Method for decoupling the yield and productivity of non-catabolic compounds produced by a host cell

Abstract

The present invention provides compositions and methods for decoupling the yield and productivity of isoprenoid compounds produced in host cells. In some embodiments, the yield and productivity are decoupled by genetically modifying the host cell to reduce the flux through the citric acid cycle (TCA). In other embodiments, the yield and productivity are decoupled by reducing the ATP level in the host cell.

Description

BACKGROUND OF THE INVENTION

[0001] Yield and productivity are two major cost drivers in any biomanufacturing process. For the biomanufacture of non-catabolic compounds such as isoprenoids, productivity and yield are typically functions of other cell-related rates, e.g., sugar and / or oxygen consumption rates. Designing a fermentation process to produce the optimal combination of productivity and yield at the lowest cost requires close characterization of this relationship. For isoprenoid production, under standard fermentation conditions, yield is always negatively correlated with cell-specific oxygen and sugar uptake rates and productivity, i.e., the faster the cells producing isoprenoids take up oxygen and sugar, the lower the yield of isoprenoids. This relationship is referred to as "rate-yield coupling". This inverse coupling of yield and productivity is problematic because yield and productivity are two key cost drivers in isoprenoid production. Due to rate-yield coupling, any attempt to increase productivity by increasing oxygen and / or sugar transfer rates will result in a concomitant decrease in yield, thus offsetting the cost-benefit of the increased productivity. Eliminating the inverse correlation between yield and productivity would be beneficial because high yield and high productivity could be achieved simultaneously, thus maximizing the efficiency of isoprenoid production (maximizing the isoprenoid product per fermentation cost). SUMMARY OF THE INVENTION

[0002] The present invention generally relates to methods for decoupling the relationship between yield and productivity during the production of non-catabolic compounds during fermentation of a host cell producing a non-catabolic compound.

[0003] In one aspect, the present invention provides a method for decoupling the relationship between the yield and productivity of a non-catabolic compound produced in a host cell capable of manufacturing a non-catabolic compound, the method comprising the step of reducing ATP utilization during the fermentation process.

[0004] In one embodiment, ATP utilization is reduced by adding one or more ATP-consuming agents. In another embodiment, the one or more ATP-consuming agents are weak organic acids. In certain embodiments, the weak organic acids are selected from sorbic acid, acetic acid, benzoic acid, and propionic acid. In a preferred embodiment, the weak organic acid is benzoic acid.

[0005] In other embodiments of the present invention, ATP utilization is reduced by overexpression of one or more ATP-dissipating enzymes. In one embodiment, one or more ATP-dissipating enzymes are selected from Saccharomyces cerevisiae SSB1 and ATP-diphosphohydrolase. In another embodiment, ATP utilization is reduced by overexpression of one or more ATP-uncoupling enzymes. In certain embodiments, one or more ATP-uncoupling enzymes are selected from NADH oxidase (NOX) and alternative oxidase (AOX).

[0006] In further embodiments of the present invention, ATP levels are reduced by expression of a futile cycle in the host cell. In certain embodiments, the futile cycle is selected from the simultaneous overexpression of phosphofructokinase and fructose-1,6-bisphosphatase and the simultaneous overexpression of phosphoenolpyruvate carboxykinase and pyruvate carboxylase.

[0007] In one embodiment of the method of the present invention, the non-catabolic compound is selected from amino acids, fatty acids, isoprenoids and polyketides. In certain embodiments, the non-catabolic compound is an isoprenoid. In certain embodiments, the isoprenoid is selected from hemiterpenes, monoterpenes, diterpenes, triterpenes, tetraterpenes, sesquiterpenes and polyterpenes. In other embodiments, the isoprenoid is selected from abietadiene, amorpha-4,11-diene (a precursor of artemisinin), carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene and valencene. In a preferred embodiment, the isoprenoid is β-farnesene.

[0008] In one embodiment of the method of the present invention, the host cell is selected from bacterial cells, plant cells and yeast cells. In certain embodiments, the host cell is a yeast cell. In a preferred embodiment, the yeast cell is Saccharomyces cerevisiae.

[0009] In another aspect of the present invention, there is provided a method for decoupling the yield and productivity of non-catabolic compounds produced in a host cell capable of producing isoprenoids, the method comprising the step of reducing the carbon flux through the tricarboxylic acid (TCA) cycle in the host cell.

[0010] In one embodiment of the method of the present invention, carbon flux through the TCA cycle is reduced by inhibition of one or more TCA enzymes. In another embodiment, the one or more TCA enzymes are downregulated. In another embodiment, the TCA enzyme is selected from citrate synthase, aconitate hydratase, NAD-dependent isocitrate dehydrogenase, 2-ketoglutarate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydralase, peroxisomal malate dehydrogenase, and pyruvate carboxylase. In a preferred embodiment, the TCA enzymes are pyruvate carboxylase and citrate synthase.

[0011] Brief Description of the Drawings

[0012] Figure 1 It is a graph of the farnesene yield at different specific oxygen uptake values (qO2) of cells.

[0013] Figure 2 It is a graph of the farnesene yield at different specific sugar uptake values (qS) of cells.

[0014] Figure 3 It is a graph of the farnesene yield at different productivity values.

[0015] Figure 4 It is a graph of the specific sugar uptake value (qS) at different oxygen uptake values (qO2) of cells.

[0016] Figure 5 Plotted is the ratio of α-ketoglutarate to isocitrate at different oxygen uptake rates.

[0017] Figure 6 It is a schematic diagram of the modification of the genome-scale model, which enables simulation of the effect of ATP production on product yield by eliminating hydrolysis or futile cycles that are potential sinks for ATP. (NGAM is "Non-growth associated maintenance".)

[0018] Figure 7 Plotted is data from the simulation of farnesene yield as a function of qS at different assumed fluxes through the TCA cycle. The actual experimental data are plotted as black circles on the simulation values.

[0019] Figure 8 It is a set of graphs showing the effect of different benzoic acid concentrations (X-axis = mM benzoic acid) on specific sugar uptake (qS) (upper graph on the Y-axis), growth rate (second graph from the top on the Y-axis), specific farnesene productivity (qP) (third graph from the top on the Y-axis), and calculated product yield (qP / qS) (bottom graph on the Y-axis).

[0020] Figure 9 Shows the farnesene yields of the following three strains at different specific oxygen uptake rates (qO2): Y21901 (control); Y22021 (control); and Y31655 (Y22021 overexpressing NOX).

[0021] Figure 10 Shows the farnesene yields of the following four strains at different specific oxygen uptake rates (qO2): Y21601 (control); Y27662 (PYC1 downregulated); Y29438 (PYC1 and CIT1 downregulated); and Y39666 (CIT1 downregulated). Detailed implementation manners

[0022] In this article, the term "productivity" or "q" P in relation to the biomanufacture of isoprenoid products refers to the number of moles of isoprenoid products produced per gram of cell dry weight per hour.

[0023] In this article, the term "yield" or "Y" P / S in relation to the biomanufacture of isoprenoid products refers to the rate of formation of isoprenoid products relative to the sugar consumption rate.

[0024] In this article, the term "q" S or "sugar consumption" refers to the rate of sugar consumption during fermentation, expressed as the number of moles of sugar consumed per gram of cell dry weight per hour.

[0025] In this article, the term "q" O2 or "oxygen consumption" refers to the rate of oxygen consumption during fermentation, expressed as the number of moles of O 2 consumed per gram of cell dry weight per hour.

[0026] In this article, the term "rate-yield coupling" refers to the inverse correlation between yield and productivity during fermentation-based isoprenoid production.

[0027] In this article, the term "weak organic acid" or "(WOA)" refers to an organic acid with a pKa of 4.0 or greater. Non-limiting examples of WOA include sorbic acid, acetic acid, benzoic acid, and propionic acid.

[0028] In this article, the term "futile cycle" refers to at least two metabolic cycles or pathways that, when operating simultaneously in opposite directions, have no other effect except to dissipate energy in the form of ATP hydrolysis.

[0029] As used herein, the term "ATP dissipation reaction" refers to a biochemical reaction that hydrolyzes ATP without utilizing the energy for any physiological process.

[0030] As used herein, the term "ATP uncoupling reaction" refers to a biochemical reaction that uncouples the oxidation of NADH or proton transport from ATP generation.

[0031] As used herein, the term "heterologous" means not normally present in nature. The term "heterologous nucleotide sequence" refers to a nucleotide sequence that is not normally found in a given cell in nature. Thus, a heterologous nucleotide sequence can be: (a) foreign to its host cell (i.e., "exogenous" to the cell); (b) naturally present in the host cell (i.e., "endogenous") but present in the cell in an unnatural amount (i.e., more or less than the amount naturally present in the host cell); or (c) naturally present in the host cell but located outside its natural locus.

[0032] As used herein, "functional disruption" of a target gene (e.g., one or more genes of the TCA pathway) refers to altering the target gene in a manner that reduces the activity of the protein encoded by the target gene in the host cell. In some embodiments, the activity of the protein encoded by the target gene is eliminated in the host cell. In other embodiments, the activity of the protein encoded by the target gene is reduced in the host cell. Functional disruption of the target gene can be achieved by deleting all or part of the gene such that gene expression is eliminated or reduced or the activity of the gene product is eliminated or reduced. Functional disruption of the target gene can also be achieved by mutating the regulatory elements of the gene (e.g., the promoter of the gene) such that expression is eliminated or reduced, or by mutating the coding sequence of the gene such that the activity of the gene product is eliminated or reduced. In some embodiments, functional disruption of the target gene results in the removal of the complete open reading frame of the target gene.

[0033] As used herein, the term "parent cell" refers to a cell having the same genetic background as the host cells disclosed herein, except that it does not contain a specific heterologous nucleotide sequence and serves as a starting point for introducing the heterologous nucleotide sequence resulting in the production of the host cells disclosed herein.

[0034] As used herein, the term "biosynthetic enzyme" refers to an enzyme that functions in a biosynthetic pathway leading to the production of a naturally occurring molecule.

[0035] Genetically modified microorganisms producing isoprenoids

[0036] Host cell

[0037] Host cells useful in the practice of the present invention include archaea (archae), prokaryotic cells or eukaryotic cells.

[0038] Suitable prokaryotic hosts include, but are not limited to: any one of a variety of Gram-positive bacteria, Gram-negative bacteria, or gram-variable bacteria. Examples include, but are not limited to, cells belonging to the following genera: Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Arthrobacter, Azobacter, Bacillus, Brevibacterium, Chromatium, Clostridium, Corynebacterium, Enterobacter, Erwinia, Escherichia, Lactobacillus, Lactococcus, Mesorhizobium, Methylobacterium, Microbacterium, Phormidium, Pseudomonas, Rhodobacter, Rhodopseudomonas, Rhodospirillum, Rhodococcus, Salmonella, Scenedesmun, Serratia, Shigella, Staphlococcus, Strepromyces, Synnecoccus, and Zymomonas.Examples of prokaryotic strains include, but are not limited to: Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium beigerinckii, Enterobacter sakazakii, Escherichia coli, Lactococcus lactis, Mesorhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, and Staphylococcus aureus. In a specific embodiment, the host cell is an Escherichia coli cell.

[0039] Suitable archaeal hosts include, but are not limited to, cells belonging to the following genera: Aeropyrum, Archaeglobus, Halobacterium, Methanococcus, Methanobacterium, Pyrococcus, Sulfolobus, and Thermoplasma. Examples of archaeal strains include, but are not limited to: Archaeoglobus fulgidus, Halobacterium sp., Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Thermoplasma acidophilum, Thermoplasma volcanium, Pyrococcus horikoshii, Pyrococcus abyssi, and Aeropyrum pernix.

[0040] Suitable eukaryotic hosts include, but are not limited to: fungal cells, algal cells, insect cells, and plant cells. In some embodiments, yeasts that can be used in the methods of the present invention include yeasts that have been deposited in a microorganism depository (e.g., IFO, ATCC, etc.) and belong to the following genera: Aciculoconidium, Ambrosiozyma, Arthoascus, Arxiozyma, Ashbya, Babjevia, Bensingtonia, Botryoascus, Botryozyma, Brettanomyces, Bullera, Bulleromyces, Candida, Citeromyces, Clavispora, Cryptococcus, Cystofilobasidium, Debaryomyces, Dekkara, Dipodascopsis, Dipodascus, Eeniella, Endomycopsella, Eremascus, Eremothecium, Erythrobasidium, Fellomyces, Filobasidium, Galactomyces, Geotrichum, Guilliermondella, Hanseniaspora, Hansenula, Hasegawaea, Holtermannia, Hormoascus, Hyphophichia, Issatchenkia, Kloeckera, Kloeckeraspora, Kluyveromyces, Kondoa, Kuraishia, Kurtzmanomyces, Leucosporidium, Lipomyces, Lodderomyces, Malassezia, Metschnikowia, Mrakia, Myxozyma, Nadsonia, Nakazawaea, Nematospora,Ogataea, Oosporidium, Pachysolen, Phachytichospora, Phaffia, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Saitoella, Sakaguchia, Saturnospora, Schizoblastosporion, Schizosaccharomyces, Schwanniomyces, Sporidiobolus, Sporobolomyces, Sporopachydermia, Stephanoascus, Sterigmatomyces, Sterigmatosporidium, Symbiotaphrina, Sympodiomyces, Sympodiomycopsis, Torulaspora, Trichosporiella, Trichosporon, Trigonopsis, Tsuchiyaea, Udeniomyces, Waltomyces, Wickerhamia, Wickerhamiella, Williopsis, Yamadazyma, Yarrowia, Zygoascus, Zygosaccharomyces, Zygowilliopsis, and Zygozyma, etc.

[0041] In some embodiments, the host microorganism is Saccharomyces cerevisiae, Pichia pastoris, Schizosaccharomyces pombe, Dekkera bruxellensis, Kluyveromyces lactis (formerly known as "Saccharomyces lactis"), Kluyveromyces marxianus, Arxula adeninivorans, or Hansenula polymorpha (also known as "Pichia angusta"). In some embodiments, the host microorganism is a Candida strain, such as Candida lipolytica, Candida guilliermondii, Candida krusei, Candida pseudotropicalis, or Candida utilis.

[0042] In a specific embodiment, the host microorganism is Saccharomyces cerevisiae. In some embodiments, the host is a Saccharomyces cerevisiae strain selected from Baker’s yeast, CBS 7959, CBS 7960, CBS 7961, CBS 7962, CBS 7963, CBS 7964, IZ-1904, TA, BG-1, CR-1, SA-1, M-26, Y-904, PE-2, PE-5, VR-1, BR-1, BR-2, ME-2, VR-2, MA-3, MA-4, CAT-1, CB-1, NR-1, BT-1, and AL-1. In some embodiments, the host microorganism is a Saccharomyces cerevisiae strain selected from PE-2, CAT-1, VR-1, BG-1, CR-1, and SA-1. In a specific embodiment, the Saccharomyces cerevisiae strain is PE-2. In another specific embodiment, the Saccharomyces cerevisiae strain is CAT-1. In another specific embodiment, the Saccharomyces cerevisiae strain is BG-1.

[0043] In some embodiments, the host microorganism is a microorganism suitable for industrial fermentation (e.g., bioethanol fermentation). In certain embodiments, the microorganism is accustomed to surviving under high solvent concentrations, high temperatures, broad substrate utilization, nutrient limitation, osmotic stress due to sugars and salts, acidity, sulfite, and bacterial contamination, or combinations thereof, which are recognized stress conditions of industrial fermentation environments.

