maltose-responsive promoter and a fusion protein comprising a target protein fused within a maltose-dependent degradation determinant frame. Such systems are capable of using the maltose content in the fermentation medium as a switch for the production of non-catabolite compounds. Controlling the timing of non-catabolite compound production occurs only when needed, redirecting carbon flux during non-production phases towards cell maintenance and cell biomass. This more efficient use of carbon significantly reduces the metabolic burden on host cells, improves cell growth, increases the stability of heterologous genes, reduces strain degradation, and contributes to improved overall cell health and viability.

[0273] In some embodiments, the fermentation method comprises a two-step process / method utilizing maltose as a switch to achieve "off" and "on" phases. In the first step (i.e., the "construction" phase, step (a)) where compound generation is not required, genetically modified host cells are grown in a growth or "construction" medium containing maltose in an amount sufficient to induce gene expression under the control of a maltose-responsive promoter, and the induced gene product is used to negatively regulate the generation of non-catabolite compounds. After transcription of the fusion DNA construct under the control of the maltose-responsive promoter, the stability of the fusion protein is controlled post-translationally. In the second step (i.e., the "generation" phase, step (b)), the fermentation is carried out in a medium containing a carbon source, wherein maltose is absent or present in a sufficiently low amount to reduce or inactivate the activity of the maltose-responsive promoter and destabilize the fusion protein. As a result, the amount of heterologous non-catabolite compounds generated by the host cells is turned on or increased.

[0274] In other embodiments, a maltose-responsive promoter can be operatively linked to one or more heterologous nucleic acids encoding one or more enzymes of a biosynthetic pathway. The presence of an activated amount of maltose in the culture medium increases the expression of the one or more enzymes of the biosynthetic pathway. Additionally or alternatively, in some embodiments, the one or more heterologous nucleic acids encoding the enzyme can be fused within a nucleic acid frame encoding a maltose-dependent degradation determinant. In these embodiments, the presence of a sufficient amount of maltose in the culture medium will increase the expression of one or more enzymes of the biosynthetic pathway, and the fusion enzyme is stabilized in the presence of maltose. In this manner, the maltose-responsive promoter and the maltose-dependent degradation determinant can be linked as positive regulators of the generation of non-catabolite compounds.

[0275] 6.3 Maltose-responsive promoter

[0276] On the other hand, the present invention provides maltose-responsive promoters for use in the compositions and methods described herein to promote the transcription of operatively linked DNA-coding sequences in the presence of maltose. In some embodiments, unmodified maltose-responsive promoters (e.g., pMAL promoters) derived from the regulatory networks of maltose fermentation systems of various organisms can be used to control the transcription of operatively linked DNA-coding sequences. In other embodiments, synthetic maltose-responsive promoters can be used to control the transcription of operatively linked DNA-coding sequences. As described in detail below, the synthetic maltose-responsive promoters provided by the present invention offer certain advantages because they reduce “leakage” of gene expression under uninduced conditions (e.g., in the absence of maltose) compared to unmodified maltose-responsive promoters.

[0277] 6.3.1. pMAL promoter

[0278] In some embodiments, maltose-responsive promoters that can be used in the methods and compositions provided by this invention promote transcription of operatively linked DNA coding sequences in the presence of maltose. In some embodiments, any maltose-responsive promoter known in the art can be used to regulate the expression of enzymes capable of generating non-catabolite compounds. In some embodiments, the maltose-responsive promoter is 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 pMAL promoter includes modified forms of these promoters that increase or decrease promoter activity compared to an unmodified pMAL promoter. An exemplary modified pMAL promoter includes pMAL32_v1 (SEQ ID NO:78).

[0279] Other useful maltose-responsive promoters that can be used in the methods and compositions provided by this invention can be derived from the regulatory network of the maltose fermentation system of *Saccharomyces cerevisiae*. Maltose fermentation in *Saccharomyces cerevisiae* requires the presence of at least one of five unconnected MAL loci: MAL1, MAL2, MAL3, MAL4, and MAL6. Each of these loci consists of a gene complex involved in maltose metabolism; said complex includes maltose permease (MALx1, where x represents one of the five loci), maltase responsible for the intracellular hydrolysis of the sugar (MALx2), and a positive regulatory protein that induces the transcription of two prior genes in the presence of maltose (MALx3). See Cheng & Michels, J. Bacteriol. 173:1817-1820 (1991); Dubin et al., J. Bacteriol. 164:605-610 (1985); Chang et al., Curr. Genet. 14:201-209 (1988); Higgins et al., Appl. Environ. Microbiol. 65:680-685 (1999). At the MAL6 locus, the activator is encoded by the MAL63 gene. Mal63p is the DNA-binding transcriptional activator required for the MAL structural genes encoding maltose permease and maltase in a maltose-dependent manner.

[0280] The MAL activator intermediate complex is stable in the absence of the inducer maltose, but the addition of maltose results in the release of the inducible MAL activator from the active form of the complex, which is capable of DNA binding and transcriptional activation. See Ran, F. and Michels., CA, J. Biol. Chem. 285(18):13850-13862 (2010). The binding sites of the MAL63 protein in the MAL61-62 promoters of different transcriptions have been characterized as upstream activation sequences of the MAL gene. See 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 herein by reference in their entirety.

[0281] At the MAL1, MAL2, MAL3, and MAL4 loci, the activators are encoded by the MAL13, MAL23, MAL33, and MAL43 genes, respectively. (Vidgren et al., Appl. Environ. Microbiol. 71(12):7864-7857(2005). Mal13p, Mal23p, Mal33p, and Mal43p are DNA-binding transcription activators encoded by those genes required for maltose-dependent induction of the MAL structural genes.

[0282] Other maltose-responsive promoters that can be used in the methods and compositions provided by this invention are derived from the regulatory network of the maltose / maltodextrin metabolic system in *E. coli*. The malT nucleic acid encodes MalT, one of four maltose-responsive promoters (P...). PQ P EFG P KBM and P SThe combination of malT and mal promoters produces tightly regulated expression systems, which have been shown to act as strong promoters induced by added maltose. See, for example, Schleif, “Two Positively Regulated Systems, ara and mal,” pp. 1300-1309 in Escherichia coliand Salmonella Cellular and Molecular Biology, Second Edition, Neidhardt et al., eds., ASMPress, Washington, DC, 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 herein by reference in their entirety.

[0283] Other maltose-responsive promoters that can be used in the methods and compositions provided in this invention include those in Berkner et al., “Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarious,” Extremophiles 14:249-259 (2010); and those in U.S. Patent No. 5,824,545.

[0284] 6.3.2. Synthesis of maltose-responsive promoters

[0285] In some embodiments, useful maltose-responsive promoters include synthetic maltose-responsive promoters. In some embodiments, genes operatively linked to a natural, unmodified maltose-responsive promoter (e.g., pMAL32) can express gene products at low levels even in the absence of maltose. Furthermore, when cells are cultured under conditions that promote low cell growth rates in the absence of maltose, the expression of genes operatively linked to a natural maltose-responsive promoter can be positively regulated. See Example 7.14 and Figures 14A and 14B.

[0286] Therefore, this invention provides a synthetic maltose-responsive promoter that, compared to a naturally occurring unmodified maltose-responsive promoter, reduces the leakage expression of gene products in the absence of maltose or in the presence of sufficiently low amounts of maltose. In some embodiments, a synthetic maltose-responsive promoter is constructed using a galactose-inducible pGAL promoter by removing at least one or all Gal4p binding sites and inserting one or more binding sites of a Mal operon activator (i.e., a Mal transcription activator). For example, all four Gal4p binding sites can be removed from the pGAL1 promoter, and various copy numbers of the binding sites of the Mal operon activator (e.g., the binding sites of Malx3p as shown in Figure 13, including, for example, Mal13p, Mal23p, Mal33p, Mal43p, and Mal63p) can be inserted into the modified pGAL1 promoter. In some embodiments, a single binding site of the Mal transcription activator can be inserted into the modified pGAL promoter. In some embodiments, two, three, four, five, six, seven, eight, nine, ten, or more binding sites of the Mal transcription activator can be inserted into a modified pGAL promoter. These modified pGAL promoters, having had their Gal4p binding sites removed and the binding sites of the inserted Mal transcription activator removed, are referred to in this invention as pGMAL promoters.

[0287] In some implementations, the pGAL1_GAL10 promoter (SEQ ID NO: 82) can be used as a background promoter to generate the pGMAL promoter. In some implementations, the pGAL1 promoter is derived from a different GAL1_GAL10 promoter (Johnston & Davis, Mol. CellBiol. 4(8): 1440-1448 (1984)). The pGAL1_GAL10 promoter includes four Gal4p binding sites (located at positions 216 to 232, 235 to 251, 253 to 269, and 317 to 333 on SEQ ID NO: 82). All of these Gal4p binding sites can be removed from the pGAL1_GAL10 promoter, and one or more Mal transcription activator binding sites can be added to generate additional synthetic maltose-responsive promoters. These synthetic promoters derived from the pGAL1_GAL10 promoter include, for example, pGMAL_v5 (SEQ ID NO: 35), pGMAL_v6 (SEQ ID NO: 36), pGMAL_v7 (SEQ ID NO: 37), pGMAL_v9 (SEQ ID NO: 38), pGMAL_v10 (SEQ ID NO: 39), pGMAL_v11 (SEQ ID NO: 40), pGMAL_v12 (SEQ ID NO: 41), pGMAL_v13 (SEQ ID NO: 42), pGMAL_v14 (SEQ ID NO: 43), pGMAL_v15 (SEQ ID NO: 44), pGMAL_v16 (SEQ ID NO: 45), pGMAL_v17 (SEQ ID NO: 46), and pGMAL_v18 (SEQ ID NO: 47).

[0288] In some implementations, the pGAL2 promoter (SEQ ID NO: 83) can be used as a background promoter to generate the pGMAL promoter. The pGAL2 promoter includes four Gal4p binding sites and two overlapping Gal4p binding sites (located at positions 230 to 246, 344 to 360, 363 to 379, 427 to 443, and 432 to 448 of SEQ ID NO: 83). At least one or all of these Gal4p binding sites can be removed from the pGAL2 promoter, and one or more Mal transcription activator binding sites can be added to generate an additional synthetic maltose-responsive promoter. These synthetic promoters derived from the pGAL2 promoter include, for example, pG2MAL_v1 (SEQ ID NO: 48), pG2MAL_v2 (SEQ ID NO: 49), pG2MAL_v3 (SEQ ID NO: 50), pG2MAL_v5 (SEQ ID NO: 51), pG2MAL_v6 (SEQ ID NO: 52), pG2MAL_v7 (SEQ ID NO: 53), pG2MAL_v8 (SEQ ID NO: 54), pG2MAL_v9 (SEQ ID NO: 55), and pG2MAL_v10 (SEQ ID NO: 56).

[0289] In some implementations, the pGAL7 promoter (SEQ ID NO: 79) can be used as a background promoter to generate the pGMAL promoter. The pGAL7 promoter includes two Gal4p binding sites (located at positions 471 to 487 and 558 to 574 of SEQ ID NO: 79). At least one or all of these Gal4p binding sites can be removed from the pGAL7 promoter, and one or more Mal transcription activator binding sites can be added to generate additional synthetic maltose-responsive promoters. These synthetic promoters derived from the pGAL7 promoter include, for example, pG7MAL_v2 (SEQ ID NO: 57), pG7MAL_v4 (SEQ ID NO: 58), pG7MAL_v6 (SEQ ID NO: 59), pG7MAL_v8 (SEQ ID NO: 60), and pG7MAL_v9 (SEQ ID NO: 61).

[0290] In some embodiments, a heterozygous promoter can be constructed by combining two or more sequences from pGAL1, pGAL2, and pGAL7. From the heterozygous promoter, at least one or all Gal4p binding sites can be removed, and one or more MAL transcription activator binding sites can be added to generate additional synthetic maltose-responsive promoters. These synthetic promoters derived from heterozygous promoters include, for example, pG172_MAL_v13 (SEQ ID NO: 62), pG271_MAL_v12 (SEQ ID NO: 63), pG721_MAL_v11 (SEQ ID NO: 64), and pG712_MAL_v14 (SEQ ID NO: 65).

[0291] In some implementations, pGCY1, pGAL80, or other pGAL promoters can be used as background promoters to generate the pGMAL promoter. The sequences of these promoters and the Gal4p binding site are well-known. See, for example, the Yeast Genome Database (http: / / www.yeastgenome.org / ). The nucleotide sequence of the Gal4p binding site is also well-known; it can be removed and replaced by a MAL transcription activator binding site.

[0292] In some embodiments, the synthetic promoter may comprise a portion of the pGMAL promoter sequence disclosed in this invention, wherein the portion retains promoter function, rather than the entire sequence associated with the SEQ ID number. In some embodiments, some nucleotide bases in the middle or at the end of the disclosed promoter sequence may not be essential to its promoter function. Therefore, in some embodiments, the synthetic maltose-responsive promoter may typically comprise at least about 200, 250, 300, 350, 400, 450, 475, 500, 525, 550, 575, 600, 625, 650, 575, 600, 625, 650 nucleotides or more of the specific sequence described in this invention, which retains promoter function. For example, portions of these sequences may include binding sites for transcription activators and other transcriptional regulators to retain promoter function. In some embodiments, the synthetic maltose-responsive promoter sequence disclosed in this invention may be further modified, for example, by adding or removing binding sites for MAL transcription activators. In other embodiments, the synthetic maltose-responsive promoter sequence disclosed in this invention may further include additional sequences, such as linker sequences at the N' and / or C' ends of the promoter sequence. For example, a 24 or 36 nucleotide linker sequence may be added to the pGMAL sequence provided in this invention to provide sufficient space between the promoter sequence and the coding sequence.