[0044] HMGR using NADH

[0045] In another aspect, provided herein is a genetically modified host cell capable of producing isoprenoids, comprising one or more heterologous nucleotide sequences encoding acetylaldehyde dehydrogenase, acetylating (ADA, EC 1.2.1.10), and one or more heterologous nucleotide sequences encoding one or more enzymes of a biosynthetic pathway for isoprenoids, wherein said one or more enzymes of the biosynthetic pathway include an enzyme using NADH. Without being bound by theory, it is believed that the increased intracellular NADH pool generated by ADA during the conversion of acetaldehyde to acetyl-CoA is utilized by the NADH-using biosynthetic enzymes, thereby contributing to the restoration of the intracellular redox balance while increasing the yield of acetyl-CoA-derived products.

[0046] In some embodiments, the above-mentioned enzyme using NADH is a non-native enzyme of the biosynthetic pathway. For example, the enzyme using NADH can replace the native NADPH-using enzyme of the biosynthetic pathway. In other embodiments, the enzyme using NADH is co-expressed with the native NADPH-using enzyme of the biosynthetic pathway. In some embodiments, the genetically modified host cell comprises HMGR that can utilize only NADH as a cofactor.

[0047] In some embodiments, the genetically modified host cell is capable of producing isoprenoids and the cell comprises one or more heterologous nucleotide sequences encoding one or more enzymes of the mevalonate (MEV) pathway for the production of isopentenyl pyrophosphate, wherein the one or more enzymes include HMG-CoA reductase (HMGR) that uses NADH. HMG-CoA reductase catalyzes the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate, and HMG-CoA reductase consists of two classes of HMGrs, namely class I and class II HMGrs. Class I includes enzymes from eukaryotes and most archaea, and class II includes HMG-CoA reductases from certain prokaryotes and archaea. In addition to sequence differences, these two classes of enzymes also differ in cofactor specificity. Unlike class I enzymes that specifically utilize NADPH, class II HMG-CoA reductases differ in their ability to distinguish between NADPH and NADH. See, for example, Hedl et al., Journal of Bacteriology 186(7):1927-1932 (2004). Cofactor specificities of selected class II HMGRs are provided below.

[0048] Table 1 - Cofactor specificities of selected class II HMGRs

[0049] Source Coenzyme specificity <![CDATA[K m NADPH (μM)]]> <![CDATA[K m NADH (μM)]]> P.mevalonii NADH 80 Archaeoglobus fulgidus (A.fulgidus) NAD(P)H 500 160 Staphylococcus aureus (S.aureus) NAD(P)H 70 100 Enterococcus faecalis (E.faecalis) NADPH 30

[0050] HMGRs useful in the compositions and methods provided herein include HMGRs that are capable of using NADH as a cofactor, e.g., HMGRs from P. mevalonii, Archaeoglobus fulgidus or Staphylococcus aureus. In certain embodiments, the HMGR is capable of using only NADH as a cofactor, e.g., HMGRs from P. mevalonii, S. pomeroyi or Delftia acidovorans.

[0051] In some embodiments, the HMGR that uses NADH is from Pseudomonas mevalonii. The sequence of the wild-type mvaA gene of Pseudomonas mevalonii encoding HMGR (E.C. 1.1.1.88) has been previously reported, see: Beach and Rodwell, J. Bacteriol. 171:2994-3001 (1989). Representative mvaA nucleotide sequences of Pseudomonas mevalonii include Genbank accession number M24015 and SEQ ID NO: 3 provided herein. Representative HMGR protein sequences of Pseudomonas mevalonii include Genbank accession number AAA2583 and SEQ ID NO: 4 provided herein.

[0052] In some embodiments, the HMGR that uses NADH is from Silicibacter pomeroyi. Representative HMGR nucleotide sequences of Silicibacter pomeroyi include SEQ ID NO: 5 provided herein. Representative HMGR protein sequences of Silicibacter pomeroyi include Genbank accession number YP_164994 and SEQ ID NO: 6 provided herein.

[0053] In some embodiments, the HMGR that uses NADH is from Delftia acidovorans. Representative HMGR nucleotide sequences of Delftia acidovorans include SEQ ID NO: 7 provided herein. Representative HMGR protein sequences of Delftia acidovorans include Genbank accession number YP_001561318 and SEQ ID NO: 8 provided herein.

[0054] HMGRs that use NADH and can also be used in the compositions and methods provided herein include those molecules referred to as "derivatives" of any of the NADH-using HMGRs described herein (e.g., from P. mevalonii, S. pomeroyi, and Delftia acidovorans). Such "derivatives" have the following characteristics: (1) it has substantial homology with any of the NADH-using HMGRs described herein; (2) it is capable of using NADH as a cofactor to catalyze the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate. A derivative of an NADH-using HMGR is said to have "substantial homology" with the NADH-using HMGR if its amino acid sequence is at least 80%, more preferably at least 90%, and most preferably at least 95% identical to the amino acid sequence of the NADH-using HMGR.

[0055] In some embodiments, the NADH-using HMGR has a selectivity for NADH as a cofactor over NADPH. In some embodiments, the NADH-using HMGR has a K m NADH :K m NADPH ratio for NADH as a cofactor that is higher than that for NADPH. In some embodiments, for example, by site-directed mutagenesis of the cofactor-binding pocket, the gene of the NADH-using HMGR is altered to make its selectivity for NADH higher than its selectivity for NAPDH. Methods for engineering NADH selectivity are described in Watanabe et al., Microbiology 153:3044-3054 (2007); methods for determining the cofactor specificity of HMGR are described in Kim et al., Protein Sci. 9:1226-1234 (2000), and the entire contents of these two references are incorporated herein by reference.

[0056] In some embodiments, the NADH-using HMGR is derived from a host species that naturally contains a mevalonate degradation pathway, e.g., a host species that catabolizes mevalonate as its sole carbon source. In these embodiments, the NADH-using HMGR, which normally catalyzes the oxidative acylation of internalized (R)-mevalonate to (S)-HMG-CoA in its native host cell, is used in a genetically modified host cell containing a mevalonate biosynthetic pathway to catalyze the reverse reaction, i.e., the reductive deacylation of (S)-HMG-CoA to (R)-mevalonate. Prokaryotes capable of growing on mevalonate as their sole carbon source are described in the following articles: Anderson et al., J. Bacteriol., 171(12):6468-6472 (1989); Beach et al., J. Bacteriol. 171:2994-3001 (1989); Bensch et al., J. Biol. Chem. 245:3755-3762; Fimongnari et al., Biochemistry 4:2086-2090 (1965); Siddiqi et al., Biochem. Biophys. Res. Commun. 8:110-113 (1962); Siddiqi et al., J. Bacteriol. 93:207-214 (1967); and Takatsuji et al., Biochem. Biophys. Res. Commun. 110:187-193 (1983), the entire contents of which are incorporated herein by reference.

[0057] Methods of preparing genetically modified cells

[0058] The methods provided herein include methods for generating a host cell that has been genetically engineered to contain ADA and / or a NADH-using biosynthetic enzyme. Expression of ADA and / or a NADH-using biosynthetic enzyme in the host cell can be achieved by introducing a nucleic acid comprising a nucleotide sequence encoding ADA and / or a NADH-using biosynthetic enzyme under the control of regulatory elements into the host cell, wherein the regulatory elements permit expression in the host cell. In some embodiments, the nucleic acid is an extrachromosomal plasmid. In other embodiments, the nucleic acid is a chromosomal integration vector that can integrate the nucleotide sequence into the chromosome of the host cell.

[0059] The nucleic acids encoding these proteins can be introduced into host cells by any method known to those skilled in the art, but are not limited thereto (for example, see: Hinnen et al. (1978) Proc. Natl. Acad. Sci. USA 75: 1292-3; Cregg et al. (1985) Mol. Cell. Biol. 5: 3376-3385; Goeddel et al., eds, 1990, Methods in Enzymology, vol. 185, Academic Press, Inc., CA; Krieger, 1990, Gene Transfer and Expression--A Laboratory Manual, Stockton Press, NY; Sambrook et al., 1989, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY). Exemplary techniques include, but are not limited to: spheroplasting, electroporation, PEG 1000-mediated transformation, and lithium acetate or lithium chloride-mediated transformation.

[0060] The copy number of an enzyme in a host cell can be altered by modifying the transcription of the gene encoding the enzyme. This can be achieved, for example, by modifying the copy number of the nucleotide sequence encoding the enzyme (e.g., by using an expression vector containing a higher or lower copy number of the nucleotide sequence, or by introducing additional copies of the nucleotide sequence into the genome of the host cell or by deleting or disrupting the nucleotide sequence in the genome of the host cell), by changing the order of the coding sequences on the polycistronic mRNA of an operon or by breaking the operon into individual genes each having its own control elements, or by increasing the strength of a promoter or operon operably linked to the nucleotide sequence. Alternatively or additionally, the copy number of an enzyme in a host cell can be altered by modifying the translation level of the mRNA encoding the enzyme. This can be done, for example, by modifying the stability of the mRNA, modifying the sequence of the ribosome binding site, modifying the distance or sequence between the ribosome binding site and the start codon of the enzyme coding sequence, modifying the entire intercistronic region located "upstream" of or adjacent to the 5'-side of the start codon in the enzyme coding region, using hairpin and specialized sequences to stabilize the 3'-end of the mRNA transcript, modifying the codon usage of the enzyme, altering the expression of rare codon tRNAs used in the biosynthesis of the enzyme, and / or increasing the stability of the enzyme (e.g., by mutation of its coding sequence).

[0061] The activity of an enzyme in a host cell can be altered in a variety of ways, including, but not limited to: expressing a modified form of the enzyme that exhibits increased or decreased solubility in the host cell, expressing an altered form of the enzyme lacking a domain through which its activity is inhibited, expressing a modified form of the enzyme having a higher or lower Kcat or a lower or higher Km for a substrate, or expressing an enzyme that is more or less affected by feedback or feed-forward regulation by another molecule in the pathway.

[0062] In some embodiments, the nucleic acid for genetically modifying a host cell comprises one or more selectable markers that can be used to select the transformed host cells and to impose a selection pressure on the host cells to maintain the foreign DNA.

[0063] In some embodiments, the above selectable marker is an antibiotic resistance marker. Illustrative examples of antibiotic resistance markers include, but are not limited to: BLA, NAT1, PAT, AUR1-C, PDR4, SMR1, CAT, mouse dhfr, HPH, DSDA, KAN Rand SHBLE gene products. The BLA gene product from Escherichia coli confers resistance to β-lactam antibiotics (e.g., narrow-spectrum cephalosporins, cephamycin, and carbapenems (ertapenem), cefamandole, and cefoperazone) and all anti-gram-negative-bacterium penicillins except temocillin; the NAT1 gene product from Streptomyces noursei confers resistance to nourseothricin; the PAT gene product from Streptomyces viridochromogenes Tu94 confers resistance to bialophos; the AUR1-C gene product from Saccharomyces cerevisiae confers resistance to Auerobasidin A (AbA); the PDR4 gene product confers resistance to cerulenin; the SMR1 gene product confers resistance to sulfometuron methyl; the CAT gene product from the Tn9 transposon confers resistance to chloramphenicol; the mouse dhfr gene product confers resistance to methotrexate; the HPH gene product from Klebsiella pneumoniae confers resistance to Hygromycin B; the DSDA gene product from Escherichia coli allows cells to grow on plates with D-serine as the sole nitrogen source; the KAN R gene from the Tn903 transposon confers resistance to G418; and, the SHBLE gene product from Streptoalloteichus hindustanus confers resistance to Zeocin (bleomycin). In some embodiments, the antibiotic resistance marker is deleted after isolation of the genetically modified host cells disclosed herein.

[0064] In some embodiments, selectable markers rescue auxotrophy in genetically modified microorganisms (e.g., nutritional auxotrophy). In such embodiments, the parental microorganism contains a functional disruption of one or more gene products that function in the amino acid or nucleotide biosynthetic pathway and, when non-functional, renders the parental cell unable to grow in culture without supplementation of one or more nutrients. Such gene products include, but are not limited to: HIS3, LEU2, LYS1, LYS2, MET15, TRP1, ADE2, and URA3 gene products in yeast. The auxotrophic phenotype can be rescued by transforming the parental cell with an expression vector or chromosomal integration construct encoding a functional copy of the disrupted gene product, and the resulting genetically modified host cell can be selected based on the loss of the auxotrophic phenotype of the parental cell. Using the URA3, TRP1, and LYS2 genes as selectable markers has significant advantages because both positive and negative selections are possible. Positive selection is carried out by nutritional defect complementation of URA3, TRP1, and LYS2 mutations, while negative selection is based on specific inhibitors that block the growth of prototrophic strains but allow the growth of URA3, TRP1, and LYS2 mutants, namely 5-fluoro-orotic acid (FOA), 5-fluoroanthranilic acid, and aminoadipic acid (aAA), respectively. In other embodiments, selectable marker rescue can be used to rescue other non-lethal defects or phenotypes that can be identified by known selection methods.

[0065] TCA cycle enzymes

[0066] The tricarboxylic acid (TCA) cycle, also known as the "citric acid cycle (CAC)" or the "Krebs cycle", is a series of biochemical reactions used to generate energy in the form of ATP by the oxidation of acetyl coenzyme A derived from carbohydrates, fats, and proteins. The TCA also provides precursors for the production of certain amino acids as well as the reducing agent NADH. In one embodiment of the present invention, the rate and yield of isoprenoid production are decoupled by downregulating the activity of one or more enzymes that are directly or indirectly involved in the TCA cycle (e.g., by providing carbon to the TCA cycle).

[0067] In some embodiments, the TCA enzyme is citrate synthase, which is also known as citrate condensing enzyme, CoA-acetylating citrate oxaloacetate-lyase, citric-condensing enzyme, citrogenase, oxaloacetate transacetase, CIT1, CIT3, (comprising the amino acid sequence NP5.101) or NP 014398.1, EC 2.3.3.1, EC 2.3.3.8 and EC 2.3.3.3. Citrate synthase catalyzes the conversion of oxaloacetate, acetyl coenzyme A (acetyl-CoA) and water into citrate and coenzyme A.

[0068] In some embodiments, the TCA enzyme is aconitate hydratase, which is also known as cis-aconitase, aconitase, ACO1, (comprising the amino acid sequence NP 013407.1), and EC 4.2.1.3. Aconitate hydratase catalyzes the conversion of citrate to isocitrate through the cis-aconitate intermediate.

[0069] In some embodiments, the TCA enzyme is NAD-dependent isocitrate dehydrogenase, which is also known as IDH2, IDH1, EC 1.1.1.42, EC 1.1.1.41, EC 1.1.1.286 and (comprising the amino acid sequence NP 014779.1 or NP 014361.1). NAD-dependent isocitrate dehydrogenase catalyzes the conversion of isocitrate to 2-oxoglutarate, carbon dioxide and NADH in a NAD-dependent manner.

[0070] In some embodiments, the TCA enzyme is 2-ketoglutarate dehydrogenase complex, which is also known as dihydrolipoamide dehydrogenase, KGD2, KGD1, LPD1, EC 1.2.4.2, EC 2.3.1.61 and (comprising amino acid sequences NP010432.3, NP 012141.1 or NP 116635.1). The 2-ketoglutarate dehydrogenase complex catalyzes the conversion of 2-ketoglutarate and coenzyme A to succinyl-CoA, carbon dioxide and NADH in a NAD-dependent manner.

[0071] In some embodiments, the TCA enzyme is succinyl-CoA ligase, which is also known as LSC2, LSC1, EC 6.2.1.4, EC 6.2.1.5, EC 2.8.3.18 and (comprising amino acid sequences NP 011670.3 or NP014785.3). Succinyl-CoA ligase catalyzes the conversion of succinyl-CoA, ADP and phosphate to succinate, ATP and coenzyme A.

[0072] In some embodiments, the TCA enzyme is minor succinate dehydrogenase, which is also known as SDH1, SDH2, SDH3, SDH4, succinate dehydrogenase, EC 1.3.5.4, EC1.3.5.1 and (comprising amino acid sequences NP 012774.1, NP 013059.1 or NP012781.1). Succinate dehydrogenase catalyzes the conversion of succinate and ubiquinone to fumarate and ubiquinol.

[0073] In some embodiments, the TCA enzyme is fumarate hydralase, which is also known as FUM1, EC 4.2.1.2 and (comprising amino acid sequence NP 015061.1). Fumarate hydralase catalyzes the conversion of fumarate and water to malic acid.

[0074] In some embodiments, the TCA enzyme is peroxisomal malate dehydrogenase, which is also known as MDH3, mitochondrial malate dehydrogenase, MDH1, cytoplasmic malate dehydrogenase, MDH2, EC 1.1.1.37, EC 1.1.5.4, and (including amino acid sequences NP010205.1, NP 014515.2, or NP012838.1). Peroxisomal malate dehydrogenase catalyzes the conversion of malate and NAD to oxaloacetate and NADH.

[0075] In some embodiments, the TCA enzyme is pyruvate carboxylase, which is also known as PYC1, PYC2, EC 4.1.1.32, EC.4.1.1.49, and (including amino acid sequences of NP011453.1 or NP 09777.1). Pyruvate carboxylase catalyzes the conversion of pyruvate, bicarbonate, and ATP to phosphate, oxaloacetate, and ADP.

[0076] Futile Cycle

[0077] In one aspect of the present invention, by introducing one or more futile cycles into a host cell producing isoprenoids, the coupling relationship between the yield and productivity of isoprenoids in the host cell can be decoupled. A futile cycle includes at least two metabolic cycles or pathways that, when operating simultaneously in opposite directions, have no other effect except for dissipating energy in the form of ATP hydrolysis. Therefore, introducing one or more futile cycles into the host cell reduces the ATP level of the cell, thereby decoupling the yield and productivity of isoprenoid production.

[0078] In one embodiment, the futile cycle includes overexpression of phosphofructokinase and fructose-1,6-bisphosphatase. Phosphofructokinase catalyzes the conversion of D-fructose-6-phosphate to D-fructose-1,6-bisphosphate. In contrast, fructose-1,6-bisphosphatase (EC3.1.3.11) catalyzes the hydrolysis of D-fructose-1,6-bisphosphate to D-fructose-6-phosphate in a reaction that consumes one molecule of ATP. Therefore, the simultaneous expression of these two enzymes results in the dissipation of ATP. For example, see U.S. Patent Applications Nos. US20150322461 and US20120088290, the entire contents of which are incorporated herein by reference.

[0079] In another embodiment, the futile cycle comprises the simultaneous overexpression of phosphoenolpyruvate carboxykinase and pyruvate carboxylase. Phosphoenolpyruvate carboxykinase catalyzes the conversion of oxaloacetate to phosphoenolpyruvate, while pyruvate carboxylase catalyzes the reverse reaction. Each enzyme hydrolyzes one ATP molecule in each reaction. However, the net cycle also produces one ATP molecule. Thus, each cycle reaction consumes one net ATP molecule. See, for example, U.S. Patent Applications Nos. US20150322461 and US20120088290.

[0080] ATP-dissipating enzyme

[0081] In one aspect of the invention, the yield and productivity of isoprenoids can be uncoupled in an isoprenoid-producing host cell by the expression of an enzyme that dissipates ATP without any other physiological effect.

[0082] In one embodiment, overexpression of the Saccharomyces cerevisiae SSB1 gene or a fragment thereof in an isoprenoid-producing host cell results in uncoupling of the yield and productivity of isoprenoids. The SSB1 gene encodes a chaperone protein that hydrolyzes an ATP molecule when binding to a nascent unfolded protein. Thus, overexpression of SSB1 or an enzymatically active fragment thereof results in ATP dissipation without any other physiological effect. See, for example, U.S. Patent Applications Nos. US20150322461 and US20120088290.

[0083] In another embodiment, overexpression of ATP-diphosphohydrolase or a fragment thereof in an isoprenoid-producing host cell results in uncoupling of the yield and productivity of isoprenoids. ATP-diphosphohydrolase is an enzyme that catalyzes the hydrolysis of the β- and γ-phosphates of ADP and ATP. Thus, overexpression of ATP-diphosphohydrolase or an enzymatically active fragment thereof results in ATP dissipation without any other physiological effect. See, for example, U.S. Patent Applications Nos. US20150322461 and US20120088290.

[0084] In another embodiment, overexpression of NADH oxidase; EC 1.6.3.4 (NOX) or a functional fragment thereof in an isoprenoid-producing host cell results in uncoupling of the yield and productivity of isoprenoids. NOX transfers hydrogen directly to O 2Reducing NADH to NAD+ without generating ATP. NOX reduces the intracellular ATP concentration by bypassing the native electron transport chain, which would otherwise generate ATP upon oxidation of NADH to NAD+. Thus, overexpression of NOX or its functional fragment results in ATP dissipation without any other physiological effects.

[0085] In another embodiment, overexpression of alternative oxidase (AOX) or its functional fragment in an isoprenoid-producing host cell results in decoupling of isoprenoid yield and productivity. The electron flow from ubiquinol to AOX leading to the reduction of O 2 to H 2 O is not coupled to proton transport, thus reducing the driving force for ATP synthase to produce ATP. Thus, overexpression of AOX or its functional fragment results in ATP dissipation without any other physiological effects.

[0086] ATP Depleting Agents

[0087] In some embodiments, the ATP level in the host cell is reduced by adding one or more ATP depleting agents. An ATP depleting agent is a compound or molecule that can reduce the ATP level in the host cell when the host cell is cultured in a medium containing the ATP depleting agent. In some embodiments, the ATP depleting agent is a reagent that decouples electron transport from ATP production. In a preferred embodiment, the ATP depleting agent is a weak organic acid. Non-limiting illustrative examples of weak organic acids are acetic acid, propionic acid, sorbic acid, and benzoic acid. The host cell can be cultured in a medium containing a weak organic acid in an amount (concentration) sufficient to reduce the ATP level so as to decouple the yield and productivity of non-catabolic compounds. In some embodiments, the amount of the weak organic acid is 0.25 mM or more. In certain embodiments, the host cell medium has at least 0.25 mM, 0.3 mM, 0.35 mM, 0.40 mM, 0.45 mM, 0.5 mM, 0.55 mM, 0.6 mM, 0.65 mM, 0.70 mM, 0.75 mM, 0.80 mM, 0.85 mM, 0.90 mM, 0.95 mM, 1.0 mM, 2.0 mM, 3.0 mM, 4.0 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, or 10.0 mM.

[0088] MEV pathway

[0089] In some embodiments, the host cell further comprises one or more heterologous enzymes that function in a biosynthetic pathway to produce cytosolic isoprenoids. Production of high levels of cytosolic isoprenoids can be affected by targeted genetic engineering of the host cell. Many enzymes are known to function in the production of cytosolic isoprenoids or the utilization of cytosolic acetyl-CoA and its precursors, and any one of these enzymes can be manipulated to alter the level of cytosolic isoprenoids in the host cell.

[0090] In some embodiments, the host cell comprises one or more heterologous enzymes of the MEV pathway. In some embodiments, the host cell comprises a heterologous mevalonate kinase. In other embodiments, the host cell comprises a heterologous HMG-CoA reductase. In some embodiments, the host cell comprises a heterologous IPP isomerase. In some embodiments, the host cell comprises a heterologous polyprenyl synthase. In some embodiments, the host cell comprises a heterologous FPP synthase. In some embodiments, the host cell comprises a heterologous terpene synthase. In some embodiments, the host cell comprises a heterologous farnesene synthase.

[0091] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense two molecules of acetyl-CoA to form acetoacetyl-CoA (e.g., acetyl-CoA thiolase). Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (NC_000913 REGION:2324131..2325315; Escherichia coli), (D49362; Paracoccus denitrificans), and (L20428; Saccharomyces cerevisiae).

[0092] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can condense acetoacetyl-CoA with another molecule of acetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (e.g., HMG-CoA synthase). Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_001145. Complement 19061.20536; Saccharomyces cerevisiae), (X96617; Saccharomyces cerevisiae), (X83882; Arabidopsis thaliana), (AB037907; Kitasatospora griseola), (BT007302; Homo sapiens), and (NC_002758, locus tag SAV2546, GeneID 1122571; Staphylococcus aureus).

[0093] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme that can convert HMG-CoA to mevalonic acid (e.g., HMG-CoA reductase). Exemplary examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NM_206548; Drosophila melanogaster), (NC_002758, locus tag SAV2545, GeneID 1122570; Staphylococcus aureus), (NM_204485; Gallus gallus), (AB015627; Streptomyces sp. KO 3988), (AF542543; Nicotiana attenuata), (AB037907; Kitasatospora griseola), (AX128213, providing a sequence encoding a truncated HMGR; Saccharomyces cerevisiae), and (NC_001145: Complement (115734.118898; Saccharomyces cerevisiae).

[0094] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., mevalonate kinase) that can convert mevalonate to mevalonate 5-phosphate. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (L77688; Arabidopsis thaliana) and (X55875; Saccharomyces cerevisiae).

[0095] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., phosphomevalonate kinase) that can convert mevalonate 5-phosphate to mevalonate 5-pyrophosphate. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (AF429385; Hevea brasiliensis), (NM_006556; Homo sapiens), and (NC_001145. Complement 712315.713670; Saccharomyces cerevisiae).

[0096] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., mevalonate pyrophosphate decarboxylase) that can convert mevalonate 5-pyrophosphate to IPP. Illustrative examples of nucleotide sequences encoding such an enzyme include, but are not limited to: (X97557; Saccharomyces cerevisiae), (AF290095; Enterococcus faecium), and (U49260; Homo sapiens).

[0097] In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding more than one enzyme of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding two enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding an enzyme that can convert HMG-CoA to mevalonic acid and an enzyme that can convert mevalonic acid to mevalonate 5-phosphate. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding three enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding four enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding five enzymes of the MEV pathway. In some embodiments, the host cell comprises one or more heterologous nucleotide sequences encoding six enzymes of the MEV pathway.

[0098] In some embodiments, the host cell produces C 5 isoprenoids. These compounds are derived from one isoprene unit and are also referred to as hemiterpenes. An example of a hemiterpene is isoprene. In other embodiments, the isoprenoids are C 10 isoprenoids. These compounds are derived from two isoprene units and are also referred to as monoterpenes. Examples of monoterpenes include limonene, citranellol, geraniol, menthol, perillyl alcohol, linalool, thujone, and myrcene. In other embodiments, the isoprenoids are C 15 isoprenoids. These compounds are derived from three isoprene units and are also referred to as sesquiterpenes. Examples of sesquiterpenes include periplanone B, ginkgolide B, the artemisinin precursor amorphadiene, artemisinin, artemisinic acid, valencene, nootkatone, epi-cedrol, epi-aristolochene, farnesol, gossypol, sanonin, periplanone, forskolin, and patchoulol (also referred to as patchouli alcohol). In other embodiments, the isoprenoids are C 20Isoprenoids. These compounds are derived from four isoprene units and are also called diterpenes. Examples of diterpenes are casbene, eleutherobin, paclitaxel, prostratin, pseudoopterosin, and taxadiene. In other examples, isoprenoids are C 20+ Isoprenoids. These compounds are derived from more than four isoprene units and include: triterpenes (C 30 isoprenoid compounds), for example, arbruside E, bruceantin, testosterone, progesterone, cortisone, digitoxin and squalene; tetraterpenes (C 40 Isoprenoid compounds), for example, β-carotene; and polyterpenes (C 40+ Isoprenoid compounds), for example, polyisoprene. In some embodiments, the isoprenoid is selected from abietadiene, amorphadiene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchoulol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene. Isoprenoid compounds also include, but are not limited to, carotenoids (e.g., lycopene, α- and β-carotene, α- and β-cryptoxanthin, bixin, zeaxanthin, astaxanthin and lutein), steroids, and compounds composed of isoprenoids modified with other chemical groups, for example, mixed terpene-alkaloids and coenzyme Q-10.

[0099] In some embodiments, the host cell further comprises a heterologous nucleotide sequence encoding an enzyme (e.g., IPP isomerase) that can convert IPP produced via the MEV pathway into DMAPP. Examples of nucleotide sequences encoding such enzymes include, but are not limited to: (NC_000913, 3031087..3031635; Escherichia coli) and (AF082326; Haematococcus pluvialis).

[0100] In some embodiments, the host cell further comprises a heterologous nucleotide sequence encoding a polyprenyl synthase that can condense IPP and / or DMAPP molecules to form polyisoprenoid compounds containing more than five carbons.

[0101] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., GPP synthase) that can condense one molecule of IPP with one molecule of DMAPP to form one molecule of geranyl pyrophosphate (“GPP”). Examples of nucleotide sequences encoding such enzymes include, but are not limited to: (AF513111; Abies grandis), (AF513112; Abies grandis), (AF513113; Abies grandis), (AY534686; Antirrhinum majus), (AY534687; Antirrhinum majus), (Y17376; Arabidopsis thaliana), (AE016877, locus AP11092; Bacillus cereus; ATCC 14579), (AJ243739; Citrus sinensis), (AY534745; Clarkia breweri), (AY953508; Ipspini), (DQ286930; Lycopersicon esculentum), (AF182828; Mentha x piperita), (AF182827; Mentha x piperita), (MPI249453; Mentha x piperita), (PZE431697, locus CAD24425; Paracoccus zeaxanthinifaciens), (AY866498; Picrorhiza kurrooa), (AY351862; Vitis vinifera), and (AF203881, locus AAF12843; Zymomonas mobilis).