[0293] In some embodiments, synthetic or naturally derived promoters do not need to have the exact sequence disclosed in this invention to maintain their promoter function in genetically modified host cells. Although many promoter sequences are highly conserved, even for the same promoter, sequence differences exist in strains or species. Therefore, in some embodiments, this invention provides synthetic or naturally derived promoters having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the promoter sequences disclosed in this invention.

[0294] Furthermore, as shown in Figures 14A and 14B, each of these pGMAL promoters possesses different promoter strengths and characteristics. For example, when maltose-induced, pGMAL_v15 has a strength comparable to the natural promoter pMAL32. In another embodiment, pGMAL_v12 is a stronger promoter than pGMAL_v15, with a strength comparable to promoter pTDH3. Any suitable pGMAL promoter can be selected for the compositions and methods provided by this invention, depending on the promoter strength required for gene expression.

[0295] In some implementations, promoters other than the pGAL promoter can be used as background promoters to generate synthetic maltose-responsive promoters. The choice of background promoter may depend on the choice of host cell, the desired expression level, etc. Therefore, in some implementations, for any selected background promoter, its endogenous transcription activator binding site can be removed, and a MAL transcription activator binding site can be inserted. For example, the MAL transcription activator binding site sequence from the bidirectional promoter pMAL12 can be incorporated into the background promoter (with the natural transcription activator binding site removed) to generate a synthetic maltose-responsive promoter. Exemplary sequences of the four MAL transcription activator binding sites from pMAL12 include the following 11 or 12 base pairs of nucleotide sequences and their reverse complementary sequences:

[0296] pMAL12_1: GATAATATTTC (SEQ ID NO: 97);

[0297] pMAL12_2: GAAAATTTCGC (SEQ ID NO: 98);

[0298] pMAL12_3:GTTAAAGTTTAC (SEQ ID NO: 99);

[0299] pMAL12_4: GAAATTTCGC (SEQ ID NO: 100);

[0300] pMAL12_1r: GAAATATTATC (SEQ ID NO: 101);

[0301] pMAL12_2r: GCGAAATTTC (SEQ ID NO: 102);

[0302] pMAL12_3r:GTAAACTTTAAC (SEQ ID NO: 103); and

[0303] pMAL12_4r: GCGAAAATTTC (SEQ ID NO: 104).

[0304] In some implementations, the MAL transcription activator binding site sequence from the bidirectional promoter pMAL32 can be incorporated into the background promoter (excluding the natural transcription activator binding site) to generate a synthetic maltose-responsive promoter. An exemplary sequence of the MAL transcription activator binding site from pMAL32 includes the following 11 or 12 base pairs of nucleotides and their reverse complementary sequence:

[0305] pMAL32_1: TATAATATTTC (SEQ ID NO: 105);

[0306] pMAL32_2: GAAAATTTCGC (identical to pMAL12_2; SEQ ID NO: 98);

[0307] pMAL32_3:GTTTAAGTTTAC (SEQ ID NO: 106);

[0308] pMAL32_4:GAAGTTTTTCGC (SEQ ID NO: 107);

[0309] pMAL32_1r: GAAATATTATA (SEQ ID NO: 108);

[0310] pMAL32_2r: GCGGAAATTTTC (identical to the second one in pMAL12_2r; SEQ ID NO: 102);

[0311] pMAL32_3r:GTAAACTTAAAC (SEQ ID NO: 109); and

[0312] pMAL32_4r: GCGAAAACTTC (SEQ ID NO: 110).

[0313] These short 11 or 12-base-pair fragments representing MAL transcription activator binding sites share at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% sequence identity with each other. Furthermore, as shown in Figure 13B, these short 11 or 12-base-pair fragments representing MAL transcription activator binding sites contain one of the following sequence motifs: DAADDTTTH, DWADDTTTH, DAADDTTWH, or DWADDTTWH.

[0314] The symbol for a sequence motif has the following meanings:

[0315] A = adenine nucleotide;

[0316] T = nucleotide thymine;

[0317] W = Nucleotide A (adenine) or T (thymine);

[0318] D = nucleotide G (guanine) or A (adenine) or T (thymine); and

[0319] H = nucleotide A (adenine), C (cytosine), or T (thymine).

[0320] Therefore, in some embodiments, the synthetic maltose-responsive promoter comprises a sequence motif selected from the group consisting of DAADDTTTH, DWADDTTTH, DAADDTTWH, DWADDTTWH, and combinations thereof. In some embodiments, any or a combination of these sequence motifs is incorporated into a background promoter having its natural transcriptional activator binding site removed.

[0321] In some embodiments, the synthetic maltose-responsive promoter comprises a core promoter and one or more MAL transcription activator binding sites. As used in this invention, the core promoter refers to the smallest portion of the promoter required to properly initiate transcription of a selected DNA sequence operatively linked to it. The term "core promoter" refers to a promoter element that provides basic transcription. It optionally comprises a TATA cassette or a TATA-like cassette and is complexed with RNA polymerase. In some embodiments, the synthetic maltose-responsive promoter comprises one or more copies of the MAL transcription activator binding site described in this invention. In some embodiments, the MAL transcription activator binding site comprises a sequence motif selected from the group consisting of DADDDTTTH, DWADDTTTH, DADDDTTWH, DWADDTTWH, and combinations thereof.

[0322] In some embodiments, under uninduced conditions (e.g., host cells cultured in a maltose-free medium), the promoter activity of a synthetic maltose-responsive promoter is less than that of a natural maltose-responsive promoter, wherein one or more MAL transcription activator binding sites (e.g., pMAL31, pMAL11, pMAL12, etc.) are derived from the natural maltose-responsive promoter. For example, when host cells containing a reporter gene (e.g., GFP) operatively linked to a synthetic maltose-responsive promoter are cultured in a maltose-free medium, under uninduced conditions, reporter gene expression under the synthetic maltose-responsive promoter is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or more, less than reporter gene expression operatively linked to a natural maltose-responsive promoter derived from one or more MAL transcription activator binding sites.

[0323] The MAL transcription activator binding site sequences described in this invention are merely exemplary; MAL transcription activator binding sites from other maltose-responsive promoters can be inserted into synthetic maltose-responsive promoters. In some embodiments, the synthetic promoter may contain one or any combination of the inserted MAL transcription activator binding sites. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more binding sites of the MAL transcription activator may be inserted into the synthetic promoter. Generally, the higher the copy number of the MAL transcription activator binding sites in the synthetic promoter, the greater the increase in promoter strength.

[0324] 6.3.3. Application of maltose-responsive promoters in heterologous nucleic acid expression and non-catabolite generation

[0325] In some embodiments, while the maltose-responsive promoters described in this invention can be used in conjunction with the maltose-dependent degradation determinants described in Section 6.2.6 above, the maltose-responsive promoters can also be used in the absence of maltose-dependent degradation determinants to promote the transcription of any operablely linked gene in the presence of maltose. For example, any gene known to promote cell growth during the cell biomass building phase can be operablely linked to a synthetic maltose-responsive promoter, such that maltose in the culture medium during this phase can activate the transcription of these genes. During the generation phase, maltose can be removed from the culture medium, thereby negatively regulating the expression of these cell growth-promoting genes operablely linked to the synthetic maltose-responsive promoter to redirect cellular resources to the generation of the desired product. Depending on the level of the desired gene product, a suitable maltose-responsive promoter with appropriate promoter strength can be selected from the compositions and methods provided in this invention.

[0326] Therefore, the present invention provides a heterologous nucleic acid encoding a target gene, said target gene being operatively linked to a maltose-responsive promoter or a portion thereof that retains promoter function. In some embodiments, the maltose-responsive promoter is a synthetic maltose-responsive promoter generated from a background promoter (which is unresponsive to maltose), and its natural transcription activator binding site is replaced by a MAL transcription activator binding site. In some embodiments, the synthetic maltose-responsive promoter is a pGMAL promoter. In some embodiments, the target gene operatively linked to the synthetic maltose-responsive promoter may include a transcriptional regulatory factor. For example, the transcriptional regulatory factor may include Gal80p or Gal4p. In some embodiments, the target gene operatively linked to the maltose-responsive promoter may include a gene that contributes to cell growth rate during the biomass construction phase of a fermentation process. In some embodiments, the target gene operatively linked to the maltose-responsive promoter includes a gene encoding a pathway enzyme. The application of the synthetic maltose-responsive promoter provided by the present invention is not limited to the fermentation environment, but can also be used as an inducible promoter to regulate the expression of any gene.

[0327] In some embodiments, a maltose-responsive promoter can be used in a fermentation environment in the method for preparing heterologous non-catabolite compounds. The method provided by this invention utilizes genetically modified host cells containing heterologous nucleic acids encoding one or more enzymes of an enzymatic pathway for preparing heterologous non-catabolite compounds. In some embodiments, the expression of the one or more enzymes is directly controlled by the maltose-responsive promoter of this invention. That is, each of the one or more heterologous nucleic acid sequences encoding the one or more enzymes of the enzymatic pathway is operatively linked (i.e., located at 3') to the maltose-responsive promoter, and the maltose-responsive promoter drives the expression of each of the one or more heterologous nucleic acids in the presence of maltose.

[0328] In other embodiments, the expression of one or more enzymes in the enzyme pathway is indirectly regulated by a maltose-responsive promoter. For example, indirect regulation of one or more enzymes in the pathway can be achieved by operatively linking a maltose-responsive promoter to a single heterologous transcription factor, the expression of which in turn directly regulates the expression of the one or more pathway enzymes (e.g., all enzyme members). The GAL regulator in yeast described above provides an exemplary regulatory network of activators, repressors, and promoters that can be used in combination with the maltose-responsive promoters described in this invention.

[0329] In some embodiments, one or more GAL4-activated promoters, such as pGAL1, pGAL7, pGAL10, pGCY1, and / or pGAL80, are operatively linked to and used to drive the expression of said one or more enzymes in the enzymatic pathway for the preparation of heterologous non-catabolite compounds. In some embodiments, the host cell further comprises 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 nucleic acid encoding GAL80 under the control of the maltose-responsive promoter described in this invention, and the expression of the GAL80 gene product is induced in the presence of maltose. Gal80p, in turn, interacts with Gal4p and inhibits Gal4p transcriptional activity. When maltose is removed or sufficiently depleted so that GAL80 expression is no longer induced, the inhibition of Gal4p by Gal80p is released, and the expression of said one or more enzymes in the enzymatic pathway for the preparation of heterologous non-catabolite compounds is freely activated.

[0330] In the above embodiments, if desired, one or more growth-promoting genes may be placed under the control of a maltose-responsive promoter to further utilize a maltose switch to separate the cell growth phase from the generation phase of heterologous non-catabolite compounds. In some embodiments, one or more genes promoting the generation of heterologous non-catabolite compounds may be operatively linked to a GAL4-activated promoter, allowing them to be expressed co-expressed with one or more enzymes of the enzymatic pathway used to prepare the heterologous non-catabolite compounds.

[0331] In other embodiments, the natural pGAL4 promoter is replaced by a heterologous nucleic acid containing a maltose-responsive promoter. In some embodiments, the host cell contains a heterologous nucleic acid encoding Gal4p, which is operatively linked to the heterologous nucleic acid containing the maltose-responsive promoter. In one embodiment, the maltose-responsive promoter is operatively linked to the coding sequence of Gal4p, and the coding sequence of one or more enzymes (e.g., all enzyme members) of the enzymatic pathway for preparing heterologous non-catabolite compounds is operatively linked to the 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 pGAL1. In some embodiments, the GAL4-responsive promoter is pGAL7. In some embodiments, the GAL4-responsive promoter is pGAL10. In some embodiments, the GAL4-responsive promoter is pGCY1. In some implementations, the GAL4-responsive promoter is pGAL80.

[0332] A detailed description of methods for generating non-catabolite compounds using maltose-responsive promoters is described in Section 6.5 and in U.S. Patent Publication Nos. 2015-0299713 and WO2015 / 020649, which are incorporated herein by reference in their entirety for all purposes. In the compositions and methods described herein, any suitable maltose-responsive promoter (synthetic or natural) can be used to express any target gene and / or to generate non-catabolite compounds.

[0333] 6.4 Inhibited and Uninhibited Maltose Content

[0334] Maltose is a disaccharide formed from two glucose molecules, as shown below. It has the chemical formula C2. 12 H 22 O 11 Its molecular weight is 343 g / mol.

[0335]

[0336] In some embodiments, in addition to or replacing maltose (as shown above and its isomers), other substrates that function similarly to maltose in the methods of the present invention can be used to stabilize maltose-dependent degradation determinants and / or to induce maltose-responsive promoters. For example, substrates that specifically bind to MBP, MBP mutants, and maltose-dependent degradation determinants and are suitable as ligands thereto can be selected from the group consisting of maltose, maltodextrin, macromolecular α(1→4)-linked dextran (e.g., maltotriose), or combinations thereof. In some embodiments, suitable analogues or derivatives of these substrates (i.e., maltose, maltodextrin, and macromolecular α(1→4)-linked dextran) can also be used to stabilize maltose-dependent degradation determinants (or to induce maltose-responsive promoters). In this invention, the terms "analyte" and "derivative" are used interchangeably to refer to a chemical substance that is structurally and functionally related to another substance, in which case it maintains the ability to specifically bind to maltose-binding proteins and maltose-dependent degradation determinants to stabilize maltose-dependent degradation determinants and / or induce maltose-responsive promoters. In some embodiments, unlike maltose, they are not metabolized by host cells. Examples of maltose analogs and derivatives include maltose derivatives such as methyl-α-maltose glycoside and 5-thiomaltose. Other examples of maltose analogs and derivatives include maltoheptaose (β-cyclodextrin), maltitol, maltohexaose, maltetritol, maltohexatonic acid, maltotetraose, etc. These and other substrates that specifically bind to MBP, MBP mutants, and maltose-dependent degradation determinants and stabilize maltose-dependent degradation determinants and / or induce maltose-responsive promoters are collectively referred to as "maltose-based inducers".