[0102] In some embodiments, the host cell comprises a heterologous nucleotide sequence encoding an enzyme (e.g., FPP synthase) that can condense two molecules of IPP with one molecule of DMAPP, or add an IPP molecule to a GPP molecule to form a farnesyl pyrophosphate (“FPP”) molecule. Examples of nucleotide sequences encoding such enzymes include, but are not limited to: (ATU80605; Arabidopsis thaliana), (ATHFPS2R; Arabidopsis thaliana), (AAU36376; Artemisia annua), (AF461050; Bos taurus), (D00694; Escherichia coli K-12), (AE009951, locus AAL95523; Fusobacterium nucleatum subsp. nucleatum ATCC 25586), (GFFPPSGEN; Gibberella fujikuroi), (CP000009, locus AAW60034; Gluconobacter oxydans 621H), (AF019892; Helianthus annuus), (HUMFAPS; Homo sapiens), (KLPFPSQCR; Kluyveromyces lactis), (LAU15777; Lupinus albus), (LAU20771; Lupinus albus), (AF309508; Mus musculus), (NCFPPSGEN; Neurospora crassa), (PAFPS1; Parthenium argentatum), (PAFPS2; Parthenium argentatum), (RATFAPS; Rattus norvegicus), (YSCFPP; Saccharomyces cerevisiae), (D89104; Schizosaccharomyces pombe), (CP000003, locus AAT87386; Streptococcus pyogenes), (CP000017, locus AAZ51849;Streptococcus pyogenes (NC_008022, locus YP_598856; Streptococcus pyogenes MGAS10270) (NC_008023, locus YP_600845; Streptococcus pyogenes MGAS2096) (NC_008024, locus YP_602832; Streptococcus pyogenes MGAS10750) (MZEFPS; Zea mays) (AE000657, locus AAC06913; Aquifex aeolicus VF5) (NM_202836; Arabidopsis thaliana) (D84432, locus BAA12575; Bacillus subtilis) (U12678, locus AAC28894; Bradyrhizobium japonicum USDA 110) (BACFDPS; Geobacillus stearothermophilus) (NC_002940, locus NP_873754; Haemophilus ducreyi 35000HP) (L42023, locus AAC23087; Haemophilus influenzae Rd KW20) (J05262; Homo sapiens) (YP_395294; Lactobacillus sakei subsp. Sakei 23K) (NC_005823, locus YP_000273; Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130) (AB003187; Micrococcus luteus) (NC_002946, locus YP_208768; Neisseria gonorrhoeae FA 1090) (U00090, locus AAB91752; Rhizobium sp. NGR234) (J05091; Saccharomyces cerevisae) (CP000031, locus AAV93568;Silicibacter pomeroyi DSS-3), (AE008481, locus AAK99890; Streptococcus pneumoniae R6) and (NC_004556, locus NP_779706; Xylella fastidiosa Temecula 1).;

[0103] In some embodiments, the host cell further comprises a heterologous nucleotide sequence encoding an enzyme that can combine IPP and DMAPP or IPP and FPP to form geranylgeranyl pyrophosphate (“GGPP”). Examples of nucleotide sequences encoding such enzymes include, but are not limited to: (ATHGERPYRS; Arabidopsis thaliana), (BT005328; Arabidopsis thaliana), (NM_119845; Arabidopsis thaliana), (NZ_AAJM01000380, locus ZP_00743052; Bacillus thuringiensis serovar israelensis, ATCC 35646 sq1563), (CRGGPPS; Catharanthus roseus), (NZ_AABF02000074, locus ZP_00144509; Fusobacterium nucleatum subsp. vincentii, ATCC 49256), (GFGGPPSGN; Gibberella fujikuroi), (AY371321; Ginkgo biloba), (AB055496; Hevea brasiliensis), (AB017971; Homo sapiens), (MCI276129; Mucor circinelloides f.lusitanicus), (AB016044; Mus musculus), (AABX01000298, locus NCU01427; Neurospora crassa), (NCU20940; Neurospora crassa), (NZ_AAKL01000008, locus ZP_00943566; Ralstonia solanacearum UW551), (AB118238; Rattus norvegicus), (SCU31632; Saccharomyces cerevisiae), (AB016095; Synechococcus elongates), (SAGGPS; Sinapis alba), (SSOGDS; Sulfolobus acidocaldarius), (NC_007759, locus YP_461832; Syntrophus aciditrophicus SB), (NC_006840, locus YP_204095; Vibrio fischeri ES114), (NM_112315; Arabidopsis thaliana), (ERWCRTE; Pantoea agglomerans,), (D90087, locus BAA14124; Pantoea ananatis), (X52291, locus CAA36538; Rhodobacter capsulatus), (AF195122, locus AAF24294; Rhodobacter sphaeroides), and (NC_004350, locus NP_721015; Streptococcus mutans UA159).

[0104] In some embodiments, the host cell further comprises a heterologous nucleotide sequence encoding an enzyme that can modify polyisoprene to form hemiterpenes, monoterpenes, sesquiterpenes, diterpenes, triterpenes, tetraterpenes, polyterpenes, steroid compounds, carotenoids, or modified isoprenoid compounds.

[0105] In some embodiments, the heterologous nucleotide encodes carene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AF461460, REGION 43.1926; Picea abies) and (AF527416, REGION: 78.1871; Salvia stenophylla).

[0106] In some embodiments, the heterologous nucleotide encodes geraniol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AJ457070; Cinnamomum tenuipilum), (AY362553; Ocimum basilicum), (DQ234300; Perilla frutescens strain 1864), (DQ234299; Perilla citriodora strain 1861), (DQ234298; Perilla citriodora strain 4935), and (DQ088667; Perilla citriodora).

[0107] In some embodiments, the heterologous nucleotide encodes linalool synthase. Examples of suitable nucleotide sequences include, but are not limited to: (AF497485; Arabidopsis thaliana), (AC002294, Locus AAB71482; Arabidopsis thaliana), (AY059757; Arabidopsis thaliana), (NM_104793; Arabidopsis thaliana), (AF154124; Artemisia annua), (AF067603; Clarkia breweri), (AF067602; Clarkia concinna), (AF067601; Clarkia breweri), (U58314; Clarkia breweri), (AY840091; Lycopersicon esculentum), (DQ263741; Lavandula angustifolia), (AY083653; Mentha citrate), (AY693647; Ocimum basilicum), (XM_463918; Oryza sativa), (AP004078, Locus BAD07605; Oryza sativa), (XM_463918, Locus XP_463918; Oryza sativa), (AY917193; Perilla citriodora), (AF271259; Perilla frutescens), (AY473623; Picea abies), (DQ195274; Picea sitchensis), and (AF444798; Perilla frutescens var. crispa cultivar No. 79).

[0108] In some embodiments, the heterologous nucleotide encodes limonene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (+)-limonene synthase (AF514287, REGION: 47.1867; Citrus limon) and (AY055214, REGION: 48.1889; Agastache rugosa) and (-)-limonene synthase (DQ195275, REGION: 1.1905; Picea sitchensi), (AF006193, REGION: 73.1986; Abies grandis) and (MHC4SLSP, REGION: 29.1828; Mentha spicata).

[0109] In some embodiments, the heterologous nucleotide encodes myrcene synthase. Examples of suitable nucleotide sequences include, but are not limited to: (U87908; Abies grandis), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (NM_127982; Arabidopsis thaliana TPS10), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (AF271259; Perilla frutescens), (AY473626; Picea abies), (AF369919; Picea abies) and (AJ304839; Quercus ilex).

[0110] In some embodiments, the heterologous nucleotide encodes an ocimene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (AY195607; Antirrhinum majus), (AY195609; Antirrhinum majus), (AY195608; Antirrhinum majus), (AK221024; Arabidopsis thaliana), (NM_113485; Arabidopsis thaliana ATTPS-CIN), (NM_113483; Arabidopsis thaliana ATTPS-CIN), (NM_117775; Arabidopsis thaliana ATTPS03), (NM_001036574; Arabidopsis thaliana ATTPS03), (NM_127982; Arabidopsis thaliana TPS10), (AB110642; Citrus unshiu CitMTSL4), and (AY575970; Lotus corniculatus var. Japonicus).

[0111] In some embodiments, the heterologous nucleotide encodes an α-pinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (+)α-pinene synthase (AF543530, REGION: 1.1887; Pinus taeda), (-)α-pinene synthase (AF543527, REGION: 32.1921; Pinus taeda), and (+) / (-)α-pinene synthase (AGU87909, REGION: 6111892; Abies grandis).

[0112] In some embodiments, the heterologous nucleotide encodes a β-pinene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (-)β-pinene synthase (AF276072, REGION: 1.1749; Artemisia annua) and (AF514288, REGION: 26.1834; Citrus limon).

[0113] In some embodiments, the heterologous nucleotide encodes sabinene synthase. Examples of suitable nucleotide sequences include, but are not limited to: AF051901 from Salvia officinalis, REGION: 26.1798.

[0114] In some embodiments, the heterologous nucleotide encodes γ-terpinene synthase. Examples of suitable nucleotide sequences include, but are not limited to: (AF514286 from Citrus limon, REGION: 30.1832) and (AB110640 from Citrus unshiu, REGION 1.1803).

[0115] In some embodiments, the heterologous nucleotide encodes terpinolene synthase. Examples of suitable nucleotide sequences include, but are not limited to: (AY693650 from Oscimum basilicum) and (AY906866 from Pseudotsuga menziesii, REGION: 10.1887).

[0116] In some embodiments, the heterologous nucleotide encodes amorpha-4,11-diene synthase, a precursor of artemisinin. An example of a suitable nucleotide sequence is SEQ ID NO.37 in US Patent Application No. 2004 / 0005678.

[0117] In some embodiments, the heterologous nucleotide encodes α-farnesene synthase. Examples of suitable nucleotide sequences include, but are not limited to: DQ309034 from Pyrus communis cultivar d'Anjou (pear; gene name AFS1) and AY182241 from Malus domestica (apple; gene name AFS1). Pechouus et al., Planta 219(1):84-94 (2004).

[0118] In some embodiments, the heterologous nucleotide encodes α-farnesene synthase. Examples of suitable nucleotide sequences include, but are not limited to: GenBank accession number AF024615 from Mentha xpiperita (mint; gene Tspa11), and AY835398 from Artemisia annua. Picaud et al., Phytochemistry 66(9):961-967 (2005).

[0119] In some embodiments, the heterologous nucleotide encodes a farnesol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: GenBank accession number AF529266 from Zea mays and YDR481C from Saccharomyces cerevisiae (gene Pho8). Song, L., Applied Biochemistry and Biotechnology 128:149-158 (2006).

[0120] In some embodiments, the heterologous nucleotide encodes a nerolidol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: AF529266 from Zea mays (corn; gene tps1).

[0121] In some embodiments, the heterologous nucleotide encodes a patchoulol synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: AY508730 REGION:1.1659 from Pogostemon cablin.

[0122] In some embodiments, the heterologous nucleotide encodes a nootkatone synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: AF441124 REGION:1.1647 from Citrus sinensis and AY917195 REGION:1.1653 from Perilla frutescens.

[0123] In some embodiments, the heterologous nucleotide encodes an abietadiene synthase. Illustrative examples of suitable nucleotide sequences include, but are not limited to: (U50768; Abies grandis) and (AY473621; Picea abies).

[0124] Method for producing isoprenoids

[0125] In some embodiments in which the genetically modified host cell comprises a heterologous nucleotide sequence encoding an NADH-using HMGR, the genetically modified host cell produces an increased amount of isoprenoid compounds compared to a host cell that does not comprise the heterologous nucleotide sequence encoding an NADH-using HMGR, but is otherwise identical. In some embodiments, the increased amount is at least 10%, measured, for example, in grams per liter of cell culture, milligrams per gram of cell dry weight, based on per unit volume of cell culture, based on per unit cell dry weight, based on per unit volume of cell culture per unit time, or based on per unit cell dry weight per unit time.

[0126] In some embodiments where the genetically modified host cell comprises a heterologous nucleotide sequence encoding ADA and a heterologous nucleotide sequence encoding an NADH-utilizing HMGR, the genetically modified host cell produces increased amounts of isoprenoid compounds compared to: (i) a host cell that does not comprise the heterologous nucleotide sequence encoding ADA but is otherwise genetically identical; (ii) a host cell that does not comprise the heterologous nucleotide sequence encoding an NADH-utilizing HMGR but is otherwise genetically identical; or (iii) a host cell that does not comprise the heterologous nucleotide sequence encoding ADA or the heterologous sequence encoding an NADH-utilizing HMGR but is otherwise genetically identical. In some embodiments, the increased amount is at least 10%, measured, for example, in grams per liter of cell culture, milligrams per gram of cell dry weight, per unit volume of cell culture, per unit cell dry weight, per unit volume of cell culture per unit time, or per unit cell dry weight per unit time.

[0127] The method generally comprises culturing a host cell in a suitable culture medium comprising a carbon source under suitable conditions. Suitable conditions and suitable culture media for microbial growth are well known in the art. In some embodiments, the carbon source is a monosaccharide, disaccharide, polysaccharide, non-fermentable carbon source, or a combination of one or more thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol. In some embodiments, the suitable culture medium is supplemented with one or more additional reagents, such as, for example, an inducing compound (e.g., when one or more nucleotide sequences encoding a gene product are under the control of an inducible promoter), a repressing compound (e.g., when one or more nucleotide sequences encoding a gene product are under the control of a repressing promoter), or a selection agent (e.g., an antibiotic for selecting a microorganism comprising a genetic modification).

[0128] Examples

[0129] Example 1: Yield is a function of cell-specific rate

[0130] In a 125 ml flask, a single colony was inoculated in 15 ml of 2% sucrose, 1% maltose, 2 g / L lysine LGM, and 50 mM succinate at pH 5.0, and then grown at 28 °C with shaking at 200 r.p.m. until the OD600 was between 4 and 9 and the residual glucose was between 3 and 6 g l-1. 1 - 1.50% glycerol was added to the culture to a concentration of 20%, and then, 1 ml vials of the cell suspension were stored at -80 °C. 1 - 2 vials of cells were thawed and grown in a medium containing 3 g l-1 yeast extract, 7 g l-1 NH4H2PO4, 1 g l-1 KH2PO4, 0.5 g l-1 MgSO4·7H2O, 50 mM succinate at pH 5.0, 4% sucrose, 2% maltose, 2 g / L lysine, and trace metal and vitamin solutions for 24 h, and then, subcultured in the same medium for 24 h until the OD600 reading was 0.1. 25 ml of the culture was used to inoculate a 0.5 liter fermenter (Sartorius, Germany) containing 225 ml of fermentation medium which contained 15 g l-1 NH4H2PO4, 20 g l-1 total reducing sugars (TRS) from sucrose syrup (Florida Crystals, West Palm Beach), and trace metal and vitamin solutions. The fermenter temperature was cycled between 30 - 34 °C and the pH was maintained at 5.0 by adding NH 4 OH. During the initial batch phase, the fermenter was aerated at a rate of 0.5 volume per volume per minute (VVM) and gradually stirred to maintain 30% dissolved oxygen. After the initial sugars were consumed, the increase in dissolved oxygen triggered a pulse of Florida cane sugar syrup (about 800 g glucose equivalent per liter, (also known as total reducing sugars (TRS))) at a rate of 10 g TRS per liter per hour at a dose of 10 g TRS per liter. Between pulses, the feed rate was reduced to 1 - 5 g TRS per liter per hour. When the dissolved oxygen reached a peak, the high feed rate was restored, indicating that the residual carbon had been depleted; after adding a certain amount of sugar, the high feed rate was terminated. As the cell density increased, the dissolved oxygen reached 0% and the pulse dose was increased to 50 g TRS / L. As the volume increased, the oxygen transfer rate was maintained at a specific rate by adjusting the agitation. Here, an algorithm (feed rate algorithm) that alternates between high and low feed rates was used to dynamically adjust the feed rate to meet the demand. During the low feed rate period, the cells consumed the sugar and any overflow metabolites accumulated during the high feed rate. The increase in dissolved oxygen then triggered the restoration of the high feed rate. The length of time spent at the low feed rate reflects the degree to which the cells were overfed or underfed during the previous high feed rate pulse; this information was monitored and used to adjust the high feed rate up or down to keep the low feed rate within a defined range. Over time, the feed rate was matched to the cell's demand for sugar. The feed rate algorithm ensures divanene, biomass, and CO2 The net accumulation of fermentation products other than this was minimal. The process lasted for 8 - 13 days. The fermenter was filled and emptied. The accumulated broth was removed daily and the biomass and farnesene concentration were measured. Concentrated solutions of NH 4 H 2 PO 4 , trace metals and vitamins were added regularly to maintain steady - state concentrations.

[0131] Oxygen delivery is usually between 100 and 120 mmol / L / hr, also known as the oxygen transfer rate or OTR. Different oxygen transfer rates (20, 110, 180, 225) during the fermentation process were achieved through a combination of variable stirring rates, air flow rates and feed rates. Once the peak biomass level (grams of cell dry weight or gDCW) was reached during the production phase, the cells were under micro - aerobic conditions and the dissolved oxygen (dO 2 ) was almost zero; in other words, the oxygen uptake rate (OUR) was equal to the oxygen transfer rate (OTR).

[0132] Fermentation yield: The farnesene yield (Ysp) was calculated as the weight of farnesene produced divided by the amount of total reducing sugar (TRS, or glucose equivalent) added to the fermenter. The number of grams of farnesene produced divided by the number of grams of total reducing sugar added, expressed as a percentage, is also simply referred to as the "yield". gDW or gDCW is called grams of dry weight, which is a measure of the cell biomass or cell mass in the fermenter.

[0133] The specific oxygen uptake rate (qO2) is the specific oxygen consumption rate of the biomass in the fermenter, expressed as mmol / O2 / gDCW / h. It is also known as the specific oxygen utilization rate (sOUR).

[0134] The specific sugar uptake rate (qS) is the specific sugar consumption rate of the biomass in the fermenter, expressed as mmol / TRS / gDCW / h.

[0135] Farnesene was quantified as previously described (Sandoval, C.M. et al., (2014) Metab Eng vol.25, pp.215 - 226).

[0136] Yield and productivity are two major cost drivers in any biomanufacturing process. For the biomanufacture of non - catabolic products, the cell - specific production rate (q P , in units of moles of product / gram of cell dry weight / h) and the yield (Y P / S , or product formation rate / sugar consumption rate) are usually functions of other cell rates, e.g., the specific growth rate (1 / h) or (h -1 ), q S (moles of sugar consumed / gram of cell dry weight / h) or q O2(moles of O2 consumed / gram dry weight / h). Designing a fermentation process that achieves the optimal combination of yield and productivity to achieve the lowest cost production requires a close characterization of this relationship.

[0137] For isoprenoid production, we have observed that the yield (Y P / S ) is always inversely correlated with the cell-specific oxygen and sugar uptake rates and the volumetric productivity. In other words, the faster our cells take up oxygen and sugar, the lower the yield of isoprenoids. We refer to this relationship as "rate-yield coupling". Figures 1 to 3 This phenomenon is shown for a single farnesene-producing strain grown at different oxygen transfer rates (the rate at which oxygen is delivered to the bioreactor and taken up by the cells). As the cell-specific sugar uptake, oxygen uptake, and productivity increase, the yield decreases. Figure 4 Indicates that: using a feed rate algorithm, the sugar uptake rate is proportional to the oxygen uptake rate.

[0138] Figure 3 The inverse correlation between yield and productivity shown in

[0139] Example 2: Computational modeling and metabolomics assays indicate that ATP can drive the coupling between rate and yield

[0140] To evaluate the possible mechanistic causes of rate-yield coupling, we analyzed the absolute concentrations of central metabolites from glycolysis, the TCA cycle, the pentose phosphate pathway, and the isoprenoid pathway in fermentation samples from a single strain operating at low OTR (30 mmol O 2 / L / h) or high OTR (180 mmol O 2 / L / hr). Samples were taken from the bioreactor using a rapid sampling protocol, where samples were quenched immediately after being removed from the tank to capture the metabolic state. While most central metabolites were measured at similar absolute concentrations under low and high OTR conditions, two metabolites (isocitrate and α-ketoglutarate) stood out due to their very different concentrations (see Figure 5)。Interestingly, these two metabolites represent consecutive steps in the TCA cycle: isocitrate can be converted to α-ketoglutarate by the action of isocitrate dehydrogenase. We observed that the concentration of isocitrate decreased and the concentration of α-ketoglutarate increased under high OTR conditions relative to low OTR conditions, indicating that the flux rate through this step increased with high OTR conditions relative to other steps.

[0141] It has been previously determined that excess ATP produced in catabolic pathways can be detrimental to the rate and yield of biological processes. In terms of rate, this is because elevated ATP concentrations inhibit glycolysis. However, excess ATP also reduces the yield of biological processes because excess ATP drives the formation of biomass, which acts as a carbon sink and reduces product yield. The approximate stoichiometry for this is that for every 1 mol of biomass formed, 1.5 mol of excess ATP is removed or cleared from the system.

[0142] To determine whether the higher flux through the TCA cycle at high OTR could explain the lower farnesene yield, we developed a new computational modeling framework based on a comprehensive genome-scale metabolic model (see Figure 6 ) to evaluate the impact of ATP formation. Typical genome-scale models allow excess ATP to be hydrolyzed or "wasted" through futile reactions that may not occur in vivo, while our new model was constructed such that a small amount of ATP is allocated to non-growth-associated maintenance (or NGAM, assumed to be 0.4 mmol gDW-1 h-1.), and the rest is strictly associated with biomass formation, as is typically observed empirically. No futile cycles are allowed, and we assume that 1.5 mol of ATP is required for every 1 mol of biomass formed.

[0143] Using this model, we examined the potential impact of tricarboxylic acid cycle (TCA) flux on isoprenoids. Our model shows that the increase in TCA cycle flux from 20 OTR to 180 OTR can indeed explain the observed rate-yield coupling effect. In other words, the rate-yield coupling effect can be explained by assuming that ATP produced at a higher rate from the TCA cycle "sinks" into biomass at a rate of 1.5 mol of ATP per mol of biomass (assuming a conservative estimate of approximately 5.667 mol of ATP / mol of PYR entering the TCA cycle).

[0144] The characteristics of the relevant simulations plotted above lead to the following conclusions:

[0145] ● For the qS ratio through PTA at 180 OTR, simulations that do not fall within the 95% confidence interval (or CI) calculated by 13C analysis are excluded (*we assume it does not change drastically with OTR).

[0146] ● The TCA cycle activity is up to 25% of the incoming qS. Our best estimate of the TCA cycle flux as a proportion of the incoming qS at 180 OTR is ~12% (at approximately qS ~1.4).

[0147] Materials and methods of Example 2.

[0148] Rapid sampling and absolute quantification of metabolite concentrations.

[0149] Definitions: 13C IDMS refers to carbon-13 isotope dilution mass spectrometry; MSTFA refers to N-trimethylsilyl-N-methyl trifluoroacetamide; MRM mode refers to the multiple reaction monitoring mode.

[0150] The absolute intracellular concentrations of isocitrate and α-ketoglutarate were obtained according to the procedure originally described by Canelas and Wahl. Briefly, the broth in the tank was sampled into methanol at -80 °C using a custom-made rapid sampling device, vortexed, and weighted. The biomass was poured into a rapid filtration device and washed with 100% -80 °C methanol. The filtrate was added to a 50 mL centrifuge tube containing 30 mL of 75% v / v ethanol and 200 μL of the '13C IDMS' internal standard extract. The mixture was boiled at 95 °C for 3 min and then placed back on dry ice. Then, the extraction tube was evaporated to dryness in a CentriVap and resuspended in 600 μL of water. Before "MSTFA" derivatization and analysis, the water was filtered and dried again by lyophilization.

[0151] Absolute intracellular quantification of isocitrate and α-ketoglutarate was achieved by comparing the signal ratios from the sample extracts with a reliable standard (Sigma) using an Agilent 7000 triple quadrupole GC / MS in the "MRM mode". The absolute concentrations were normalized to the cell dry weight (measured at the time of extraction).

[0152] Modeling:

[0153] A genome-scale model representing yeast metabolism was generated according to the standard procedure. All reactions from the publicly available reconstruction iTO977 were incorporated into the starting model. In addition, we added the "second-generation" farnesene pathway, including the following 6 reactions:

[0154] ■ Alternative HMG-CoA reductase (or NADH HMGR)

[0155] ○'s_3_hydroxy_3_methylglutaryl_coa_c + 2.0nadh_c <=> 2.0nad_c + r_mevalonate_c + coenzyme_a_c',

[0156] ■ Acetaldehyde dehydrogenase, acetylating (or ADA)

[0157] ○'nad_c + coenzyme_a_c + acetaldehyde_c --> acetyl_coa_c + nadh_c',

[0158] ■ Phosphoketolase acting on F6P (or PK - f6p)

[0159] ○'phosphate_c + beta_d_fructofuranose_6_phosphate_c --> h2o_c + acetyl_phosphate_c + d_erythrose_4_phosphate_c',

[0160] ■ Phosphoketolase acting on X5P (or PK - x5p)

[0161] ○'phosphate_c + d_xylulose_5_phosphate_c --> h2o_c + acetyl_phosphate_c + d_glyceraldehyde_3_phosphate_c'

[0162] ■ Phosphotransacetylase (or PTA)

[0163] ○'coenzyme_a_c + acetyl_phosphate_c <=> phosphate_c + acetyl_coa_c',

[0164] ■ Farnesene synthesis (or FS)

[0165] ○'_2_trans6_trans_farnesyl_diphosphate_c --> diphosphate_c + beta_farnesene_c',

[0166] The model has been verified to produce farnesene, with a maximum theoretical yield of ~29.5% (g farnesene / g sugar). During this process, we deactivated the following reactions to prevent uncontrolled free cycling between NAD / NADH / NADP / NADPH:

[0167] ■nadp_specific_glutamate_dehydrogenase_1

[0168] ■methylenetetrahydrofolate_dehydrogenase_nad_

[0169] Add a reaction named "atp_drain_flux_for_constant_maintanence_requirements" that simply represents the hydrolysis of ATP to ADP and limit it to a constant value of 0.4 mmol gDW-1h-1.

[0170] Set the default environmental conditions to work with the model object of cobrapy module version 0.3.2 by applying a custom function written in Python. The growth medium is set to "glucose_aerobic_minimal", and functionally this allows the uptake of glucose at a rate of 1 mmol gDW-1h-1, and there is no limitation on the uptake of o2, nh3, phosphate, sulfate, and water.

[0171] Next, a set of reactions called the CORE farnesene biosynthetic pathway is defined. These reactions include: 'acetyl_coa_acetyltransferase'; 'glucokinase_glk1'; 'inorganic_pyrophosphatase'; 'dimethylallyltranstransferase'; 'atp_drain_flux_for_constant_maintanence_requirements; geranyltranstransferase'; 'phosphomevalonate_kinase'; 'hydroxymethylglutaryl_coa_synthase'; 'isopentenyl_diphosphate_delta_isomerase'; 'diphosphomevalonate_decarboxylase'; 'galactose_transporter'; 'farnesene_synthase'; 'exchange_of_betafarnesene_c'; 'exchange_of_phosphate_e'; 'exchange_of_alphadglucose_e'; 'exchange_of_h2o_e'; 'glucose_6_phosphate_isomerase; phosphofructokinase_1'; 'fructose_bisphosphate_aldolase'; 'triosephosphate_isomerase'; 'enolase_1'; 'transport_of_h2o_extracellular'; 'phosphoglycerate_kinase'; 'phosphoglycerate_mutase_1_1'; 'pyruvate_kinase_1'; 'pyruvate_decarboxylase_isozyme_1'; 'acetaldehyde_dehydrogenase_acetylating_'; '_3_hydroxy_3_methylglutaryl_coenzyme_a_reductase_1'; '_3_hydroxy_3_methylglutaryl_coenzyme_a_reductase_1_NADH';'mevalonate_kinase'; 'exchange_of_co2_e';'phosphoketolase_f6p'; 'phosphoketolase_x5p'; 'phosphotransacetylase'; 'transaldolase'; 'transketolase_1'; 'glucose_6_phosphate_1_dehydrogenase'; 'probable_6_phosphogluconolactonase_1'; '_6_phosphogluconate_dehydrogenase_decarboxylating_1'; 'transketolase_1_1'; 'ribose_5_phosphate_isomerase'; 'ribulose_phosphate_3_epimerase'; 'transport_of_carbon_dioxide_extracellular'; and 'glyceraldehyde_3_phosphate_dehydrogenase_1'。;

[0172] The following reaction representing the alternative HMG-CoA reductase (or NADH HMGR) is restricted to have a flux of 0. This is because there is evidence that most of the flux is carried by the native Sc.HMGR using NADPH vs. NADH:

[0173] '_3_hydroxy_3_methylglutaryl_coenzyme_a_reductase_1_NADH'

[0174] No flux is allowed outside of these core reactions.

[0175] Since no flux is allowed outside of the core reactions, any excess NADH will make the simulation infeasible. Therefore, a reaction was added to allow the excess NADH produced to be converted to ATP (assuming a stoichiometry of 1:1):

[0176] NADH + PI + ADP → NAD + ATP + H2O

[0177] We also allow for a controlled free cycle between NAD / NADH / NADP / NADPH through the udhA reaction.

[0178] NADPH + NAD <=> NADP + NADH because this is the heterologous enzyme present in our top farnesene producer. This means that excess NADPH can be converted to NADH and then to ATP using the reactions described previously.

[0179] We also introduced a reaction that mimics the loss of pyruvate to the TCA cycle.

[0180] 'PYR_leak_to_TCA’:

[0181] PYR + 5.6667 ADP + 5.6667 Pi = 3 CO2 + 5.6667 ATP + 5.6667 H2O

[0182] Finally, we added another reaction that hydrolyzes ATP, called the "CUSTOM_NGAM" reaction:

[0183] ATP + H 2 O → Pi + ADP

[0184] Once this constrained model was fully constructed, we combined it with another copy of the same yeast genome-scale metabolic model, but in this copy, all reactions were unconstrained except for one key exception. Importantly, the flux through the CUSTOM_NGAM reaction in the first model (the only possible ATP emission and the conversion of NADH to ATP) was strictly coupled to the biomass formation reaction in the second (usually unconstrained) model. The default biomass reaction (in the second model) was set to "biomass_1060_biomass". The flux through this reaction was reported as mu or the growth rate, with units of 1 / h. See Figure 6 , which intuitively describes the model setup and interactions. The two models shared an assigned maximum glucose uptake of 1 mmol gDW-1 h-1, so if the first model produced excess energy, the cost would be that some sugar had to be sent to the second model to "sink" into growth (biomass formation).

[0185] The coupling constraint is as follows:

[0186] Growth in (unconstrained) model ≥ CUSTOM_NGAM * (1. / 1000.) * (1. / mol_atp_per_c_mol_biomass) * 12.0107 * 2

[0187] Where:

[0188] ■ The unit of Growth in (unconstrained) model is: h-1

[0189] ■ The unit of CUSTOM_NGAM is: mmol ATP gDW-1 h-1

[0190] ■ The term “(1. / 1000.)” is converted from mmol to mol. Therefore, after applying this term, we have the unit: mol ATP gDW-1 h-1.

[0191] ■ mol_atp_per_c_mol_biomass = 1.4647 (unitless). Therefore, after applying this term, we have the unit: cmol biomass gDW-1 h-1.

[0192] ■ 12.0107 is the atomic mass of carbon (12.0107 g / 1 cmol). Therefore, after applying this term, we have the unit: g Carbon gDW-1 h-1.