[0337] In some embodiments, although maltose is described throughout the invention as an MBP mutant and a ligand for maltose-dependent degradation determinants, and as an inducer for maltose-responsive promoters, any suitable maltose-based inducer (e.g., maltose, maltodextrin, and analogs or derivatives of macromolecular α(1→4)-linked dextran) may be used instead of maltose. Therefore, any disclosure relating to maltose as described in this invention also applies to other maltose-based inducers. Similarly, any discussion regarding the amount of inhibited and non-inhibited maltose applies to other maltose-based inducers.

[0338] In some embodiments, the “inducible” amount of maltose is sufficient to induce the desired high expression level of the coding sequence operably linked to the maltose-responsive promoter and / or preserve the stability of the maltose-dependent degradation determinant and / or fusion protein. In some embodiments, the “inducible” amount of maltose is a sufficient amount to “contact” the maltose-dependent degradation determinant with maltose or to stabilize the maltose-dependent degradation determinant (or its fusion protein). In some embodiments, the “inducible” amount of maltose is an amount that activates or enhances the activity of the maltose-responsive promoter compared to promoter activity without maltose. In some embodiments, the “non-inducible” amount of maltose, compared to the “inducible” amount of maltose present in the culture medium, is an amount of maltose below which the expression of the coding sequence operably linked to the maltose-responsive promoter is not induced or reduced. In some implementations, the “non-inducible” amount of maltose is the amount of maltose that reduces the activity of maltose-responsive promoters and / or reduces the stability of maltose-dependent degradation determinants (and their fusion proteins) compared to the “inducible” amount of maltose present in the culture medium.

[0339] The “inducible” and “non-inducible” amounts of maltose used in the methods provided by this invention can be determined for any genetically modified host cell capable of generating heterologous noncatabolite compounds as described above. In some embodiments, the non-inducible amount of maltose is determined by gene expression profiling, i.e., maltose titration, with the amount of maltose in the culture medium used for the fermentation process continuously increasing. For example, the genetically modified host cell population can be divided into multiple subpopulations and cultured in parallel, wherein each subpopulation is grown in a culture medium containing different amounts, such as increased amounts of maltose (including no maltose), and the expression of reporter genes or the production of noncatabolite compounds is analyzed after a defined time period.

[0340] In some embodiments, a maltose-responsive promoter (and / or a maltose-dependent degradation determinant) is linked in the presence of maltose to achieve a “off” state for the generation of non-catabolite compounds. The maltose titration comprises at least two concentrations of maltose, thereby minimizing the stability of the amount of non-catabolite compounds generated by the host cell; that is, no further decrease in compound generation is observed with increasing maltose concentration. In some embodiments, the “inhibitory” amount of maltose is at least a minimum amount at which the amount of non-catabolite compounds generated by the host cell is at its lowest level (e.g., approximately zero). This amount may also be referred to as the “saturated” or “optimal” amount of maltose for inhibiting the generation of non-catabolite compounds in a particular host cell. In some such embodiments, even when the amount of compound generation is low, the “inhibitory” amount of maltose may include any concentration of maltose at which the generation of non-catabolite compounds has been reduced relative to the “on” state. In some embodiments, in this configuration of the switch, the “non-inhibitory” amount of maltose is any amount of maltose below the “inhibitory” amount. In some embodiments, the non-inhibitory amount of maltose is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times, or more than about 100 times, less than the inhibitory amount of maltose. In a specific embodiment, the non-inhibitory amount of maltose is less than about 0.8% (w / v) of the culture medium. In another specific embodiment, the non-inhibitory amount of maltose is about 0% (w / v) of the culture medium.

[0341] In a specific embodiment, as described above, for a given host cell, the inhibitory amount of maltose is an optimal or saturated amount, and the non-inhibitory amount is maltose-free. In another specific embodiment, the inhibitory amount of maltose is at least about 0.25%, and the non-inhibitory amount is maltose-free. In another specific embodiment, the inhibitory amount of maltose is about 0.25% to 3% of maltose, and the non-inhibitory amount is maltose-free. In another specific embodiment, the inhibitory amount of maltose is about 0.5% to 1% of maltose, and the non-inhibitory amount is maltose-free. In another specific embodiment, the inhibitory amount of maltose is at least about 3% of maltose, and the limit is maltose-free.

[0342] In some embodiments, a maltose-responsive promoter (and / or a maltose-dependent degradation determinant) is linked in the presence of maltose to achieve an "off" state for the generation of non-catabolite compounds, wherein the inhibition level of maltose in the culture medium is at least about 0.1% (weight of maltose per volume of culture medium). In some embodiments, the inhibition level of maltose in the culture medium is at least about 0.25%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 0.5%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 0.75%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 1.0%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 1.25%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 1.5%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 1.75%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 2.0%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 2.25%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 2.5%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 2.75%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 3.0%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 3.25%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 3.5%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 3.75%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 4.0%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 4.25%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 4.5%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 4.75%. In some embodiments, the inhibition level of maltose in the culture medium is at least about 5.0%. In some embodiments, the inhibition level of maltose in the culture medium is between about 5% and 50%. In some embodiments, the inhibition level of maltose in the culture medium is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50%.

[0343] In some embodiments, the non-inhibitory amount of maltose is an amount that is at least about 2, 10, 100, 1000, 10,000, or about 100,000 times lower than the inhibitory amount of maltose determined according to the above method. In some embodiments, the non-inhibitory amount of maltose is an amount that is at least about 2, 10, 100, 1000, 10,000, or 100,000 times lower than the saturated amount of maltose determined according to the above method. In some embodiments, the non-inhibitory amount of maltose is less than about 50%, less than about 20%, less than about 10%, less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.1%, less than about 0.01%, or less than about 0.001% of the inhibitory amount of maltose determined according to the above method. In some embodiments, the non-inhibitory amount of maltose is less than about 50%, less than about 20%, less than about 10%, less than about 1%, less than about 0.1%, less than about 0.01%, or less than about 0.001% of the maltose saturation amount determined according to the above method. In a specific embodiment, the non-inhibitory amount of maltose is about 0 mg / L (0%), i.e., maltose-free. Therefore, in this specific embodiment, the host cells are grown in a cell culture medium containing no external source of maltose during the generation phase.

[0344] In some embodiments, a maltose-responsive promoter (and / or a maltose-dependent degradation determinant) is linked in the presence of maltose to achieve an "on" state for the generation of non-catabolite compounds, wherein the maltose titration comprises at least two concentrations of maltose, thereby maximally stabilizing the generation of non-catabolite compounds by the host cell, i.e., no further increase in the amount of compound generation is observed with increasing maltose concentration. In some embodiments, the "non-inhibitory" amount of maltose is at least a minimum amount at which the amount of non-catabolite compounds generated by the host cell is at its highest level. In this configuration of the switch, the amount may also be referred to as the "saturation" or "optimal" amount of maltose for inducing the generation of non-catabolite compounds in a particular host cell. In some such embodiments, even when the amount of compound generation is at a suboptimal level, the "inducing" or "non-inhibitory" amount of maltose may include any concentration of maltose at which the amount of non-catabolite compound generation has increased relative to the "off" state. In some embodiments, in this configuration of the switch, the "non-inducible" or "inhibitory" amount of maltose is any amount of maltose less than the "inducible" or "non-inhibitory" amount. In some embodiments, the non-inducible or inhibitory amount of maltose is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times, or more than about 100 times, less than the inducible or non-inhibitory amount of maltose. In a specific embodiment, the non-inducible amount of maltose is less than about 0.8% (w / v) of the culture medium. In another specific embodiment, the non-inducible amount of maltose is about 0% (w / v) of the culture medium.

[0345] In a specific embodiment, as described above, for a given host cell, the inducing amount of maltose is an optimal or saturation level, and the non-inducing amount is maltose-free. In another specific embodiment, the inducing amount of maltose is at least about 0.25%, and the non-inducing amount is maltose-free. In another specific embodiment, the inducing amount of maltose is from about 0.25% to 3% of maltose, and the non-inducing amount is maltose-free. In another specific embodiment, the inducing amount of maltose is from about 0.5% to 1% of maltose, and the non-inducing amount is maltose-free. In yet another specific embodiment, the inducing amount of maltose is at least about 3% of maltose, and the limit is maltose-free.

[0346] In some embodiments, a maltose-responsive promoter (and / or a maltose-dependent degradation determinant) is linked in the presence of maltose to achieve an "on" state for the generation of non-catabolite compounds, wherein the maltose induction amount in the culture medium is at least about 0.1% (weight of maltose per volume of culture medium). In some embodiments, the maltose induction amount in the culture medium is at least about 0.25%. In some embodiments, the maltose induction amount in the culture medium is at least about 0.5%. In some embodiments, the maltose induction amount in the culture medium is at least about 0.75%. In some embodiments, the maltose induction amount in the culture medium is at least about 1.0%. In some embodiments, the maltose induction amount in the culture medium is at least about 1.25%. In some embodiments, the maltose induction amount in the culture medium is at least about 1.5%. In some embodiments, the maltose induction amount in the culture medium is at least about 1.75%. In some embodiments, the maltose induction amount in the culture medium is at least about 2.0%. In some embodiments, the maltose induction content in the culture medium is at least about 2.25%. In some embodiments, the maltose induction content in the culture medium is at least about 2.5%. In some embodiments, the maltose induction content in the culture medium is at least about 2.75%. In some embodiments, the maltose induction content in the culture medium is at least about 3.0%. In some embodiments, the maltose induction content in the culture medium is at least about 3.25%. In some embodiments, the maltose induction content in the culture medium is at least about 3.5%. In some embodiments, the maltose induction content in the culture medium is at least about 3.75%. In some embodiments, the maltose induction content in the culture medium is at least about 4.0%. In some embodiments, the maltose induction content in the culture medium is at least about 4.25%. In some embodiments, the maltose induction content in the culture medium is at least about 4.5%. In some embodiments, the maltose induction content in the culture medium is at least about 4.75%. In some embodiments, the maltose induction content in the culture medium is at least about 5.0%. In some embodiments, the maltose induction amount in the culture medium is between about 5% and 50%. In some embodiments, the maltose induction amount in the culture medium is about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or about 50%.

[0347] In some embodiments, in the presence of maltose, a maltose-responsive promoter (and / or a maltose-dependent degradation determinant) is linked to achieve an "on" state for the generation of non-catabolite compounds, wherein the inhibitory amount of maltose is at least about 2, 10, 100, 1000, 10,000, or about 100,000 times lower than the non-inhibitory amount of maltose determined according to the above method. In some embodiments, the inhibitory amount of maltose is at least about 2, 10, 100, 1000, 10,000, or about 100,000 times lower than the saturated amount of maltose determined according to the above method. In some embodiments, the amount of maltose inhibition is less than about 50%, less than about 20%, less than about 10%, less than about 1%, less than about 0.5%, less than about 0.2%, less than about 0.1%, less than about 0.01%, or less than about 0.001% of the amount of maltose non-inhibitory as determined by the above methods. In some embodiments, the amount of maltose inhibition is less than about 50%, less than about 20%, less than about 10%, less than about 1%, less than about 0.1%, less than about 0.01%, or less than about 0.001% of the amount of maltose saturation as determined by the above methods. In a specific embodiment, the amount of maltose inhibition is about 0 mg / L (0%), i.e., maltose-free. Therefore, in this specific embodiment, the host cells are grown in a cell culture medium containing no external source of maltose during the construction phase.

[0348] While the terms "inducible," "non-inducible," "inhibitory," and "non-inhibitory" for maltose have been primarily discussed within the context of methods for generating non-catabolizable compounds, these terms and their meanings apply to any compositions and methods provided by this invention. For example, an "inducible" amount of maltose comprises a sufficient quantity of maltose to induce stability in a fusion protein containing a target protein fused within a maltose-dependent degradation determinant frame. In another embodiment, a "non-inducible" amount of maltose comprises a sufficiently low quantity of maltose or none at all, making the fusion protein unstable.

[0349] 6.5 Using maltose-dependent degradation determinants and / or maltose-responsive promoters to generate non-catabolite compounds

[0350] In some embodiments of the fermentation method provided by the present invention, maltose conditions are manipulated by combining a maltose-responsive promoter and / or a maltose-dependent degradation determinant, such that the amount of non-catabolite compounds generated during the construction phase (step (a) of the above method) is less than about 50, 40, 30, 20, or 10% of the maximum amount of non-catabolite compounds generated by the genetically modified host, for example, when host cells are cultured during the construction phase (step (b) of the above method), the amount of non-catabolite compounds generated is about 50, 40, 30, 20, or 10%.

[0351] The duration of the build-up and generation phases in a fermentation process can vary and depends on factors such as the host cell growth rate and its intrinsic growth rate; and other culture conditions such as pH and temperature, depending on the specific requirements of the host cell, the fermentation process, and the overall process. However, since certain negative selection pressures associated with the generation of non-catabolite compounds are alleviated in the “off” state, any duration of the build-up phase is expected to provide some benefit to the final productivity of the fermentation.

[0352] In some embodiments, the construction phase is carried out for a period of time sufficient to generate cellular biomass capable of supporting the production of non-catabolite compounds during the construction phase. In some embodiments, the construction phase is carried out for a period of time sufficient to allow the cell population present at inoculation to undergo multiple doublings until the desired cell density is reached. In some embodiments, the construction phase is carried out for a period of time sufficient to allow the host cell population in the fermentation vessel or container in which the construction phase is carried out to reach a cell density (OD) between approximately 0.01 and 400. 600 In some implementations, the construction phase is performed until an OD of at least 0.01 is achieved. 600 In some implementations, the construction phase is carried out until an OD of at least about 0.1 is reached. 600 In some implementations, the construction phase is carried out until an OD of at least approximately 1.0 is reached. 600 In some implementations, the construction phase is carried out until at least about 10 OD is reached. 600 In some implementations, the construction phase is carried out until at least approximately 100 OD is reached. 600 In some implementations, the build phase is performed until an OD between approximately 0.01 and 100 is reached. 600 In some implementations, the build phase is performed until an OD of approximately 0.1 to 10 is reached. 600 In some implementations, the construction phase is carried out until an OD of approximately 1 to 100 is reached. 600 In other implementations, the construction phase lasts for at least approximately 12, 24, 36, 48, 60, 72, 84, 96 hours, or more than approximately 96 hours.

[0353] In some embodiments, the generation phase is carried out for a period of time sufficient to generate the desired amount of non-catabolite compound. In some embodiments, the generation phase is carried out for at least about 12, 24, 36, 48, 60, 72, 84, 96 hours or more than about 96 hours.

[0354] In some implementations, the generation phase lasts for approximately 3 to 20 days. In some implementations, the generation phase lasts for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 days or more.

[0355] In some embodiments, the method for generating non-catabolizable compounds includes culturing host cells in separate construction and generation media. For example, the method may include culturing genetically modified host cells during a construction phase, wherein the cells are cultured under non-generation conditions to generate an inoculum, and then transferring the inoculum to a second fermentation medium under conditions suitable for inducing compound generation, maintaining steady-state conditions during the second fermentation phase to generate a cell culture containing the non-catabolizable product. In some embodiments, maltose is present in the construction medium, while maltose is absent in the fermentation medium, thus generating a cell culture containing the non-catabolizable product. Any residual maltose transferred along with the cells in the construction medium will be metabolized by the cells during the fermentation phase.

[0356] In some embodiments, the method provided by the present invention is sufficient to generate one or more non-catabolite compounds in amounts greater than about 10 grams per liter of fermentation medium. In some such embodiments, non-catabolite-derived compounds are generated in amounts 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.

[0357] In some embodiments, the method provided by the present invention is sufficient to generate one or more non-catabolite compounds in an amount greater than about 50 mg per gram of stem cell weight. In some embodiments, the amount of the recombinant non-catabolite compound generated is about 50 to about 1500 mg, more than about 100 mg, more than about 150 mg, more than about 200 mg, more than about 250 mg, more than about 500 mg, more than about 750 mg, or more than about 1000 mg per gram of stem cell weight.

[0358] In some embodiments, the implementation of the method provided by the present invention results in an increase in the amount of non-catabolite compounds generated from a genetically modified population of host cells, compared to the amount generated by a method that does not include a generation phase in the culture of host cells under non-generation conditions. In some embodiments, implementation of the method results in the generation of one or more non-catabolite compounds in an amount 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1,000 times or more, based on a unit volume of cell culture.

[0359] In some embodiments, implementation of the method results in the generation of one or more non-catabolite compounds in an amount 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1,000 times or more, based on the weight of each unit of stem cells.

[0360] In some embodiments, implementation of the method results in the generation of one or more non-catabolite compounds in an amount 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1,000 times or more, based on a unit volume of cell culture per unit time.

[0361] In some embodiments, implementation of the method results in the generation of one or more non-catabolite compounds in an amount 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 times, at least about 2.5 times, at least about 5 times, at least about 10 times, at least about 20 times, at least about 30 times, at least about 40 times, at least about 50 times, at least about 75 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400 times, at least about 500 times, or at least about 1,000 times or more, based on the weight of each unit of stem cells per unit time.

[0362] 6.6 Stabilization constructs and their applications in coupled cell growth and the generation of non-catabolite compounds

[0363] On the other hand, the compositions and methods provided by this invention can counteract the potential negative impact of spontaneous mutations on the production of heterologous noncatabolite compounds by genetically modified host cells. As mentioned above, the production of heterologous noncatabolite compounds from genetically modified host cells typically requires a net input of ATP, NADPH, and carbon, and usually requires a large supply of oxygen to help balance the redox reactions of the system. This environment makes evolution tend towards lower-level products, higher biomass producing more favorable genotypes, and mutations occur spontaneously in host cells, including loss of mutations. Therefore, mutant host cells with reduced product yields have an "adaptive advantage" compared to the original high-product-yielding parent cells because more metabolic resources of the mutant host cells are used to build biomass. This results in mutants of low-product-yielding or no-product-yielding cells (referred to as "broken strains") with higher growth rates outnumbering mutants of the original high-product-yielding parent cells with slower growth rates. The different growth rates of these cells, in turn, lead to mutants, with low or no product yields, which eventually take over the cell population in the fermenter (a process known as “strain degradation”) and cause a significant decrease in product yield over time.

[0364] To stabilize the production of heterologous noncatabolite compounds, in some embodiments, the expression of nucleic acids encoding proteins affecting cell growth is coupled to the production of heterologous noncatabolite compounds. For example, nucleic acids encoding proteins affecting cell growth and one or more nucleic acids encoding enzymes in the biosynthetic pathway for producing noncatabolite compounds are operatively linked to their respective promoters, which are co-regulated by the same transcription factor. In other words, these nucleic acids, which are not normally regulated by the same transcription factor, are co-regulated as regulators. As a result, any spontaneous mutation that negatively affects the expression or stability of the transcription factor will negatively affect the expression of all nucleic acids in the regulator. When the expression of genes affecting cell growth in the regulator is reduced due to spontaneous mutations, this will lead to unfavorable growth conditions for these mutant cells compared to parental cells with high product yields. Therefore, the expression of regulators co-regulated by the same transcription factor is more stable against mutations that may reduce the production of heterologous noncatabolite compounds.

[0365] Due to its stabilizing effect on the formation of non-catabolizable compounds, the phrase "stabilizing construct" used in this invention refers to a nucleic acid encoding a protein that affects cell growth, said nucleic acid being operatively linked to a promoter regulated by a common transcriptional regulator of the target regulator (i.e., a group of target genes regulated as a unit). Furthermore, in some embodiments, the term "heterologous nucleic acid encoding a protein affecting cell growth" refers to a nucleic acid encoding a protein affecting cell growth that is operatively linked to a heterologous promoter rather than its endogenous promoter. Similarly, in some embodiments, the term "heterologous nucleic acid encoding an enzyme of a biosynthetic pathway" is used to refer to a nucleic acid encoding an enzyme that is operatively linked to a heterologous promoter rather than its endogenous promoter.

[0366] In some embodiments, promoters that respond to common transcriptional regulators of the target regulator are used to drive the expression of a conditionally essential gene in a metabolic pathway whose end product can be consumed by genetically modified host cells. For example, the conditionally essential gene could be the LYS9 gene in a biosynthetic pathway that produces lysine as the end product. In this embodiment, the conditionally essential gene in the metabolic pathway is a gene that affects cell growth, as functional disruption of the conditionally essential gene would affect cell growth or viability. Cell populations containing this gene design require high expression of the target regulator (when cultured in the absence of externally added lysine), and therefore, the expression of the regulator is more stable against mutations that may reduce regulator expression. This method of regulator stability is compatible with host cells containing gene designs with gene switches that conditionally reduce regulator expression when needed, for example, during the biomass build-up phase (e.g., the GAL regulator shown in Figure 17). During the build-up phase, nutritional deficiencies caused by reduced expression of the conditionally essential gene can be compensated for by providing the target metabolite (e.g., lysine) to the growth medium. Such exemplary implementations are shown in Figure 17 and are described in further detail below.

[0367] The exemplary embodiments shown in Figure 17 relate to the enzymatic generation of heterologous noncatabolite compounds via a biosynthetic pathway, while the compositions and methods provided by the present invention have broader applications. For example, they can be used to generate any heterologous noncatabolite compound, such as a target protein encoded by a heterologous nucleic acid in a host cell. Examples of target heterologous proteins include antibodies, vaccines, antibiotics, hormones (e.g., insulin or human growth factor), etc. Similar to noncatabolite compounds generated by enzymes via a biosynthetic pathway, the generation of a large amount of the target heterologous protein puts pressure on the metabolic resources of the host cell. This environment can favor the evolution of mutant cells with a genotype that produces lower-grade products and higher biomass, leading to unstable generation of the heterologous target protein. Therefore, the expression of the stabilizing constructs provided by the present invention can be coupled with the expression of nucleic acids encoding any target heterologous protein to stabilize its generation during fermentation. Any discussion relating to the enzymatic generation of noncatabolite compounds via the biosynthetic pathway described in the present invention also applies to the generation of any target heterologous protein that is also a noncatabolite compound.

[0368] 6.6.1. Co-linking of transcriptional regulators with the generation of cell growth and non-catabolite compounds

[0369] Any suitable transcriptional regulator can be used to couple the expression of nucleic acids encoding proteins that affect cell growth with the generation of non-catabolite compounds (or any target protein). In one embodiment, the transcriptional regulator of the GAL regulator can be used to couple the expression of one or more heterologous nucleic acids encoding one or more enzymes of a biosynthetic pathway with the expression of heterologous nucleic acids encoding proteins that affect cell growth. In one embodiment, the transcriptional activator Gal4p can be used as a co-transcriptional regulator to regulate the expression of all heterologous nucleic acids in the regulator. In another embodiment, the transcriptional repressor Gal80p can be used as a co-transcriptional repressor to inhibit the expression of all heterologous nucleic acids in the regulator. In some embodiments, both Gal4p and Gal80p are used as co-transcriptional regulators to co-regulate the expression of all heterologous nucleic acids in the regulator.

[0370] The use of transcriptional regulators in GAL regulators is merely exemplary; any suitable transcriptional regulator can be used to co-regulate and control the expression of nucleic acids in the regulator. For example, MAL transcriptional activators can be used as co-transcriptional regulators to regulate the expression of nucleic acids in the regulator, wherein each nucleic acid is operatively linked to a maltose-responsive promoter. In another embodiment, Pho regulator activators can be used as co-transcriptional regulators to regulate the expression of nucleic acids in the regulator when each nucleic acid is operatively linked to a Pho regulator promoter.

[0371] In some implementations, artificial transcription regulators can be used to co-regulate the expression of nucleic acids in regulators. For example, LexA fused with an activation domain can be used as an artificial transcription regulator. Transcription regulators typically consist of separable functional domains: a DNA-binding domain that interacts with a specific DNA sequence; and an activation domain that interacts with other proteins to mimic promoter transcription. The separable functional domains of transcription regulators can be replaced with natural and non-natural counterparts to generate artificial transcription regulators.

[0372] The selection of common transcriptional regulators for the transcription and expression of heterologous nucleic acids that couple proteins encoding one or more enzymes that affect cell growth and biosynthetic pathways (or any target protein) will depend on the host cell, the required level of heterologous non-catabolite compounds to be produced, and other parameters related to the genetically modified host cell.

[0373] 6.6.2. Nucleic acids encoding proteins that affect cell growth are operatively linked to co-regulated promoters as stabilization constructs.

[0374] Any suitable nucleic acid encoding a protein that affects cell growth can be used in a stabilization construct to express one or more enzymes in a biosynthetic pathway that couples with a common transcriptional regulator to produce a non-catabolite compound (or any target protein). As used in this invention, the terms "nucleic acid encoding a protein that affects cell growth" or "gene that affects cell growth" refer to a nucleic acid encoding a protein that affects the cell growth (e.g., growth rate, cell biomass, or cell viability) of a genetically modified host cell. Any spontaneous mutations that negatively affect the production of common transcriptional regulators will also negatively affect the expression of the nucleic acid encoding the protein that affects cell growth, thus negatively impacting cell growth. Therefore, genetically modified host cells containing this stabilization construct will be more stable against mutations that may reduce the production of non-catabolite compounds.

[0375] In some embodiments, a heterologous promoter is operatively linked to a nucleic acid encoding a protein that affects cell growth by replacing or substituting an endogenous promoter in the host cell genome. In other embodiments, the heterologous promoter operatively linked to the nucleic acid encoding a protein that affects cell growth can be integrated into the chromosome at a different location, while its endogenous counterpart is functionally disrupted. These methods are merely exemplary, and other suitable methods can be used to incorporate the stabilization construct into genetically modified host cells.

[0376] 6.6.2.1 Use of previously identified essential genes in the stabilization construct

[0377] In some implementations, the nucleic acids encoding proteins that affect cell growth include essential genes, which are necessary for life to be sustained under optimal conditions where all nutrients are available. These essential genes are available in a database of essential genes (DEGs), publicly available at http: / / tubic.tju.edu.cn / deg / or http: / / www.essentialgene.org. The essential genes in the DEG database have been identified using genome-wide gene inactivation techniques. Essential genes have been identified in the genomes of various organisms, such as Arabidopsis thaliana, Aspergillus gumigatus, Caenorhabditis elegans, Danio rerio, Drosophila melanogaster, Homo sapiens, Mus musculus, Saccharomyces cerevisiae, and Schizosacchromyces pombe. See, for example, Meinke et al. (2008) Trends Plant Sci 13:483-91; Hu et al. (2007) PloS Pathog.3:e24; Kamath et al. (2003) Nature 421:231-7; Amsterdam et al. (2004) Proc Natl Acad Sci USA, 101:12792-7; Spradling et al. al. (1999) Genetics 153:135-77; Liao (2008) Proc Natl Acad Sci USA 105:1987-92; Georgiet al. (2013) PLoS genetics 9.5:e1003484; Liao (2007) Trends Genet 23:378-81; Giaever et al. al.(2002)Nature 418:387-91; and Kim et al. Nature Biotech 28.6 (2010) 617-623. The functions encoded by essential genes are considered fundamental to life and therefore likely universal to all cells.

[0378] The DEG database contains many types of essential genes. These include genes involved in protein synthesis, secretion, and quality control; genes involved in cell membrane formation and cell division; genes involved in metabolism and biosynthesis; genes involved in DNA replication and chromosome maintenance; genes involved in RNA synthesis and degradation, and so on.