[0193] ■ The number “2” is included as the last term because assuming the carbon content of the cell is approximately 50% of the dry weight (PMID10482783), 1 g of carbon can make up approximately 2 g of DCW. After applying this term, we have gDW gDW-1 h-1, and the gDW cancels out leaving h-1 (matching the unit on the left side of the mu or growth rate equation).

[0194] To generate the final result, the farnesene yield in the first (constrained) model was optimized for the following parameter values of qS:

[0195] qS (equally spaced from 20 to 180 OTR): [0.5, 0.92, 1.34, 1.76, 2.18, 2.6]

[0196] At each qS, we simulated all possible flux splits to the TCA cycle (from 0 flux to the maximum to the TCA cycle) (see Figure 7 ). To generate additional variation, we also simulated the flux of the unknown phosphotransacetylase (second-generation farnesene pathway) from 0 to the maximum at each fixed qS and TCA cycle flux. As Figure 7 shown, the experimental data indicate that the flux through the TCA cycle (as part of qS) must increase.

[0197] Example 3 - Increasing Product Yield Relative to Biomass by Inefficient ATP Combustion

[0198] We hypothesize that if biomass is the preferred sink for excess ATP, then excess ATP may affect rate-yield coupling. The ATP expenditure for cell maintenance is constant regardless of the specific ATP production rate. Thus, at low cell-specific rates, the ATP available for biomass or non-catabolic compound production decreases proportionally, while at high cell-specific rates, the ATP available for biomass or non-catabolic compound production increases proportionally. If the most efficient way to consume excess ATP is for the cell to incorporate it into biomass, then the carbon available for non-catabolic compound production decreases, and the yield decreases at high cell-specific rates. Conversely, at low cell-specific rates, less excess ATP is available for forced biomass formation, and proportionally more carbon can be shunted into the production of non-catabolic compounds. This hypothesis predicts that reducing ATP levels to eliminate excess ATP (which would otherwise sink into biomass) will result in a more favorable distribution of carbon into non-catabolic compounds such as farnesene.

[0199] Benzoic acid can be used to consume ATP in the cell by forcing the cell to use ATP to pump out the excess protons that move into the cytoplasm through benzoyl cations. Strains producing farnesene were treated with different concentrations of benzoic acid, and the effects on the specific sugar uptake rate (qS), specific farnesene production rate (qP), and specific growth rate (mu) were determined. The yield (qP / qS) was calculated from the measured specific sugar uptake rate and specific farnesene production rate.

[0200] The effect of benzoic acid is shown in Figure 8 . Interestingly, while the specific growth rate decreased linearly with increasing benzoic acid concentration, the specific productivity did not change and even increased slightly at intermediate concentrations. The increase in benzoic acid concentration (and the associated increase in ATP consumption) was associated with a more favorable distribution of carbon into farnesene, as reflected in the increase in the calculated yield (qP / qS). These data are interesting and prompted us to further directly determine the rate-yield coupling in strains genetically engineered to reduce ATP yield.

[0201] The method of Example 3.

[0202] Single colonies were grown on CSM agar plates and then picked into sterile 96-well microtiter plates (1.1 mL working volume Axygen) containing 360 μL of defined liquid growth medium (LGM; see Reference Example 1, Westfall et al., 2012), which contained 50 mM succinate (pH 5.0) and 2% sucrose + 1% maltose + 2 g / L lysine, and grown at 28 °C for 72 h. 14 μL was subcultured in 360 μL of fresh defined LGM containing 50 mM succinate (pH 5.0) with a specified amount of benzoic acid and then grown at 33.5 °C for 72 h. Sucrose was ramped up to a final concentration of 8% on day 3 and then the culture (in early log phase) was diluted 26-fold into production plates containing 8% sucrose and different concentrations of benzoic acid after 6 h of incubation to avoid the lag phase. Farnesene, biomass, and total residual sugars were assayed at two points in the log growth phase (T1 and T2) to determine specific productivity, growth rate, and specific sugar uptake rate. Farnesene was measured using isopropanol whole-well extraction and quantified by UV absorbance at 220 nm with reference to a standard curve. Biomass was determined by measuring the fluorescence signal of intracellular tryptophan at an excitation wavelength of 290 nm and detection at 350 nm (UVOD). The relationship between this tryptophan signal and actual biomass is strain-dependent and was determined empirically for each strain assayed. We did this by measuring UVOD and biomass absorbance (OD) before starting the production plates; this gave the OD / UVOD conversion factor per well, which was then used to convert the UVOD signal back to biomass at the end of the production run. Another conversion we had to make when determining biomass was from optical density (OD) to cell dry weight; this was also determined empirically. To eliminate any contribution of the farnesene emulsion to the OD signal, the culture was diluted in a solution of 20% (v / v) PEG 20, 20% (v / v) ethanol, 2% (v / v) Triton X-114. The growth rate was determined by applying linear regression to LN(OD) vs time. Total reducing sugars were assayed using an enzymatic assay for sucrose, fructose, and glucose with a NADH absorbance output read at 340 nM as described in various commercial kits (such as those sold by SigmaAldrich).

[0203] Benzoic acid, acetic acid, sorbic acid, lactic acid, and propionic acid can all be used to reduce biomass yield / cause ATP consumption (when added at different levels). Many other carboxylic acids should be able to reduce biomass yield (or cause ATP consumption). The extent of ATP consumption is generally related to the pKa of the acid and the octanol-water partition coefficient (logP), both of which affect the transmembrane permeability of the molecule. At low extracellular pH values, weak acids should predominantly occur in the undissociated form, which has relatively high membrane permeability. After entering the cell by passive diffusion, the higher pH of the cytosol causes the acid to dissociate, thereby acidifying the cell and triggering an ATP-dependent proton efflux. Thus, weak acids will at least cause a temporary reduction in intracellular ATP levels. At high concentrations, ATP depletion, cytoplasmic acidification, and proton motive force dissipation may occur. This "weak acid uncoupling" mechanism is generally considered to be the main mechanism of weak organic acid toxicity. Examples of weak acids that can be used to consume cellular ATP levels are shown in Table 1 below.

[0204] Table 1 - Examples of weak organic acids that can be used to consume cellular ATP levels. The concentrations required to reduce biomass yield to 50% of the reference condition (YRC50) and the predicted concentrations of undissociated acid at pH 5.0 are shown together with the most commonly cited pKa and partition coefficients.

[0205] Acid pKa Octanol-water partition coefficient (logP) <![CDATA[YCR 50 > Undissociated concentration Acetic acid 4.75 -0.31 105.0 mM 37.7 mM Propionic acid 4.88 0.33 20.0 mM 8.6 mM Sorbic acid 4.76 1.33 1.3 mM 0.47 mM Benzoic acid 4.19 1.87 2.0 mM 0.27 mM

[0206] Example 4 - Genetic modification to reduce ATP levels reduces rate-yield coupling in the bioreactor

[0207] The farnesene-producing strain was modified by engineering overexpression of NOX (NADH oxidase) under the TDH3 promoter. The oxidation of NADH by NOX prevents NADH from being used as an electron donor for ATP synthase to synthesize ATP in the mitochondria. Thus, overexpression of NOX reduces intracellular ATP levels. This strain, Y31655, was run in bioreactors set at different OTRs together with control strains (Y21901, Y22021) that were not engineered for NOX. The specific OUR (qO2) is a function of the OTR divided by the total biomass present in the bioreactor. The rate-yield coupling effect was determined by plotting the product yield against the specific oxygen uptake rate from each bioreactor condition. In Figure 9 it, for the control strains, the relationship between yield and specific rate is shown in two different grey shades, while for the strain overexpressing NOX, it is shown in black. The rate-yield coupling slope of the strain overexpressing NOX was significantly reduced (halved) compared to the control. This experiment shows that ATP levels affect rate-yield coupling; a reduction in intracellular ATP levels is associated with a reduction in the coupling between yield and specific rate. This is consistent with our hypothesis that rate-yield coupling is driven by excess ATP that sinks into the biomass, siphoning off carbon from farnesene production.

[0208] Materials and Methods

[0209] Details regarding bioreactor conditions are provided in Example 1.

[0210] The coding sequence of NADH oxidase from Lactococcus lactis (defined as the nucleotide sequence spanning the start codon to the stop codon of the NADH oxidase gene) was fused at its 5' end to the native Saccharomyces cerevisiae TDH3 promoter and at its 3' end to the native Saccharomyces cerevisiae TDH3 gene terminator. The TDH3 promoter was defined as the nucleotide sequence ~830 bp immediately upstream of the TDH3 start codon. The TDH3 terminator was defined as the nucleotide sequence ~300 bp immediately downstream of the TDH3 gene stop codon. The TDH3 promoter-NOX-TDH3 terminator construct was integrated into the native GAS4 locus using flanking homologous sequences (approximately 500 bp upstream and 500 bp downstream of the GAS4 gene) according to standard yeast molecular genetic techniques.

[0211] Example 5 - Reducing the flux-reduction rate-yield coupling to the TCA cycle

[0212] To determine the effect of reducing the flux through the TCA cycle (which is the major electron source for ATP synthesis in mitochondria) on the rate-yield coupling, strains were prepared in which PYC1 or CIT1 was downregulated either alone or in combination. Pyc1 converts cytoplasmic pyruvate to oxaloacetate, which can be transported into the mitochondria and enter the TCA cycle. Cit1 is the rate-limiting enzyme of the TCA cycle. Downregulation of these two enzymes should significantly reduce the carbon flux into the TCA cycle and reduce the production of ATP by ATP synthase in the mitochondria. We designed the downregulation of CIT1 or PYC1 by replacing the native promoter of CIT1 or PYC1 with a synthetic promoter that is active in the presence of maltose (e.g., in seed-construction conditions) but inactive in the absence of maltose (e.g., in production conditions). We measured the rate-yield coupling in the following farnesene-producing strains: Y27662 (engineered to downregulate PYC1), Y39666 (engineered to downregulate CIT1), Y29438 (engineered to downregulate CIT1 and PYC1), and the non-engineered control Y21601. Strikingly, in the strain in which both PYC1 and CIT1 were downregulated (Y29438), the rate appeared to be almost completely decoupled from the yield. This observation demonstrates that the rate can be decoupled from the yield and identifies a solution for constructing strains that can maintain high yield and high specific rate compatible with large-scale manufacturing. This solution is an observation from modeling and experimental evidence (both detailed above) that indicates that excess ATP is the major driver for carbon conversion to biosynthetic reactions.

[0213] Materials and Methods: Same as the NOX experiment.

[0214] Downregulation of PYC1 and / or CIT1 was achieved by replacing the native promoter of each gene with a synthetic promoter that is turned off in the absence of maltose, using standard yeast molecular genetic techniques for replacing or inserting DNA sequences in the yeast genome using the host's native homologous recombination mechanism.