[0379] Examples of essential genes involved in protein synthesis in yeast include, but are not limited to, EFB1 (translation elongation factor 1β; DEG20010002; NCBINP_009398), RRN6 (a protein involved in the transcription of the 35S rRNA gene; DEG20010015; NP_009539), and RER2 (a cis-isopentenyltransferase involved in the synthesis of dolicols; DEG20010033; NP_009556).

[0380] Examples of essential genes in yeast for secretion and quality control include, but are not limited to, EXO84 (an essential protein in spliceosome assembly and exocytosis; DEG20010050; NP_009660), SEC31 (an essential phosphoprotein component of the COPII envelope of secretory pathway vesicles; DEG20010148; NP_010086), etc.

[0381] Examples of essential genes in yeast for mating and cell division include, but are not limited to, MTW1 (an essential component of the MIND kinetochore complex; DEG20010006; NP_009367), CDC24 (guanine nucleotide exchange factor; DEG20010008; NP_009359), CDC15 (a protein kinase in the mitotic exit pathway; DEG20010012; NP_009411), HTA2 (histone H2A; DEG20010013; NP_009552), CDC27 (DEG20010028; NP_009469), and CMD1 (calmodulin; DEG20010051; NP_009667). CKS1 (cyclin-dependent protein kinase regulatory subunit and aptamer; DEG20010055; NP_009693), MEC1 (genome integrity checkpoint protein; DEG20010056; NP_009694), etc.

[0382] Examples of essential genes involved in metabolism or biosynthesis in yeast include, but are not limited to, CDC19 (pyruvate kinase; DEG20010007; NP_009362), GP118 (mannosyltransferase; DEG20010034; NP_009558), TSC3 (protein stimulating serine palmitoyltransferase activity; DEG20010042; NP_116327), ALG14 (DEG20010044; NP_009626), RIB7 (diaminohydroxyphosphonoribosylaminopyrimidine deaminase; DEG20010061; NP_0097), RIB5 (riboflavin synthase; DEG20010082; NP_009815), FAD1 (flavin adenine dinucleotide synthase; DEG20010115; NP_010239), and HEM13. (Porphyrinogen III oxidase; DEG20010163; NP_010326) etc.

[0383] Examples of essential genes involved in DNA replication in yeast include, but are not limited to, RFA1 (a subunit of heterotrimeric replication protein A involved in DNA replication, repair, and recombination; DEG20010010; NP_009404), POL30 (proliferating cell nuclear antigen; DEG20010048; NP_009645), POL3 (catalytic subunit of DNA polymerase δ; DEG20010126; NP_010181), and CDC9 (DNA ligase; DEG20010144; NP_010117).

[0384] Examples of essential genes involved in RNA synthesis and degradation in yeast include, but are not limited to, TFC3 (the largest of the six subunits of RNA polymerase III transcription initiation factor; DEG20010001; NP_009400), MAK16 (an essential nucleoprotein, a component of the 66S preribosomal particle; DEG20010003; NP_009377), PRP45 (a protein required for pre-mRNA splicing; DEG20010004; NP_009370), POP5 (two RNase MRP subunits required for cleavage of pre-rRNA and nuclear RNA sep; DEG20010005; NP_009369), and PTA1 (a subunit of all-CPF required for cleavage and polyadenylation of the 3' ends of mRNA and snoRNA; involved in pre-tRNA processing; DEG20010009). NP_009356.1), SEN34 (subunit of rRNA splicing endonuclease; DEG20010011; NP_009405), ABD1 (methyltransferase; DEG20010075; NP_009795), etc.

[0385] The essential genes mentioned above are merely illustrative; many other essential genes in yeast and other organisms exist in the DEG database. Other essential genes in other organisms not included in the DEG database can be further analyzed and identified using a homologous sequence search against DEG. The functions encoded by essential genes are considered generally essential for all cells. Therefore, if a query sequence aligned using BLAST has a homologous gene in DEG, the queried gene is likely also essential. Further details regarding the essential gene database and homologous sequence searches are documented in the following references: e.g., Zhang et al., Nuc. Acids Res. 2004 Jan; 32:D271-272; and Zhang and Lin, Nuc. Acids. Res. 2009 Jan; 37:D455-D458, which are incorporated herein by reference in their entirety. In genetically modified host cells, any one or a combination of these essential genes can be used to couple their expression to the production of non-catabolite compounds using common transcriptional regulators.

[0386] 6.6.2.2 Use of conditionally essential genes in stabilization constructs

[0387] In some embodiments, nucleic acids encoding proteins that affect cell growth may include conditionally essential genes that are indispensable only under specific environmental or growth conditions. Factors crucial to or affecting cell growth can be highly dependent on a given culture medium or conditions. Examples of conditionally essential genes include auxotrophic genes, which are conditionally essential. For instance, in the absence of uracil in the culture medium, the orotidine-5'-phosphate decarboxylase gene URA3, which catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidine, is essential for the host cell. In another embodiment, in the absence of tryptophan in the culture medium, the TRP1 gene, which encodes phosphoribosyl-an-aminobenzoic acid isomerase, which catalyzes the third step in tryptophan biosynthesis, is essential for the host cell. In yet another embodiment, in the absence of lysine in the culture medium, the LYS2 gene, which encodes aminoadipic acid reductase, may become essential. Any of these conditionally essential genes can be operatively linked to a promoter regulated by a common transcription factor that also regulates the expression of one or more enzymes in a biosynthetic pathway for the production of non-catabolite compounds (or any desired heterologous protein).

[0388] Many other conditionally essential genes encode enzymes for the synthesis of essential compounds that are necessary for cell growth when host cells are grown in a culture medium lacking these compounds. Other examples of such conditionally essential genes include those encoding enzymes in biosynthetic pathways for the production of essential amino acids, fatty acids, nucleotides, etc. Host cells genetically modified with such conditionally essential genes will require high expression of regulators to produce sufficient amounts of the essential compounds. Therefore, these regulators will be more stable against mutations that may reduce the production of non-catabolite compounds.

[0389] Numerous databases exist that provide information on various biosynthetic pathways, including those used to synthesize essential compounds. These include, for example, the KEGG pathway database (see Kanehisha et al., (2002) NucleicAcids Res., 30:42-46); and the MetaCyc pathway database (see Altman et al., (2013) BNCBioinformatics 14:112). Other useful databases include the BRENDA database (see Schomburg et al. (2002) Nucleic Acids Res. 30: 47-49); the SWISS-PROT database (Bairoch and Apweiler (2000) Nucleic Acids Res. 28: 45-48); EcoCyc (Karp et al. (2002) Nucleic Acids Res. 30: 56-8); and EMP / MPW (Selkov et al. (1998) Nucleic Acids Res. 26: 43-45). Many of these pathway databases provide the nucleotide sequences of genes encoding enzymes involved in the biosynthetic pathways for the production of essential compounds. Any suitable nucleic acid encoding the enzyme involved in the synthesis of essential compounds can be used in stabilization constructs.

[0390] In some implementations, amino acid biosynthesis genes encoding essential amino acids can be used as conditionally essential genes in a stable construct. These include one or more of the following: lysine biosynthesis genes, methionine biosynthesis genes, leucine biosynthesis genes, histidine biosynthesis genes, tryptophan biosynthesis genes, etc. The biosynthetic pathways for synthesizing these amino acids are well known. The nucleic acid sequences of many of these amino acid biosynthesis genes are also known to many organisms and are publicly available. See, for example, the GenBank sequence database (maintained by the National Center for Biotechnology Information).

[0391] In some implementations, nucleic acids encoding enzymes in the lysine biosynthesis pathway can be used in the stabilization construct. In yeast, the lysine biosynthesis pathway includes many different enzymes to synthesize lysine. These include enzymes that convert 2-ketoglutarate and acetyl-CoA (acetyl-CoA) to hypercitrate (e.g., hypercitrate synthase; LYS21 or LYS20), enzymes that convert hypercitrate to hyperaconitate and hyperaconitate to hyperisocitrate (e.g., hyperaconitase; LYS4), enzymes that convert hyperisocitrate to α-ketoadipic acid (e.g., hyperisocitrate dehydrogenase; LYS12), enzymes that convert α-ketoadipic acid to L-2-aminoadipic acid (e.g., enzyme 2.6.1.39; 2-aminoadipic acid aminotransferase), enzymes that convert L-2-aminoadipic acid to L-2-aminoadipic acid 6-semialdehyde (e.g., α-aminoadipic acid reductase; LYS2), and enzymes that convert L-2-aminoadipic acid 6-semialdehyde to sacchropine (e.g., sacchropine dehydrogenase (NADP+)). Enzymes that convert yeast amino acids to L-lysine (e.g., yeast amino acid dehydrogenase (NAD+, L-lysine formation); LYS9); and enzymes that convert yeast amino acids to L-lysine (e.g., yeast amino acid dehydrogenase (NAD+, L-lysine formation); LYS1).

[0392] Other organisms may use slightly different pathways and enzymes for the biosynthesis of lysine. Biosynthetic pathways, biosynthetic enzymes, and their corresponding nucleic acid sequences in other organisms can be found in databases such as KEGG and MetaCyc. For example, nucleic acids encoding enzymes that convert 2-ketoglutarate to homocitrate (e.g., homocitrate synthase) from many different organisms can be used as conditionally essential genes in stabilization constructs to couple the cell growth of genetically modified host cells with the production of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (LYS20 / YDL182W; Saccharomyces cerevisiae)), (LYS21 / YDL131W, which is a paralog of LYS20; Saccharomyces cerevisiae)), (AGOS_ADR107W; Eremophytum gossypii)), (Ecym_8045; Cymbalariae), (KLLA0E23695g; Kluveromyces lactis)), (KLLA0F05489g; Kluveromyces lactis)), (KLTH0E12848g; Lachancea thermotolerans), (KLTH0H02486g; Lachanceathermotolerans), (Kpol_2000p11; Vanderwaltozyma polysopora), (ZYRO0A13222g; Zygosaccharomyces rouxii), etc.

[0393] In some implementations, nucleic acids encoding an enzyme that converts hypercitrate to hyperisocitrate (e.g., hypercitrate enzyme) can be used as conditionally essential genes in a stabilization construct to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (LYS4 / YDR234W; Saccharomyces cerevisiae), (AGOS_ABL106C; Eremothecium gossypii), (Ecym_5123; Cymbalariae), (KLLA0C15125g; Kluveromyces lactis), (KLTH0E10582p; Lachancea thermotolerans), (Kpol_1031p57; Venderwaltozyma polyspora), (K1705; Zygosaccharomyces rouxii), etc.

[0394] In some implementations, the nucleic acid encoding an enzyme that converts isocitrate to α-ketoadipic acid (e.g., isocitrate dehydrogenase) can be used as a conditionally essential gene in a stabilization construct to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to, (LYS12 / YIL094C; Saccharomyces cerevisiae), etc.

[0395] In some implementations, the nucleic acid encoding an enzyme that converts α-ketoadipic acid to L-2-aminoadipic acid (e.g., enzyme 2.6.1.39; aminoadipic acid aminotransferase) can be used as a conditionally essential gene in a stabilization construct to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (AADAT, KAT2, KATII; Homo sapiens), (AADAT; Pan troglodytes), (AADAT; Pan paniscus), (AADAT; Pongoabelii), (AADAT; Momascus leucogenys), etc.

[0396] In some implementations, the nucleic acid encoding an enzyme that converts L-2-aminoadipic acid to L-2-aminoadipic acid 6-semialdehyde (e.g., α-aminoadipic acid reductase) can be used as a conditionally essential gene in a stable construct to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (LYS2 / YBR115C; Saccharomyces cerevisiae), (AGOS_ADL346W; Eremothecium gossypii), (Ecym_3457; Cymbalariae), (KLLA0B09218g; Kluveromyces lactis), (KLTH0F10384g; Lachanceathermotolerans), (Kpol_1006p6; Vanderwaltozyma polyspora), (ZYRO0C16566g; Zygosaccharomyces rouxii), etc.

[0397] In some implementations, nucleic acids encoding enzymes that convert L-2-aminoadipic acid 6-galactaldehyde to succinate (e.g., succinylcholine dehydrogenase, NADP+L-glutamate formation) can be used as conditionally essential genes in stabilization constructs to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (LYS9 / YNR050C; Saccharomyces cerevisiae)), (SORBI_03g030510; Sorghum bicolor)), (AGOS_ABR116c; Eremothecium gossypii)), (Ecym_7008; Cymbalariae), (KLAA0C18744g; Kluveromyces lactis)), (KLTH0A07590g; Lachanceathermotolerans), (Kpol_1028p12; Vanderwaltozyma polyspora), (ZYRO0D17578g; Zygosaccharomyces rouxii) etc.

[0398] In some implementations, nucleic acids encoding enzymes that convert sacchropine to L-lysine (e.g., sacchropine dehydrogenase, NAD+, L-lysine formation) can be used as conditionally essential genes in stabilization constructs to couple the cell growth of genetically modified host cells with the generation of heterologous non-catabolite compounds. Exemplary examples of suitable nucleotide sequences include, but are not limited to: (LYS1; YIR034c; Saccharomyces cerevisiae)), (AGOS_ACR167c; Eremophytum gossypii)), (Ecym_5636; Eremophytum cymbalariae), (KLLA0E07987g; Zygosaccharomyces rouxii)), (KLTH0C00594g; Lachancea thermotolerans), (Kpol_1057p13; Vanderwaltozyma polyspora), (ZYRO0D00594g; Zygosaccharomyces rouxii)), etc.

[0399] The use of nucleic acids encoding enzymes in the lysine biosynthesis pathway as conditionally essential genes is merely exemplary; nucleic acids encoding enzymes in other amino acid biosynthesis pathways can be used as conditionally essential genes in stabilization constructs. These include, for example, nucleic acids encoding enzymes in the methionine biosynthesis pathway: folic acid polyglutamate synthase (e.g., MET7), N5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase (MET6), L-aspartate 4-β-transferase (HOM3); aspartate β-semialdehyde dehydrogenase (e.g., HOM2), homoserine dehydrogenase (e.g., HOM6), homoserine O-trans-acetyltransferase (e.g., MET2), O-acetylhomoserine (thio)-lyase (e.g., MET17), etc.

[0400] Other examples include nucleic acids encoding enzymes in the leucine biosynthesis pathway: small isoenzyme α-isopropylmalate synthase (LEU9), α-isopropylmalate synthase (LEU4); isopropylmalate isomerase (LEU1), β-IPM dehydrogenase (LEU2); branched-chain amino acid transaminases (e.g., BAT2), branched-chain amino acid aminotransferases (BAT1), etc.

[0401] Other examples include nucleic acids of enzymes encoding the tryptophan biosynthesis pathway: anthranilate synthases (TRP3 and TRP2), anthranilate phosphoribosyltransferase (TRP4), N-(5'-phosphoribosyl)-anthranilate isomerase (TRP), indole-3-glycerol phosphate synthase (TRP3), tryptophan synthase (TRP5), etc.

[0402] Other examples include nucleic acids of enzymes encoding the histidine biosynthesis pathway: ATP phosphoribosyltransferase (HIS), phosphoribosyl-ATP pyrophosphatase (HIS4), phosphoribosyl-AMP cyclase (HIS4), phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolamide (HIS6), imidazolium glycerol phosphate synthase (HIS7), imidazolium glycerol phosphate dehydratase (HIS3), histidine-phosphotransaminase (HIS5), histidine phosphatase (HIS2), histidine dehydrogenase (HIS4), etc.

[0403] Other examples include nucleic acids of enzymes encoding the phenylalanine biosynthesis pathway, such as branched acid mutase (ARO7), prephenyl acid dehydratase (PHA2), and aromatic amino acid aminotransferases (ARO8 and ARO9).

[0404] Other examples include nucleic acids encoding enzymes that encode the threonine biosynthesis pathway, such as homoserine kinase (THR1) and threonine synthase (THR4).

[0405] Other examples include nucleic acids encoding enzymes that encode the isoleucine biosynthesis pathway: threonine deaminase (ILV1), acetolactate synthase (ILV6, ILV2), acetylhydroxy acid reductase (ILV5), dihydroxy acid dehydratase (ILV3), branched-chain amino acid transaminase (BAT2), branched-chain amino acid transaminase (BAT1), etc.

[0406] Other examples include nucleic acids of enzymes encoding the valine biosynthesis pathway: acetolactate synthase (ILV6, ILV2), acetylhydroxy acid reductase (ILV5), dihydroxy acid dehydratase (ILV3), branched-chain amino acid transaminase (BAT2), branched-chain amino acid transaminase (BAT1), etc.

[0407] In some embodiments, nucleic acids encoding enzymes in nucleotide biosynthesis pathways may be used as conditionally essential genes in the compositions and methods of the present invention. These include, for example, nucleic acids encoding enzymes in biosynthetic pathways for the production of adenine, thymine, uracil, guanine, or cytosine. In other embodiments, nucleic acids encoding enzymes in fatty acid biosynthesis pathways may be used as conditionally essential genes in the compositions and methods of the present invention.

[0408] The nucleic acid sequences encoding these enzymes in yeast are available from the Yeast Genome Database and can be found at www.yeastgenome.org. Functional homologs from other organisms can also be obtained using a BLAST search. Any other suitable conditionally essential genes can be used to generate genetically modified host cells with conditionally auxotrophic phenotypes, which can be alleviated by the expression of conditionally essential genes.

[0409] 6.6.2.3 Screening for other gene candidates that affect cell growth

[0410] Certain nucleic acids that may not yet be identified as essential or conditionally essential genes in the DEG database may still be very important for cell growth in a specific culture medium or under selected fermentation conditions. In some embodiments, the genes affecting cell growth may include those that affect cell growth such that underexpression or absence of them will result in cells growing at a significantly slower rate compared to when they are fully expressed in a specific culture medium or under specific conditions.

[0411] Various methods can be used to screen for potential gene candidates that influence cell growth and are suitable for use in stabilization constructs. In one embodiment, gene candidates influencing cell growth can be screened using inducible promoters. The inducible promoter is operatively linked to a gene candidate influencing cell growth, and its inducer is used to turn the expression of the gene candidate influencing cell growth on or off. After culturing for an appropriate period (e.g., approximately 24, 48, or 72 hours), the cell growth phenotypes of cells under induced and non-induced conditions can be compared. Under non-induced conditions, the inducible promoter is inactivated, and the gene candidate influencing cell growth is no longer transcribed. As cells divide, the amount of protein encoded by the gene candidate influencing cell growth gradually decreases, eventually reaching a depletion state that mimics a complete loss of function mutation. Differences in cell growth phenotypes between cells cultured under induced and non-induced conditions will indicate whether the gene candidate influencing cell growth is important for cell growth.

[0412] In some implementations, if inactivation of its expression (e.g., under non-induction conditions) leads to a reduction in cell biomass (e.g., cell count or density), a gene candidate affecting cell growth is selected in the stabilization construct as the gene affecting cell growth, compared to at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more after an appropriate time period (e.g., about 24 hours, 48 ​​hours, or 72 hours) when its expression is activated (e.g., under induction conditions). Cell density or culture count under non-induction and induction conditions can be compared. For example, the optical density (OD) of induced and non-inducible cell cultures can be compared. 600In other embodiments, if cells are grown on an agar plate, cell forming units (CFRs) can be counted. In other embodiments, maximum specific growth rates can be compared. Other suitable methods for representing cell growth can be used to compare the effects of gene candidate expression on cell growth under induced and non-induced conditions.

[0413] In some implementations, gene candidates affecting cell growth are screened in combination with promoters regulated by common transcriptional regulators of the regulators. For example, if the nucleic acid encoding an enzyme in a biosynthetic pathway for the production of non-catabolite compounds is operatively linked to the pGAL promoter, then gene candidates affecting cell growth can also be operatively linked to the pGAL promoter to screen for their suitability as a stabilization construct. In this embodiment, the host cells may be further modified to functionally disrupt the expression of the repressor GAL80, thereby enabling the transcriptional activator Gal4p to activate the expression of the regulator.

[0414] In some implementations, different promoters of varying promoter strengths can be tested among gene candidates that affect cell growth. When low expression levels of gene candidates affecting cell growth are required for cell growth, the gene candidates can be operatively linked to relatively weak promoters. In other cases where high expression levels of gene candidates affecting cell growth are needed, the gene candidates can be operatively linked to strong promoters. Any naturally derived or synthetic promoters used for regulators can be used to drive the expression of gene candidates affecting cell growth. For example, if transcriptional regulators of the GAL regulator are used, any promoter of the GAL regulator, pGAL1, pGAL2, pGAL7, pGAL10, pGCY1, pGAL80, or any synthetic promoter derived therefrom, can be used during screening for gene candidates affecting cell growth.

[0415] 6.6.3. Fermentation compositions and the use of stabilization constructs to generate non-catabolite compounds

[0416] On the other hand, the present invention provides fermentation compositions generated from genetically modified host cells and methods for generating heterologous non-catabolite compounds using stabilized constructs. In some embodiments, the method of generating heterologous non-catabolite compounds includes culturing genetically modified host cells in a culture medium, the host cells comprising: (a) a heterologous nucleic acid encoding an enzyme of a biosynthetic pathway for generating the heterologous non-catabolite compound, wherein the heterologous nucleic acid is operatively linked to a first promoter; (b) a nucleic acid encoding a protein affecting cell growth, wherein the nucleic acid is operatively linked to a second promoter; and (c) a nucleic acid encoding a transcriptional regulatory factor, wherein both the first and second promoters are regulated by the transcriptional regulatory factor. In some embodiments, the fermentation composition comprises the genetically modified host cells of the present invention and the heterologous non-catabolite compounds generated therefrom in a culture medium. Typically, the genetically modified host cells are cultured in a culture medium containing a carbon source under suitable conditions for a time sufficient to generate the desired host cell biomass and / or the desired amount of non-catabolite compounds. In some implementations, the host cell contains a heterologous nucleic acid encoding a target protein as a non-catabolite compound, rather than a heterologous nucleic acid encoding an enzyme in the biosynthetic pathway for generating the non-catabolite compound.

[0417] In the compositions and methods provided by this invention, both the first promoter and the second promoter are regulated by one or more common transcriptional regulators. Since these common transcriptional regulators regulate the expression of both heterologous nucleic acids encoding enzymes of biosynthetic pathways and heterologous nucleic acids of proteins affecting cell growth, any spontaneous mutations that negatively affect the expression or stability of the transcriptional regulators will negatively affect the expression of both heterologous nucleic acids. These mutated cells will not survive or will grow at a much slower rate compared to high-yield cells; therefore, high-yield cells (e.g., parental cells with an original high-yield genotype) will dominate the cell population during fermentation. Therefore, coupling the expression of heterologous nucleic acids encoding enzymes for the production of non-catabolite compounds to the expression of proteins affecting cell growth will stabilize the high-yield genotype of the host cell, thereby ensuring the stable production of non-catabolite compounds during long-term fermentation. Similarly, coupling the expression of heterologous nucleic acids encoding target proteins to the expression of proteins affecting cell growth will stabilize the production of the target protein.

[0418] In some embodiments, at least one of the first and second promoters is heterologous to the aforementioned nucleic acids. For example, an endogenous promoter that typically drives the expression of genes in chromosomes that affect cell growth can be replaced by a heterologous promoter regulated by a common transcription factor. In another embodiment, an endogenous promoter that typically drives the expression of genes involved in biosynthetic pathways that produce non-catabolite compounds can be replaced by a heterologous promoter. In some embodiments, both the first and second promoters are heterologous to their respective nucleic acids. By utilizing heterologous promoters, these nucleic acids, which are not typically regulated by the same transcription factors, can be co-regulated as regulators in genetically modified host cells.

[0419] The choice of promoter sequence depends on the required expression level of the heterologous nucleic acid encoding the protein that affects cell growth or the enzyme in the biosynthetic pathway that generates a non-catabolite compound. In some embodiments, the first promoter sequence and the second promoter sequence are identical. In other embodiments, the first promoter sequence and the second promoter sequence are different. In some embodiments, the first promoter and the second promoter have different promoter strengths. For example, naturally derived pGAL promoters such as pGAL1, pGAL2, pGAL7, and pGAL10 are generally stronger promoters than synthetic promoters derived from these pGAL promoters (e.g., pGAL2_v3 (SEQ ID NO:84), pGAL7_v1 (SEQ ID NO:85), pGAL2_v4 (SEQ ID NO:86), pGAL1_v3 (SEQ ID NO:87), pGAL10_v3 (SEQ ID NO:88), pGAL2_v2 (SEQ ID NO:89), pGAL7_v2 (SEQ ID NO:90)). If higher expression of biosynthetic pathway enzymes is desired compared to proteins affecting cell growth, the nucleic acids encoding these enzymes can be operatively ligated to naturally derived pGAL promoters, while the nucleic acids encoding proteins affecting cell growth can be operatively ligated to weaker synthetic promoters derived from naturally derived pGAL promoters. Generally, promoters are selected to balance their promoter strengths to match the required expression of genes affecting cell growth in the host cell with the required expression of genes involved in biosynthetic pathways that produce non-catabolite compounds.

[0420] Other details relating to the construction of cell biomass and the generation of non-catabolite compounds described in Section 6.5 and other sections also apply to compositions and methods comprising the stabilized constructs provided by the present invention.

[0421] 6.6.4. Uses of Stabilized Constructs in the Constitutive Generation of Non-Catalytic Compounds

[0422] In some implementations, heterologous noncatabolite compounds are constitutively generated throughout the fermentation process. For certain heterologous noncatabolite compounds that are relatively nontoxic to cells, genetically modified host cells can tolerate the presence of these compounds throughout the fermentation process (i.e., during both the fermentation build-up and fermentation generation phases). Examples of such heterologous compounds include various acetyl-CoA-derived compounds, such as some isoprene-like compounds, fatty acids, and polyketides. Specific examples of these compounds will be further described in sections 6.11 through 6.13 below.

[0423] To constitutively generate heterologous noncatabolite compounds, the expression of heterologous nucleic acids encoding enzymes of biosynthetic pathways and proteins affecting cell growth can be regulated using any suitable transcription factors and promoters. Known gene disruption techniques can be used to reduce or eliminate the expression of any endogenous transcriptional repressors in the promoter. For example, if the heterologous nucleic acid is regulated by a transcriptional regulator of the GAL regulator, the repressor GAL80 can be functionally disrupted. By functionally disrupting the expression of GAL80, Gal80p can no longer inhibit the activity of Gal4p.

[0424] The use of transcription factors and promoters of the GAL regulator is merely exemplary, and any suitable transcription factor and promoter can be used to co-regulate and regulate the expression of regulators (i.e., nucleic acids encoding proteins that affect cell growth and nucleic acids encoding enzymes for biosynthetic pathways that generate non-catabolite compounds). For example, the MAL transcription activator can be used as a co-transcriptional regulator to drive the expression of the two heterologous nucleic acids, each operatively linked to a maltose-responsive promoter. In another embodiment, the Pho regulator activator can be used as a co-transcriptional regulator to drive the expression of the two heterologous nucleic acids, each operatively linked to a Pho regulator promoter. In yet another embodiment, artificial transcription factors can be used to activate the expression of the two heterologous nucleic acids. For example, LexA fused with an activation domain can be used as an artificial transcriptional regulator.