[0215] All publications, patents, and patent applications cited in this specification are hereby incorporated by reference as if each individual publication or patent application had been specifically and individually indicated to be incorporated by reference. Although the above invention has been described in some detail by way of illustration and example for purposes of clear understanding, it will be readily apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. SEQUENCE LISTING <110> Amyris, Inc. Penelope R. Chu Joshua Adam Lehman Thomas Jon Sherbat Chandresh Thakkar Anne Enning Dong Jiang Hanxiao <120> Method for decoupling the yield and productivity of non-catabolic compounds produced by a host cell <130> 4885PCT.1029BS <140> PCT / US2020 / 036417 <141> 2020-06-05 <150> 62 / 858,152 <151> 2019-06-06 <150> 63 / 034,883 <151> 2020-06-04 <160> 21 <170> PatentIn version 3.5 <210> 1 <211> 486 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Val Gln Arg Leu Leu Pro Gly Ala His Ile Cys Arg Arg Ser Phe 1 5 10 15 Asn Ser Ser Ala Ile Ile Lys Ser Ser Ala Leu Thr Leu Lys Glu Ala 20 25 30 Leu Glu Asn Val Ile Pro Lys Lys Arg Asp Ala Val Lys Lys Leu Lys 35 40 45 Ala Cys Tyr Gly Ser Thr Phe Val Gly Pro Ile Thr Ile Ser Ser Val 50 55 60 Leu Gly Gly Met Arg Gly Asn Gln Ser Met Phe Trp Gln Gly Thr Ser 65 70 75 80 Leu Asp Pro Glu His Gly Ile Lys Phe Gln Gly Leu Thr Ile Glu Glu 85 90 95 Cys Gln Asn Arg Leu Pro Asn Thr Gly Ile Asp Gly Asp Asn Phe Leu 100 105 110 Pro Glu Ser Met Leu Trp Leu Leu Met Thr Gly Gly Val Pro Thr Phe 115 120 125 Gln Gln Ala Ala Ser Phe Arg Lys Glu Leu Ala Ile Arg Gly Arg Lys 130 135 140 Leu Pro His Tyr Thr Glu Lys Val Leu Ser Ser Leu Pro Lys Asp Met 145 150 155 160 His Pro Met Thr Gln Leu Ala Ile Gly Leu Ala Ser Met Asn Lys Gly 165 170 175 Ser Leu Phe Ala Thr Asn Tyr Gln Lys Gly Leu Ile Gly Lys Met Glu 180 185 190 Phe Trp Lys Asp Thr Leu Glu Asp Ser Leu Asn Leu Ile Ala Ser Leu 195 200 205 Pro Leu Leu Thr Gly Arg Ile Tyr Ser Asn Ile Thr Asn Glu Gly His 210 215 220 Pro Leu Gly Gln Tyr Ser Glu Glu Val Asp Trp Cys Thr Asn Ile Cys 225 230 235 240 Ser Leu Leu Gly Met Thr Asn Gly Thr Asn Ser Ser Asn Thr Cys Asn 245 250 255 Leu Thr Ser Gln Gln Ser Leu Asp Phe Ile Asn Leu Met Arg Leu Tyr 260 265 270 Thr Gly Ile His Val Asp His Glu Gly Gly Asn Val Ser Ala His Thr 275 280 285 Thr His Leu Val Gly Ser Ala Leu Ser Asp Pro Tyr Leu Ser Tyr Ser 290 295 300 Ser Gly Ile Met Gly Leu Ala Gly Pro Leu His Gly Leu Ala Ala Gln 305 310 315 320 Glu Val Val Arg Phe Leu Ile Glu Met Asn Ser Asn Ile Ser Ser Ile 325 330 335 Ala Arg Glu Gln Glu Ile Lys Asp Tyr Leu Trp Lys Ile Leu Asn Ser 340 345 350 Asn Arg Val Ile Pro Gly Tyr Gly His Ala Val Leu Arg Lys Pro Asp 355 360 365 Pro Arg Phe Thr Ala Met Leu Glu Phe Ala Gln Lys Arg Pro Ile Glu 370 375 380 Phe Glu Asn Asp Lys Asn Val Leu Leu Met Gln Lys Leu Ala Glu Ile 385 390 395 400 Ala Pro Lys Val Leu Leu Glu His Gly Lys Ser Lys Asn Pro Phe Pro 405 410 415 Asn Val Asp Ser Ala Ser Gly Ile Leu Phe Tyr His Tyr Gly Ile Arg 420 425 430 Glu Leu Leu Phe Phe Thr Val Ile Phe Gly Cys Ser Arg Ala Met Gly 435 440 445 Pro Leu Thr Gln Leu Val Trp Asp Arg Ile Leu Gly Leu Pro Ile Glu 450 455 460 Arg Pro Lys Ser Leu Asn Leu Glu Gly Leu Glu Ala Leu Thr Lys Ala 465 470 475 480 Ser Asn Val Asn Lys Leu 485 <210> 2 <211> 479 <212> PRT <213> Saccharomyces cerevisiae <400> 2 Met Ser Ala Ile Leu Ser Thr Thr Ser Lys Ser Phe Leu Ser Arg Gly 1 5 10 15 Ser Thr Arg Gln Cys Gln Asn Met Gln Lys Ala Leu Phe Ala Leu Leu 20 25 30 Asn Ala Arg His Tyr Ser Ser Ala Ser Glu Gln Thr Leu Lys Glu Arg 35 40 45 Phe Ala Glu Ile Ile Pro Ala Lys Ala Glu Glu Ile Lys Lys Phe Lys 50 55 60 Lys Glu His Gly Lys Thr Val Ile Gly Glu Val Leu Leu Glu Gln Ala 65 70 75 80 Tyr Gly Gly Met Arg Gly Ile Lys Gly Leu Val Trp Glu Gly Ser Val 85 90 95 Leu Asp Pro Glu Glu Gly Ile Arg Phe Arg Gly Arg Thr Ile Pro Glu 100 105 110 Ile Gln Arg Glu Leu Pro Lys Ala Glu Gly Ser Thr Glu Pro Leu Pro 115 120 125 Glu Ala Leu Phe Trp Leu Leu Leu Thr Gly Glu Ile Pro Thr Asp Ala 130 135 140 Gln Val Lys Ala Leu Ser Ala Asp Leu Ala Ala Arg Ser Glu Ile Pro 145 150 155 160 Glu His Val Ile Gln Leu Leu Asp Ser Leu Pro Lys Asp Leu His Pro 165 170 175 Met Ala Gln Phe Ser Ile Ala Val Thr Ala Leu Glu Ser Glu Ser Lys 180 185 190 Phe Ala Lys Ala Tyr Ala Gln Gly Val Ser Lys Lys Glu Tyr Trp Ser 195 200 205 Tyr Thr Phe Glu Asp Ser Leu Asp Leu Leu Gly Lys Leu Pro Val Ile 210 215 220 Ala Ser Lys Ile Tyr Arg Asn Val Phe Lys Asp Gly Lys Ile Thr Ser 225 230 235 240 Thr Asp Pro Asn Ala Asp Tyr Gly Lys Asn Leu Ala Gln Leu Leu Gly 245 250 255 Tyr Glu Asn Lys Asp Phe Ile Asp Leu Met Arg Leu Tyr Leu Thr Ile 260 265 270 His Ser Asp His Glu Gly Gly Asn Val Ser Ala His Thr Thr His Leu 275 280 285 Val Gly Ser Ala Leu Ser Ser Pro Tyr Leu Ser Leu Ala Ala Gly Leu 290 295 300 Asn Gly Leu Ala Gly Pro Leu His Gly Arg Ala Asn Gln Glu Val Leu 305 310 315 320 Glu Trp Leu Phe Lys Leu Arg Glu Glu Val Lys Gly Asp Tyr Ser Lys 325 330 335 Glu Thr Ile Glu Lys Tyr Leu Trp Asp Thr Leu Asn Ala Gly Arg Val 340 345 350 Val Pro Gly Tyr Gly His Ala Val Leu Arg Lys Thr Asp Pro Arg Tyr 355 360 365 Thr Ala Gln Arg Glu Phe Ala Leu Lys His Phe Pro Asp Tyr Glu Leu 370 375 380 Phe Lys Leu Val Ser Thr Ile Tyr Glu Val Ala Pro Gly Val Leu Thr 385 390 395 400 Lys His Gly Lys Thr Lys Asn Pro Trp Pro Asn Val Asp Ser His Ser 405 410 415 Gly Val Leu Leu Gln Tyr Tyr Gly Leu Thr Glu Ala Ser Phe Tyr Thr 420 425 430 Val Leu Phe Gly Val Ala Arg Ala Ile Gly Val Leu Pro Gln Leu Ile 435 440 445 Ile Asp Arg Ala Val Gly Ala Pro Ile Glu Arg Pro Lys Ser Phe Ser 450 455 460 Thr Glu Lys Tyr Lys Glu Leu Val Lys Lys Ile Glu Ser Lys Asn 465 470 475 <210> 3 <211> 778 <212> PRT <213> Pseudomonas mevalonii <400> 3 Met Leu Ser Ala Arg Ser Ala Ile Lys Arg Pro Ile Val Arg Gly Leu 1 5 10 15 Ala Thr Val Ser Asn Leu Thr Arg Asp Ser Lys Val Asn Gln Asn Leu 20 25 30 Leu Glu Asp His Ser Phe Ile Asn Tyr Lys Gln Asn Val Glu Thr Leu 35 40 45 Asp Ile Val Arg Lys Arg Leu Asn Arg Pro Phe Thr Tyr Ala Glu Lys 50 55 60 Ile Leu Tyr Gly His Leu Asp Asp Pro His Gly Gln Asp Ile Gln Arg 65 70 75 80 Gly Val Ser Tyr Leu Lys Leu Arg Pro Asp Arg Val Ala Cys Gln Asp 85 90 95 Ala Thr Ala Gln Met Ala Ile Leu Gln Phe Met Ser Ala Gly Leu Pro 100 105 110 Gln Val Ala Lys Pro Val Thr Val His Cys Asp His Leu Ile Gln Ala 115 120 125 Gln Val Gly Gly Glu Lys Asp Leu Lys Arg Ala Ile Asp Leu Asn Lys 130 135 140 Glu Val Tyr Asp Phe Leu Ala Ser Ala Thr Ala Lys Tyr Asn Met Gly 145 150 155 160 Phe Trp Lys Pro Gly Ser Gly Ile Ile His Gln Ile Val Leu Glu Asn 165 170 175 Tyr Ala Phe Pro Gly Ala Leu Ile Ile Gly Thr Asp Ser His Thr Pro 180 185 190 Asn Ala Gly Gly Leu Gly Gln Leu Ala Ile Gly Val Gly Gly Ala Asp 195 200 205 Ala Val Asp Val Met Ala Gly Arg Pro Trp Glu Leu Lys Ala Pro Lys 210 215 220 Ile Leu Gly Val Lys Leu Thr Gly Lys Met Asn Gly Trp Thr Ser Pro 225 230 235 240 Lys Asp Ile Ile Leu Lys Leu Ala Gly Ile Thr Thr Val Lys Gly Gly 245 250 255 Thr Gly Lys Ile Val Glu Tyr Phe Gly Asp Gly Val Asp Thr Phe Ser 260 265 270 Ala Thr Gly Met Gly Thr Ile Cys Asn Met Gly Ala Glu Ile Gly Ala 275 280 285 Thr Thr Ser Val Phe Pro Phe Asn Lys Ser Met Ile Glu Tyr Leu Glu 290 295 300 Ala Thr Gly Arg Gly Lys Ile Ala Asp Phe Ala Lys Leu Tyr His Lys 305 310 315 320 Asp Leu Leu Ser Ala Asp Lys Asp Ala Glu Tyr Asp Glu Val Val Glu 325 330 335 Ile Asp Leu Asn Thr Leu Glu Pro Tyr Ile Asn Gly Pro Phe Thr Pro 340 345 350 Asp Leu Ala Thr Pro Val Ser Lys Met Lys Glu Val Ala Val Ala Asn 355 360 365 Asn Trp Pro Leu Asp Val Arg Val Gly Leu Ile Gly Ser Cys Thr Asn 370 375 380 Ser Ser Tyr Glu Asp Met Ser Arg Ser Ala Ser Ile Val Lys Asp Ala 385 390 395 400 Ala Ala His Gly Leu Lys Ser Lys Thr Ile Phe Thr Val Thr Pro Gly 405 410 415 Ser Glu Gln Ile Arg Ala Thr Ile Glu Arg Asp Gly Gln Leu Glu Thr 420 425 430 Phe Lys Glu Phe Gly Gly Ile Val Leu Ala Asn Ala Cys Gly Pro Cys 435 440 445 Ile Gly Gln Trp Asp Arg Arg Asp Ile Lys Lys Gly Asp Lys Asn Thr 450 455 460 Ile Val Ser Ser Tyr Asn Arg Asn Phe Thr Ser Arg Asn Asp Gly Asn 465 470 475 480 Pro Gln Thr His Ala Phe Val Ala Ser Pro Glu Leu Val Thr Ala Phe 485 490 495 Ala Ile Ala Gly Asp Leu Arg Phe Asn Pro Leu Thr Asp Lys Leu Lys 500 505 510 Asp Lys Asp Gly Asn Glu Phe Met Leu Lys Pro Pro His Gly Asp Gly 515 520 525 Leu Pro Gln Arg Gly Tyr Asp Ala Gly Glu Asn Thr Tyr Gln Ala Pro 530 535 540 Pro Ala Asp Arg Ser Thr Val Glu Val Lys Val Ser Pro Thr Ser Asp 545 550 555 560 Arg Leu Gln Leu Leu Lys Pro Phe Lys Pro Trp Asp Gly Lys Asp Ala 565 570 575 Lys Asp Met Pro Ile Leu Ile Lys Ala Val Gly Lys Thr Thr Thr Asp 580 585 590 His Ile Ser Met Ala Gly Pro Trp Leu Lys Tyr Arg Gly His Leu Glu 595 600 605 Asn Ile Ser Asn Asn Tyr Met Ile Gly Ala Ile Asn Ala Glu Asn Lys 610 615 620 Lys Ala Asn Cys Val Lys Asn Val Tyr Thr Gly Glu Tyr Lys Gly Val 625 630 635 640 Pro Asp Thr Ala Arg Asp Tyr Arg Asp Gln Gly Ile Lys Trp Val Val 645 650 655 Ile Gly Asp Glu Asn Phe Gly Glu Gly Ser Ser Arg Glu His Ala Ala 660 665 670 Leu Glu Pro Arg Phe Leu Gly Gly Phe Ala Ile Ile Thr Lys Ser Phe 675 680 685 Ala Arg Ile His Glu Thr Asn Leu Lys Lys Gln Gly Leu Leu Pro Leu 690 695 700 Asn Phe Lys Asn Pro Ala Asp Tyr Asp Lys Ile Asn Pro Asp Asp Arg 705 710 715 720 Ile Asp Ile Leu Gly Leu Ala Glu Leu Ala Pro Gly Lys Pro Val Thr 725 730 735 Met Arg Val His Pro Lys Asn Gly Lys Pro Trp Asp Ala Val Leu Thr 740 745 750 His Thr Phe Asn Asp Glu Gln Ile Glu Trp Phe Lys Tyr Gly Ser Ala 755 760 765 Leu Asn Lys Ile Lys Ala Asp Glu Lys Lys 770 775 <210> 4 <211> 369 <212> PRT <213> Pseudomonas mevalonii <400> 4 Met Leu Arg Asn Thr Phe Phe Arg Asn Thr Ser Arg Arg Phe Leu Ala 1 5 10 15 Thr Val Lys Gln Pro Ser Ile Gly Arg Tyr Thr Gly Lys Pro Asn Pro 20 25 30 Ser Thr Gly Lys Tyr Thr Val Ser Phe Ile Glu Gly Asp Gly Ile Gly 35 40 45 Pro Glu Ile Ser Lys Ser Val Lys Lys Ile Phe Ser Ala Ala Asn Val 50 55 60 Pro Ile Glu Trp Glu Ser Cys Asp Val Ser Pro Ile Phe Val Asn Gly 65 70 75 80 Leu Thr Thr Ile Pro Asp Pro Ala Val Gln Ser Ile Thr Lys Asn Leu 85 90 95 Val Ala Leu Lys Gly Pro Leu Ala Thr Pro Ile Gly Lys Gly His Arg 100 105 110 Ser Leu Asn Leu Thr Leu Arg Lys Thr Phe Gly Leu Phe Ala Asn Val 115 120 125 Arg Pro Ala Lys Ser Ile Glu Gly Phe Lys Thr Thr Tyr Glu Asn Val 130 135 140 Asp Leu Val Leu Ile Arg Glu Asn Thr Glu Gly Glu Tyr Ser Gly Ile 145 150 155 160 Glu His Ile Val Cys Pro Gly Val Val Gln Ser Ile Lys Leu Ile Thr 165 170 175 Arg Asp Ala Ser Glu Arg Val Ile Arg Tyr Ala Phe Glu Tyr Ala Arg 180 185 190 Ala Ile Gly Arg Pro Arg Val Ile Val Val His Lys Ser Thr Ile Gln 195 200 205 Arg Leu Ala Asp Gly Leu Phe Val Asn Val Ala Lys Glu Leu