[0425] 6.6.5. Using stabilization constructs with switches to generate non-catabolite compounds

[0426] On the other hand, the stabilization construct described in this invention can be used in combination with a switch that separates the growth phase of fermentation (i.e., the cell biomass building phase) and the generation phase of fermentation (i.e., the heterologous compound generation phase). Generation of non-catabolite compounds during the building phase may be undesirable. This results in slow cell growth and the inability to achieve optimal cell density during the generation phase. On the other hand, biomass generation during the generation phase of fermentation is undesirable because it diverts metabolic resources away from the generation of heterologous non-catabolite compounds. In some embodiments, the switch can be used in combination with a stabilization construct in genetically modified host cells to separate these two distinct metabolic phases of fermentation and maximize the generation of heterologous non-catabolite compounds.

[0427] In one embodiment, the construction phase is carried out for a period of time sufficient to generate a certain amount of cellular biomass that can support the generation of heterologous compounds during the construction phase. The construction phase is carried out for a period of time sufficient to allow the cell population present at inoculation to undergo multiple doublings until the desired cell density is reached. In some embodiments, the construction phase is carried out for a period of time sufficient to allow the host cell population in the fermentation vessel or container in which the construction phase is carried out to reach a cell density (OD) between approximately 0.01 and 400. 600 In some implementations, the construction phase is performed until an OD of at least 0.01 is achieved. 600 In some implementations, the construction phase is carried out until an OD of at least about 0.1 is reached. 600 In some implementations, the construction phase is carried out until an OD of at least approximately 1.0 is reached. 600 In some implementations, the construction phase is carried out until at least about 10 OD is reached. 600 In some implementations, the construction phase is carried out until at least approximately 100 OD is reached. 600 In some implementations, the build phase is performed until an OD between approximately 0.01 and 100 is reached. 600 In some implementations, the build phase is performed until an OD of approximately 0.1 to 10 is reached. 600 In some implementations, the construction phase is carried out until an OD of approximately 1 to 100 is reached. 600 In other implementations, the construction phase lasts for at least approximately 12, 24, 36, 48, 60, 72, 84, 96 hours, or more than approximately 96 hours.

[0428] In some embodiments, the generation phase lasts for a period of time sufficient to generate the desired amount of non-catabolite compound. In some embodiments, the generation phase lasts for at least about 12, 24, 36, 48, 60, 72, 84, or 96 hours, or more than about 96 hours. In some embodiments, the generation phase lasts for between about 3 and 20 days. In some embodiments, the generation phase lasts for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or more than about 20 days.

[0429] In embodiments where a switch is used to separate the two stages of fermentation, a conditionally essential gene can be used as a gene in the stabilization construct that influences cell growth. For example, the conditionally essential gene may encode an enzyme in a metabolic pathway whose end product is an essential compound necessary for cell growth and can be consumed by the host cell. Genetically modified host cells containing this conditionally essential gene in the stabilization construct will require high expression of the regulator to generate sufficient amounts of the essential compound. During the "off" phase of fermentation (i.e., the construction phase), where the switch shuts off the expression of the conditionally essential gene and biosynthetic pathway genes, a conditional auxotrophic symptom will result in these genetically modified host cells. This auxotrophic symptom can be compensated for by adding the essential compound to the culture medium. During the "on" phase of fermentation (i.e., the generation phase), where the switch is turned on, the expression of the conditionally essential gene and biosynthetic pathway genes for generating non-catabolite compounds is enabled, and the essential compound is not added to the culture medium. Therefore, only cells capable of expressing the conditionally essential gene and generating sufficient amounts of the essential compound can grow. This strategy is compatible with genetically modified host cells that have a genetic switch that conditionally reduces regulator expression when needed, because the auxotrophic effect caused by the reduction in regulator expression can be compensated by providing the culture medium with the essential compounds required by the auxotrophic effect.

[0430] Therefore, the present invention provides a method for generating heterologous non-catabolite compounds, wherein the method comprises: (a) a construction phase, wherein a genetically modified host cell population is cultured in a medium that restricts the generation of heterologous non-catabolite compounds, wherein the medium contains an essential compound; and (b) a generation phase, wherein the genetically modified host cell population or subpopulation is cultured under conditions that promote the generation of heterologous non-catabolite compounds without supplementation with a medium containing the essential compound (or supplementation with a sufficiently low amount of the essential compound). In this embodiment, the genetically modified host cells may comprise: (i) a heterologous nucleic acid encoding a target protein (e.g., an enzyme for a biosynthetic pathway for generating heterologous non-catabolite compounds), wherein the heterologous nucleic acid is operatively linked to a first promoter; (ii) a nucleic acid encoding a conditionally essential gene product of a biosynthetic pathway for generating the essential compound, wherein the nucleic acid is operatively linked to a second promoter; and (iii) a nucleic acid encoding a transcriptional regulatory factor, wherein both the first promoter and the second promoter are regulated by the same transcriptional regulatory factor. During this generation phase, because the essential compounds are not added to the culture medium (or are added in sufficiently low amounts), only host cells capable of expressing genetically modified conditionally essential genes can grow. This increases the selection pressure on the functional expression of transcriptional regulators, which in turn enhances the generation stability of non-catabolite compounds.

[0431] During the construction phase, any suitable amount of the essential compound can be added to the culture medium. During the construction phase, concentration limitations of the essential compound required for growth due to nutrient deficiencies in the culture medium are generally undesirable. Adding a certain amount of the essential compound allows genetically modified auxotrophic host cells containing a conditionally essential gene (whose expression is suppressed during the construction phase) to grow to the desired biomass concentration as described above. In some embodiments, the suitable amount of essential compound in the construction phase culture medium can be determined by titration with the presence of an increased amount of the essential compound in the construction phase culture medium. For example, the genetically modified host cell population can be divided into multiple subpopulations and cultured in parallel, where each subpopulation is grown in a medium containing different (e.g., increased) amounts of the essential compound (including no essential compound), and cell biomass (e.g., OD) can be measured after a defined time period. 600 reading).

[0432] In other embodiments, the appropriate amount of essential compounds in the culture medium during the construction phase can be determined by measuring cell growth rates, such as the maximum specific growth rate of genetically modified auxotrophic host cells in different concentrations of the essential compounds. The term "specific growth rate" refers to the increase in biomass or cell number per unit time. The term "maximum specific growth rate" refers to the "specific growth rate" during the exponential growth phase of culture. Typically, during the exponential growth phase, the specific growth rate remains approximately constant because the substrate (or product) does not significantly inhibit growth.

[0433] In some embodiments, the essential compound titration may comprise at least one concentration of the essential compound, thereby increasing the host cell biomass (e.g., by OD). 600 The maximum specific growth rate (or the maximum growth rate) plateaus at its maximum, meaning that no further increase in host cell biomass or maximum specific growth rate is observed with an increased amount of the essential compound. In some embodiments, the amount of the essential compound added to the culture medium during the construction phase is at least the minimum amount of the essential compound at which the host cell biomass or maximum specific growth rate reaches its maximum. This amount may also be referred to as the "saturation" amount of the essential compound.

[0434] In some embodiments, a saturation amount of the essential compound may be added to the construction phase culture medium. In some embodiments, the essential compound may be added to the construction phase culture medium in an amount greater than the saturation amount to ensure that the genetically modified host cells reach the desired cell biomass or maximum specific growth rate. For example, the essential compound added to the construction phase culture medium is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 times, or about 100 times greater than the saturation amount determined from the essential compound titration profile. In some embodiments, if it is desired to grow the genetically modified auxotrophic host cells to a lower biomass concentration or maximum specific growth rate, the essential compound may be added to the construction phase culture medium in an amount less than the saturation amount. For example, the essential compound added to the culture medium during the construction phase may be at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100 times or more less than the saturation amount determined from the titration curve of the essential compound.

[0435] Furthermore, for a given concentration of carbon source (e.g., 20 g glucose per liter of culture medium), suitable concentrations of essential compounds, such as essential amino acids, for auxotrophic strains are generally known in the art. In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.0001% (weight of the essential compound per volume of culture medium). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.0005% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.001% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.005% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.01% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.05% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.1% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 0.5% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 1% (w / v). In some embodiments, the concentration of the essential compound in the culture medium during the construction phase is at least about 5% or 10% (w / v). Culture media designed for culturing auxotrophic strains are well known in the art and are described, for example, in the Guide to Yeast Genetics and Molecular Biology, 194 (Guthrie et al., Academic Press 1990); and Introduction to Yeast Media (Sigma-Aldrich).

[0436] Following the construction phase, the culture medium for the generation phase, used to culture the genetically modified host cells, does not contain externally added essential compounds or contains only sufficiently low amounts of them. Thus, selection pressure is applied to the host cells to maintain the expression of conditionally essential genes, enabling the cells to generate the essential compounds required for cell growth to meet nutrient deficiencies. In some embodiments, the generation phase culture medium does not contain externally added essential compounds. However, in some embodiments, the essential compounds may be added to the generation phase culture medium in sufficiently low amounts. For example, the generation phase culture medium may contain essential compounds in amounts at least about 10, 100, 1000, 10,000, or about 100,000 times less than the saturation amount. In some embodiments, the generation phase culture medium contains essential compounds in amounts at least about 10, 100, 1000, 10,000, or about 100,000 times less than the amount of essential compounds in the construction phase culture medium.

[0437] In some embodiments, the essential compounds are amino acids, and the conditionally essential genes in the stabilized construct encode enzymes in the amino acid biosynthesis pathway. For example, the essential compound used to replenish the culture medium during the construction phase is lysine, and any one or a combination of the lysine biosynthesis genes LYS1, LYS2, LY4, LYS9, LYS12, LYS14, LYS20, or LYS21 can be used in the stabilized construct. In another embodiment, the essential compound used to replenish the culture medium during the construction phase is methionine. Other amino acid biosynthesis genes described in Section 6.6.2 may also be used in the stabilized construct.

[0438] In other embodiments, the essential compound used to replenish the culture medium during the construction phase may be uracil, thymine, guanine, or cytosine, and any one or combination of genes encoding enzymes in the biosynthetic pathways of said nucleotides may be used in the stabilization construct. In other embodiments, the essential compound is a fatty acid, and a conditionally essential gene in the stabilization construct encodes an enzyme in the fatty acid biosynthetic pathway.

[0439] In some embodiments, inducible promoters and their inducers can be used as switches to shut off the generation of heterologous noncatabolite compounds during the build phase and to turn on the generation during the generation phase. For example, the inducible promoter is operatively linked to a nucleic acid encoding a transcription activator that can activate the expression of two heterologous nucleic acids encoding enzymes for the biosynthetic pathway that generates noncatabolite compounds and conditionally essential gene products during the generation phase of fermentation. Examples of suitable inducible promoters include maltose-responsive promoters, which can be induced by ligands such as maltose-based inducers. Further details of maltose-responsive promoters and maltose-based inducers are set forth in Sections 6.3 and 6.4 above. Other inducible promoters suitable for the methods of the present invention include oxygen-sensitive promoters, such as the DAN1 promoter, which is inactive under aerobic conditions but highly active under anaerobic conditions. Examples of suitable oxygen-sensitive promoters are set forth, for example, in WO2015 / 020649, which is incorporated herein by reference in its entirety.

[0440] In other embodiments, an inducible promoter is operatively linked to a nucleic acid encoding a transcriptional repressor that, during the construction phase of fermentation, inhibits the expression of two heterologous nucleic acids encoding enzymes of a biosynthetic pathway for the generation of non-catabolite compounds and conditionally essential gene products. Any suitable inducible promoter, including those described above or known in the art, can be used in these embodiments to activate the transcriptional repressor.

[0441] Figure 17 illustrates an exemplary embodiment of using an inducible promoter as a switch to turn off or inhibit the expression of heterologous nucleic acids encoding enzymes in biosynthetic pathways that generate non-catabolite compounds and conditionally essential gene products. As shown in Figure 17, the nucleic acid encoding the transcriptional repressor Gal80p, a GAL regulator, is operatively linked to the maltose-responsive promoter pMAL32. When an inducible amount of maltose is added to the culture medium, Gal80p is expressed, which in turn inhibits the activation of the transcriptional activator Gal4p and the expression of one or more biosynthetic enzymes for generating heterologous compounds. Gal80p also inhibits the expression of LYS9, a conditionally essential gene encoding an enzyme in the lysine biosynthetic pathway. During the construction phase of the Gal80p-inhibited regulator expression (including the expression of conditionally essential genes), lysine is added to the culture medium to allow cell growth.

[0442] During the generation phase of the exemplary embodiment shown in Figure 17, genetically modified host cells are cultured in a medium free of maltose or lysine (or present in sufficiently low amounts). Since GAL80 is no longer expressed, the transcriptional activator Gal4p is no longer repressed, thus activating the expression of biosynthetic pathway genes to produce non-catabolite compounds. Simultaneously, Gal4p also activates the expression of LYS9, which in turn enables the lysine biosynthetic pathway to produce the essential compound lysine. Because the genetically modified cells are capable of producing lysine, these cells can be cultured in a lysine-free medium during the generation phase. The lysine-free medium increases the selection pressure for Gal4p expression during the generation phase. Therefore, any cells that acquire spontaneous mutations that negatively affect the GAL regulator will not survive, thereby allowing high-yield host cells to dominate the cell population during prolonged fermentation.

[0443] 6.7 Generation of non-catabolite compounds using maltose-dependent degradation determinants and stabilization constructs

[0444] On the other hand, the present invention provides fermentation compositions and methods for generating heterologous noncatabolite compounds using maltose-dependent degradation determinants and stabilization constructs. Maltose-dependent degradation determinants fused within the transcriptional regulatory factor frame can force the transcriptional regulatory factor to become dependent on its stability in binding to maltose. For example, in the absence of maltose in the culture medium, fusion proteins containing Gal80p fused to maltose-dependent degradation determinants will become unstable during the generation phase. Therefore, even if spontaneous mutations reactivate the expression of fusion proteins containing Gal80p, the fusion proteins will become unstable in the absence of maltose in the culture medium and will not be able to suppress the expression of the GAL regulator during the generation phase. When maltose-dependent degradation determinants are used in combination with the stabilization constructs described in this invention, the generation of heterologous noncatabolite compounds is further stabilized because both constructs can counteract any negative effects of spontaneous mutations.

[0445] Therefore, the present invention provides compositions and methods that provide at least two layers of stability in the generation of heterologous noncatabolite compounds. In one embodiment, the fermentation composition and method include culturing genetically modified host cells in a culture medium, wherein the genetically modified host cells comprise: (a) a heterologous nucleic acid encoding a fusion protein comprising a transcriptional regulatory factor fused to a maltose-dependent degradation determinant frame; (b) one or more heterologous nucleic acids encoding one or more enzymes for a biosynthetic pathway for the generation of heterologous noncatabolite compounds, each of the one or more heterologous nucleic acids being operatively linked to a promoter regulated by the fusion protein; and (c) a stabilization construct comprising a heterologous nucleic acid encoding a protein affecting cell growth, wherein the heterologous nucleic acid is operatively linked to a promoter regulated by the fusion protein. The stabilization construct in the genetically modified host cells provides growth-favorable conditions for cells with high product yields and growth-disadvantageous conditions for spontaneously mutant cells with low or no product yields, thus stabilizing the generation of heterologous noncatabolite compounds during fermentation. Furthermore, the maltose-dependent degradation determinant provides post-translational control for fusion proteins containing transcriptional regulators fused to the maltose-dependent degradation determinant frame. By manipulating the maltose content in the culture medium, the stability of the maltose-dependent degradation determinant can be controlled, thereby controlling the post-translational stability and activity levels of the transcriptional regulators. This, in turn, provides an additional stabilizing layer for the generation of heterologous non-catabolizable compounds.

[0446] In some embodiments, the fusion protein comprises a transcriptional activator fused within the maltose-dependent degradation determinant frame. In one embodiment, the transcriptional activator Gal4p is selected as the transcriptional regulator in the fusion protein. For this embodiment, host cells containing genetically modified heterologous nucleic acids as described herein can be cultured in a maltose-containing medium during the generation phase. The stable fusion protein bound to maltose can then activate the expression of heterologous nucleic acids operatively linked to a Gal4p-responsive promoter to express enzymes in biosynthetic pathways that generate non-catabolite compounds and proteins that influence cell growth. When the maltose-dependent degradation determinant is fused within the transcriptional activator frame, any endogenous transcriptional repressors of the regulator (e.g., Gal80p) can be functionally disrupted so that they do not interfere with the activity of the fusion protein.

[0447] In other embodiments, the fusion protein comprises a transcriptional repressor fused to a maltose-dependent degradation determinant frame. For example, the transcriptional repressor Gal80p is selected as the transcriptional regulator in the fusion protein. In this embodiment, host cells containing the genetically modified heterologous nucleic acid described herein can be cultured in a medium containing maltose and essential compounds during the cell biomass building phase of fermentation. Thus, the transcriptional repressor in the maltose-bound fusion protein is stable, thereby inhibiting the activity of Gal4p. This, in turn, inhibits the expression of heterologous nucleic acids encoding one or more enzymes of biosynthetic pathways that generate non-catabolite compounds and proteins that affect cell growth during the cell biomass building phase of fermentation.

[0448] In some embodiments, any maltose-dependent degradation determinants, fusion proteins, maltose-based inducers, maltose-responsive promoters, culture conditions, and other features related to the generation of non-catabolite compounds described in this invention may be used in combination with the stabilization construct to further improve the amount and stability of non-catabolite compounds generated by genetically modified host cells. Furthermore, other details relating to the generation of non-catabolite compounds and the construction of cell biomass described in Section 6.5 and other sections apply to compositions and methods incorporating the maltose-dependent degradation determinants and stabilization constructs provided in this invention.

[0449] 6.8 Genetically Modified Host Cells

[0450] This invention provides genetically modified host cells containing heterologous nucleic acids encoding the fusion protein described herein. In some embodiments, the genetically modified host cells are microorganisms (e.g., genetically modified Saccharomyces cerevisiae cells) containing heterologous nucleic acids encoding the fusion protein, wherein the fusion protein is more stable when the maltose-dependent degradation determinant is in contact with maltose compared...

Claims

1. A method for generating heterologous non-catabolizable compounds, the method comprising: (a) A population of genetically modified yeast host cells is cultured in a culture medium containing an essential compound, said essential compound being necessary for cell growth, wherein said genetically modified yeast host cells comprise: (i) a heterologous nucleic acid, wherein the heterologous nucleic acid encodes an enzyme for a biosynthetic pathway for generating the heterologous noncatabolite compound, wherein the heterologous nucleic acid is operatively linked to a first promoter; (ii) a heterologous nucleic acid encoding a conditionally essential gene product of a biosynthetic pathway for the production of the essential compound, wherein the conditionally essential gene product is essential for cell growth when yeast host cells are grown in a culture medium lacking the essential compound, wherein the essential compound is selected from amino acids, nucleotides, or fatty acids, wherein the conditionally essential gene product is an enzyme for the biosynthetic pathway for the production of the amino acid, nucleotide, or fatty acid, wherein the nucleic acid is operatively linked to a second promoter; and (iii) A nucleic acid encoding a transcriptional regulatory factor, which is operatively linked to a maltose-responsive promoter. Both the first and second promoters are regulated by the transcriptional regulatory factors, and the expression of the heterologous nucleic acid encoding the non-catabolite compound and the expression of the nucleic acid encoding the conditionally essential gene product are restricted. The first and second promoters are each selected from the group consisting of pGAL1, pGAL2, pGAL3, pGAL7, pGAL10, pGCY1, pGAL80, and the pGAL synthesis promoter. (b) The cell population or subpopulation thereof is cultured under culture conditions containing sufficiently low amounts or no essential compounds, said culture conditions promoting the expression of nucleic acids encoding the product of said conditionally essential gene, thereby generating said essential compounds necessary for cell growth, and compared to step (a), said culture conditions promoting the expression of heterologous nucleic acids encoding the enzymes of the biosynthetic pathway, thereby increasing the production of said heterologous noncatabolite compounds. The transcriptional regulatory factor mentioned above is Gal80p, a transcriptional repressor fused within the maltose-dependent degradation determinant variant frame. The maltose-dependent degradation determinant variant is encoded by an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26; amino acids 1-365 of SEQ ID NO: 16, 18, 20, 22, 24 or 26; and amino acids 1-370 of SEQ ID NO: 16, 18, 20, 22, 24 or 26.

2. The method of claim 1, wherein the genetically modified yeast host cell further comprises one or more additional heterologous nucleic acids encoding one or more additional enzymes of the biosynthetic pathway, wherein each of the one or more additional heterologous nucleic acids is operatively linked to an additional promoter, the additional promoter being regulated by the same transcriptional regulatory factor as the first and second promoters.

3. The method according to claim 1, wherein the pGAL synthesis promoter is selected from the group consisting of: pGAL2_v3 (SEQ ID NO: 84), pGAL7_v1 (SEQ ID NO: 85), pGAL2_v4 (SEQ ID NO: 86), pGAL1_v3 (SEQ ID NO: 87), pGAL10_v3 (SEQ ID NO: 88), pGAL2_v2 (SEQ ID NO: 89), pGAL7_v2 (SEQ ID NO: 90), and portions thereof that retain promoter functionality.

4. The method according to claim 1, wherein the conditionally essential gene product is an enzyme in an amino acid biosynthetic pathway for producing the amino acid, and the essential compound is an amino acid generated by the amino acid biosynthetic pathway.

5. The method according to claim 4, wherein the conditionally essential gene product is an enzyme in the lysine biosynthesis pathway, and the essential compound is lysine generated by the lysine biosynthesis pathway.

6. The method of claim 5, wherein the conditionally essential gene product is encoded by LYS1 、 LYS2 、 LYS4 、 LYS9 、 LYS12 、 LYS14 、 LYS20 or a combination thereof.

7. The method according to claim 1, wherein the conditionally essential gene product is an enzyme in the methionine biosynthesis pathway.

8. The method of claim 7, wherein the conditionally essential gene product is derived from... MET2 , MET17 , HOM2 , HOM3 , HOM6 Or a combination thereof, for encoding.

9. The method of claim 1, wherein the first promoter and the second promoter are identical.

10. The method of claim 1, wherein the first promoter and the second promoter are different.

11. The method of claim 10, wherein the first promoter and the second promoter have different promoter strengths.

12. The method according to claim 1, wherein the heterologous non-catabolizable compound is selected from the group consisting of isoprene-like compounds, fatty acids, and polyketide compounds.

13. The method of claim 1, wherein the conditionally essential gene product is not an enzyme in the biosynthetic pathway for generating the heterologous noncatabolite compound.

14. The method of claim 1, wherein the heterologous nucleic acid encoding the enzyme of the biosynthetic pathway for generating the heterologous non-catabolite compound and the first promoter are integrated via chromosome into the genome of the genetically modified yeast host cell.

15. The method of claim 1, wherein the nucleic acid encoding the conditionally essential gene product and the second promoter are integrated into the genome of the genetically modified yeast host cell via chromosome.

16. The method of claim 1, wherein the cultivation comprises: (a) Construction phase, wherein the genetically modified yeast host cell population is cultured in a medium that restricts the generation of the heterologous non-catabolite compound; followed by (b) Generation phase, wherein the host cell population or its subpopulation is cultured under culture conditions that promote the generation of the heterologous non-catabolite compound.

17. The method of claim 16, wherein the nucleic acid encoding the transcriptional regulatory factor is operatively linked to an inducible promoter.

18. The method of claim 16, wherein the transcriptional regulatory factor is a transcriptional activator, and wherein the transcriptional activator is induced in the generation phase using a maltose inducer.

19. The method of claim 16, wherein the transcriptional regulatory factor is a transcriptional repressor, and wherein the transcriptional repressor is induced by a maltose inducer during the construction phase.

20. The method of claim 17, wherein the inducible promoter is a maltose-responsive promoter.

21. The method of claim 20, wherein the maltose-responsive promoter is a synthetic maltose-responsive promoter.

22. The method of claim 20, wherein the maltose-responsive promoter is selected from the group consisting of: pMAL1 (SEQ ID NO: 29), pMAL2 (SEQ ID NO: 30), pMAL11 (SEQ ID NO: 31), pMAL12 (SEQ ID NO: 32), pMAL31 (SEQ ID NO: 33), pMAL32 (SEQ ID NO: 34), pMAL32_v1 (SEQ ID NO: 78), pGMAL_v5 (SEQ ID NO: 35), pGMAL_v6 (SEQ ID NO: 36), pGMAL_v7 (SEQ ID NO: 37), pGMAL_v9 (SEQ ID NO: 38), pGMAL_v10 (SEQ ID NO: 39), pGMAL_v11 (SEQ ID NO: 40), pGMAL_v12 (SEQ ID NO: 41), pGMAL_v13 (SEQ ID NO: 78). NO: 42), pGMAL_v14 (SEQ ID NO: 43), pGMAL_v15 (SEQ ID NO: 44), pGMAL_v16 (SEQ ID NO: 45), pGMAL_v17 (SEQ ID NO: 46), pGMAL_v18 (SEQ ID NO: 47), pG2MAL_v1 (SEQ ID NO: 48), pG2MAL_v2 (SEQ ID NO: 49), pG2MAL_v3 (SEQ ID NO: 50), pG2MAL_v5 (SEQ ID NO: 51), pG2MAL_v6 (SEQ ID NO: 52), pG2MAL_v7 (SEQ ID NO: 53), pG2MAL_v8 (SEQ ID NO: 54), pG2MAL_v9 (SEQ ID NO: 54) NO: 55), pG2MAL_v10 (SEQ ID pG7MAL_v2 (SEQ ID NO: 57), pG7MAL_v4 (SEQ ID NO: 58), pG7MAL_v6 (SEQ ID NO: 59), pG7MAL_v8 (SEQ ID NO: 60), pG7MAL_v9 (SEQ ID NO: 61), pG172_MAL_v13 (SEQ ID NO: 62), pG271_MAL_v12 (SEQ ID NO: 63), pG721_MAL_v11 (SEQ ID NO: 64), pG712_MAL_v14 (SEQ ID NO: 65), and the portions thereof that retain the functionality of the bootloader.

23. The method of claim 16, wherein the construction phase is carried out for a period of time sufficient to bring the host cell population to a cell density OD600 between 0.01 and 400.

24. The method of claim 16, wherein the construction phase is performed for a period of at least 12 hours.

25. The method of claim 16, wherein the construction phase lasts for a period of at least 24 hours.

26. The method of claim 16, wherein the construction phase is performed for a period of at least 36 hours.

27. The method of claim 16, wherein the construction phase is performed for a period of at least 48 hours.

28. The method of claim 16, wherein the construction phase is performed for a period of at least 60 hours.

29. The method of claim 16, wherein the construction phase is performed for a period of at least 72 hours.

30. The method of claim 16, wherein the construction phase is performed for a period of at least 84 hours.

31. The method of claim 16, wherein the construction phase is performed for a period of at least 96 hours.

32. The method of claim 16, wherein the construction phase is performed for a period of 96 hours or more.

33. The method of claim 16, wherein the generation phase is carried out for a period of 3 to 20 days.

34. The method of claim 16, further comprising recovering the heterologous non-catabolite compound.

35. The method according to claim 1, wherein the genetically modified yeast host cell is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae ).

36. The method according to any one of claims 1-35, wherein the maltose-based inducer is maltose.

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