Ser Lys 210 215 220 Glu Tyr Pro Asp Leu Thr Leu Glu Thr Glu Leu Ile Asp Asn Ser Val 225 230 235 240 Leu Lys Val Val Thr Asn Pro Ser Ala Tyr Thr Asp Ala Val Ser Val 245 250 255 Cys Pro Asn Leu Tyr Gly Asp Ile Leu Ser Asp Leu Asn Ser Gly Leu 260 265 270 Ser Ala Gly Ser Leu Gly Leu Thr Pro Ser Ala Asn Ile Gly His Lys 275 280 285 Ile Ser Ile Phe Glu Ala Val His Gly Ser Ala Pro Asp Ile Ala Gly 290 295 300 Gln Asp Lys Ala Asn Pro Thr Ala Leu Leu Leu Ser Ser Val Met Met 305 310 315 320 Leu Asn His Met Gly Leu Thr Asn His Ala Asp Gln Ile Gln Asn Ala 325 330 335 Val Leu Ser Thr Ile Ala Ser Gly Pro Glu Asn Arg Thr Gly Asp Leu 340 345 350 Ala Gly Thr Ala Thr Thr Ser Ser Phe Thr Glu Ala Val Ile Lys Arg 355 360 365 Leu <210> 5 <211> 360 <212> PRT <213> Silicibacter pomeroyi <400> 5 Met Leu Asn Arg Thr Ile Ala Lys Arg Thr Leu Ala Thr Ala Ala Gln 1 5 10 15 Ala Glu Arg Thr Leu Pro Lys Lys Tyr Gly Gly Arg Phe Thr Val Thr 20 25 30 Leu Ile Pro Gly Asp Gly Val Gly Lys Glu Ile Thr Asp Ser Val Arg 35 40 45 Thr Ile Phe Glu Ala Glu Asn Ile Pro Ile Asp Trp Glu Thr Ile Asn 50 55 60 Ile Lys Gln Thr Asp His Lys Glu Gly Val Tyr Glu Ala Val Glu Ser 65 70 75 80 Leu Lys Arg Asn Lys Ile Gly Leu Lys Gly Leu Trp His Thr Pro Ala 85 90 95 Asp Gln Thr Gly His Gly Ser Leu Asn Val Ala Leu Arg Lys Gln Leu 100 105 110 Asp Ile Tyr Ala Asn Val Ala Leu Phe Lys Ser Leu Lys Gly Val Lys 115 120 125 Thr Arg Ile Pro Asp Ile Asp Leu Ile Val Ile Arg Glu Asn Thr Glu 130 135 140 Gly Glu Phe Ser Gly Leu Glu His Glu Ser Val Pro Gly Val Val Glu 145 150 155 160 Ser Leu Lys Val Met Thr Arg Pro Lys Thr Glu Arg Ile Ala Arg Phe 165 170 175 Ala Phe Asp Phe Ala Lys Lys Tyr Asn Arg Lys Ser Val Thr Ala Val 180 185 190 His Lys Ala Asn Ile Met Lys Leu Gly Asp Gly Leu Phe Arg Asn Ile 195 200 205 Ile Thr Glu Ile Gly Gln Lys Glu Tyr Pro Asp Ile Asp Val Ser Ser 210 215 220 Ile Ile Val Asp Asn Ala Ser Met Gln Ala Val Ala Lys Pro His Gln 225 230 235 240 Phe Asp Val Leu Val Thr Pro Ser Met Tyr Gly Thr Ile Leu Gly Asn 245 250 255 Ile Gly Ala Ala Leu Ile Gly Gly Pro Gly Leu Val Ala Gly Ala Asn 260 265 270 Phe Gly Arg Asp Tyr Ala Val Phe Glu Pro Gly Ser Arg His Val Gly 275 280 285 Leu Asp Ile Lys Gly Gln Asn Val Ala Asn Pro Thr Ala Met Ile Leu 290 295 300 Ser Ser Thr Leu Met Leu Asn His Leu Gly Leu Asn Glu Tyr Ala Thr 305 310 315 320 Arg Ile Ser Lys Ala Val His Glu Thr Ile Ala Glu Gly Lys His Thr 325 330 335 Thr Arg Asp Ile Gly Gly Ser Ser Ser Thr Thr Asp Phe Thr Asn Glu 340 345 350 Ile Ile Asn Lys Leu Ser Thr Met 355 360 <210> 6 <211> 463 <212> PRT <213> Silicibacter pomeroyi <400> 6 Met Leu Ser Arg Ala Thr Arg Thr Ala Ala Ala Lys Ser Leu Val Lys 1 5 10 15 Ser Lys Val Ala Arg Asn Val Met Ala Ala Ser Phe Val Lys Arg His 20 25 30 Ala Ser Thr Ser Leu Phe Lys Gln Ala Asn Lys Val Glu Ser Leu Gly 35 40 45 Ser Ile Tyr Leu Ser Gly Lys Lys Ile Ser Val Ala Ala Asn Pro Phe 50 55 60 Ser Ile Thr Ser Asn Arg Phe Lys Ser Thr Ser Ile Glu Val Pro Pro 65 70 75 80 Met Ala Glu Ser Leu Thr Glu Gly Ser Leu Lys Glu Tyr Thr Lys Asn 85 90 95 Val Gly Asp Phe Ile Lys Glu Asp Glu Leu Leu Ala Thr Ile Glu Thr 100 105 110 Asp Lys Ile Asp Ile Glu Val Asn Ser Pro Val Ser Gly Thr Val Thr 115 120 125 Lys Leu Asn Phe Lys Pro Glu Asp Thr Val Thr Val Gly Glu Glu Leu 130 135 140 Ala Gln Val Glu Pro Gly Glu Ala Pro Ala Glu Gly Ser Gly Glu Ser 145 150 155 160 Lys Pro Glu Pro Thr Glu Gln Ala Glu Pro Ser Gln Gly Val Ala Ala 165 170 175 Arg Glu Asn Ser Ser Glu Glu Thr Ala Ser Lys Lys Glu Ala Ala Pro 180 185 190 Lys Lys Glu Ala Ala Pro Lys Lys Glu Val Thr Glu Pro Lys Lys Ala 195 200 205 Asp Gln Pro Lys Lys Thr Val Ser Lys Ala Gln Glu Pro Pro Val Ala 210 215 220 Ser Asn Ser Phe Thr Pro Phe Pro Arg Thr Glu Thr Arg Val Lys Met 225 230 235 240 Asn Arg Met Arg Leu Arg Ile Ala Glu Arg Leu Lys Glu Ser Gln Asn 245 250 255 Thr Ala Ala Ser Leu Thr Thr Phe Asn Glu Val Asp Met Ser Ala Leu 260 265 270 Met Glu Met Arg Lys Leu Tyr Lys Asp Glu Ile Ile Lys Lys Thr Gly 275 280 285 Thr Lys Phe Gly Phe Met Gly Leu Phe Ser Lys Ala Cys Thr Leu Ala 290 295 300 Ala Lys Asp Ile Pro Ala Val Asn Gly Ala Ile Glu Gly Asp Gln Ile 305 310 315 320 Val Tyr Arg Asp Tyr Thr Asp Ile Ser Val Ala Val Ala Thr Pro Lys 325 330 335 Gly Leu Val Thr Pro Val Val Arg Asn Ala Glu Ser Leu Ser Val Leu 340 345 350 Asp Ile Glu Asn Glu Ile Val Arg Leu Ser His Lys Ala Arg Asp Gly 355 360 365 Lys Leu Thr Leu Glu Asp Met Thr Gly Gly Thr Phe Thr Ile Ser Asn 370 375 380 Gly Gly Val Phe Gly Ser Leu Tyr Gly Thr Pro Ile Ile Asn Ser Pro 385 390 395 400 Gln Thr Ala Val Leu Gly Leu His Gly Val Lys Glu Arg Pro Val Thr 405 410 415 Val Asn Gly Gln Ile Val Ser Arg Pro Met Met Tyr Leu Ala Leu Thr 420 425 430 Tyr Asp His Arg Leu Leu Asp Gly Arg Glu Ala Val Thr Phe Leu Lys 435 440 445 Thr Val Lys Glu Leu Ile Glu Asp Pro Arg Lys Met Leu Leu Trp 450 455 460 <210> 7 <211> 1014 <212> PRT <213> Delftia acidovorans <400> 7 Met Leu Arg Phe Val Ser Ser Gln Thr Cys Arg Tyr Ser Ser Arg Gly 1 5 10 15 Leu Leu Lys Thr Ser Leu Leu Lys Asn Ala Ser Thr Val Lys Ile Val 20 25 30 Gly Arg Gly Leu Ala Thr Thr Gly Thr Asp Asn Phe Leu Ser Thr Ser 35 40 45 Asn Ala Thr Tyr Ile Asp Glu Met Tyr Gln Ala Trp Gln Lys Asp Pro 50 55 60 Ser Ser Val His Val Ser Trp Asp Ala Tyr Phe Lys Asn Met Ser Asn 65 70 75 80 Pro Lys Ile Pro Ala Thr Lys Ala Phe Gln Ala Pro Pro Ser Ile Ser 85 90 95 Asn Phe Pro Gln Gly Thr Glu Ala Ala Pro Leu Gly Thr Ala Met Thr 100 105 110 Gly Ser Val Asp Glu Asn Val Ser Ile His Leu Lys Val Gln Leu Leu 115 120 125 Cys Arg Ala Tyr Gln Val Arg Gly His Leu Lys Ala His Ile Asp Pro 130 135 140 Leu Gly Ile Ser Phe Gly Ser Asn Lys Asn Asn Pro Val Pro Pro Glu 145 150 155 160 Leu Thr Leu Asp Tyr Tyr Gly Phe Ser Lys His Asp Leu Asp Lys Glu 165 170 175 Ile Asn Leu Gly Pro Gly Ile Leu Pro Arg Phe Ala Arg Asp Gly Lys 180 185 190 Ser Lys Met Ser Leu Lys Glu Ile Val Asp His Leu Glu Lys Leu Tyr 195 200 205 Cys Ser Ser Tyr Gly Val Gln Tyr Thr His Ile Pro Ser Lys Gln Lys 210 215 220 Cys Asp Trp Leu Arg Glu Arg Ile Glu Ile Pro Glu Pro Tyr Gln Tyr 225 230 235 240 Thr Val Asp Gln Lys Arg Gln Ile Leu Asp Arg Leu Thr Trp Ala Thr 245 250 255 Ser Phe Glu Ser Phe Leu Ser Thr Lys Phe Pro Asn Asp Lys Arg Phe 260 265 270 Gly Leu Glu Gly Leu Glu Ser Val Val Pro Gly Ile Lys Thr Leu Val 275 280 285 Asp Arg Ser Val Glu Leu Gly Val Glu Asp Ile Val Leu Gly Met Ala 290 295 300 His Arg Gly Arg Leu Asn Val Leu Ser Asn Val Val Arg Lys Pro Asn 305 310 315 320 Glu Ser Ile Phe Ser Glu Phe Lys Gly Ser Ser Ala Arg Asp Asp Ile 325 330 335 Glu Gly Ser Gly Asp Val Lys Tyr His Leu Gly Met Asn Tyr Gln Arg 340 345 350 Pro Thr Thr Ser Gly Lys Tyr Val Asn Leu Ser Leu Val Ala Asn Pro 355 360 365 Ser His Leu Glu Ser Gln Asp Pro Val Val Leu Gly Arg Thr Arg Ala 370 375 380 Leu Leu His Ala Lys Asn Asp Leu Lys Glu Lys Thr Lys Ala Leu Gly 385 390 395 400 Val Leu Leu His Gly Asp Ala Ala Phe Ala Gly Gln Gly Val Val Tyr 405 410 415 Glu Thr Met Gly Phe Leu Thr Leu Pro Glu Tyr Ser Thr Gly Gly Thr 420 425 430 Ile His Val Ile Thr Asn Asn Gln Ile Gly Phe Thr Thr Asp Pro Arg 435 440 445 Phe Ala Arg Ser Thr Pro Tyr Pro Ser Asp Leu Ala Lys Ala Ile Asp 450 455 460 Ala Pro Ile Phe His Val Asn Ala Asn Asp Val Glu Ala Val Thr Phe 465 470 475 480 Ile Phe Asn Leu Ala Ala Glu Trp Arg His Lys Phe His Thr Asp Ala 485 490 495 Ile Ile Asp Val Val Gly Trp Arg Lys His Gly His Asn Glu Thr Asp 500 505 510 Gln Pro Ser Phe Thr Gln Pro Leu Met Tyr Lys Lys Ile Ala Lys Gln 515 520 525 Lys Ser Val Ile Asp Val Tyr Thr Glu Lys Leu Ile Ser Glu Gly Thr 530 535 540 Phe Ser Lys Lys Asp Ile Asp Glu His Lys Lys Trp Val Trp Asn Leu 545 550 555 560 Phe Glu Asp Ala Phe Glu Lys Ala Lys Asp Tyr Val Pro Ser Gln Arg 565 570 575 Glu Trp Leu Thr Ala Ala Trp Glu Gly Phe Lys Ser Pro Lys Glu Leu 580 585 590 Ala Thr Glu Ile Leu Pro His Glu Pro Thr Asn Val Pro Glu Ser Thr 595 600 605 Leu Lys Glu Leu Gly Lys Val Leu Ser Ser Trp Pro Glu Gly Phe Glu 610 615 620 Val His Lys Asn Leu Lys Arg Ile Leu Lys Asn Arg Gly Lys Ser Ile 625 630 635 640 Glu Thr Gly Glu Gly Ile Asp Trp Ala Thr Gly Glu Ala Leu Ala Phe 645 650 655 Gly Thr Leu Val Leu Asp Gly Gln Asn Val Arg Val Ser Gly Glu Asp 660 665 670 Val Glu Arg Gly Thr Phe Ser Gln Arg His Ala Val Leu His Asp Gln 675 680 685 Gln Ser Glu Ala Ile Tyr Thr Pro Leu Ser Thr Leu Asn Asn Glu Lys 690 695 700 Ala Asp Phe Thr Ile Ala Asn Ser Ser Leu Ser Glu Tyr Gly Val Met 705 710 715 720 Gly Phe Glu Tyr Gly Tyr Ser Leu Thr Ser Pro Asp Tyr Leu Val Met 725 730 735 Trp Glu Ala Gln Phe Gly Asp Phe Ala Asn Thr Ala Gln Val Ile Ile 740 745 750 Asp Gln Phe Ile Ala Gly Gly Glu Gln Lys Trp Lys Gln Arg Ser Gly 755 760 765 Leu Val Leu Ser Leu Pro His Gly Tyr Asp Gly Gln Gly Pro Glu His 770 775 780 Ser Ser Gly Arg Leu Glu Arg Phe Leu Gln Leu Ala Asn Glu Asp Pro 785 790 795 800 Arg Tyr Phe Pro Ser Glu Glu Lys Leu Gln Arg Gln His Gln Asp Cys 805 810 815 Asn Phe Gln Val Val Tyr Pro Thr Thr Pro Ala Asn Leu Phe His Ile 820 825 830 Leu Arg Arg Gln Gln His Arg Gln Phe Arg Lys Pro Leu Ala Leu Phe 835 840 845 Phe Ser Lys Gln Leu Leu Arg His Pro Leu Ala Arg Ser Ser Leu Ser 850 855 860 Glu Phe Thr Glu Gly Gly Phe Gln Trp Ile Ile Glu Asp Ile Glu His 865 870 875 880 Gly Lys Ser Ile Gly Thr Lys Glu Glu Thr Lys Arg Leu Val Leu Leu 885 890 895 Ser Gly Gln Val Tyr Thr Ala Leu His Lys Arg Arg Glu Ser Leu Gly 900 905 910 Asp Lys Thr Thr Ala Phe Leu Lys Ile Glu Gln Leu His Pro Phe Pro 915 920 925 Phe Ala Gln Leu Arg Asp Ser Leu Asn Ser Tyr Pro Asn Leu Glu Glu 930 935 940 Ile Val Trp Cys Gln Glu Glu Pro Leu Asn Met Gly Ser Trp Ala Tyr 945 950 955 960 Thr Glu Pro Arg Leu His Thr Thr Leu Lys Glu Thr Asp Lys Tyr Lys 965 970 975 Asp Phe Lys Val Arg Tyr Cys Gly Arg Asn Pro Ser Gly Ala Val Ala 980 985 990 Ala Gly Ser Lys Ser Leu His Leu Ala Glu Glu Asp Ala Phe Leu Lys 995 1000 1005 Asp Val Phe Gln Gln Ser 1010 <210> 8 <211> 499 <212> PRT <213> Delftia acidovorans <400> 8 Met Leu Arg Ile Arg Ser Leu Leu Asn Asn Lys Arg Ala Phe Ser Ser 1 5 10 15 Thr Val Arg Thr Leu Thr Ile Asn Lys Ser His Asp Val Val Ile Ile 20 25 30 Gly Gly Gly Pro Ala Gly Tyr Val Ala Ala Ile Lys Ala Ala Gln Leu 35 40 45 Gly Phe Asn Thr Ala Cys Val Glu Lys Arg Gly Lys Leu Gly Gly Thr 50 55 60 Cys Leu Asn Val Gly Cys Ile Pro Ser Lys Ala Leu Leu Asn Asn Ser 65 70 75 80 His Leu Phe His Gln Met His Thr Glu Ala Gln Lys Arg Gly Ile Asp 85 90 95 Val Asn Gly Asp Ile Lys Ile Asn Val Ala Asn Phe Gln Lys Ala Lys 100 105 110 Asp Asp Ala Val Lys Gln Leu Thr Gly Gly Ile Glu Leu Leu Phe Lys 115 120 125 Lys Asn Lys Val Thr Tyr Tyr Lys Gly Asn Gly Ser Phe Glu Asp Glu 130 135 140 Thr Lys Ile Arg Val Thr Pro Val Asp Gly Leu Glu Gly Thr Val Lys 145 150 155 160 Glu Asp His Ile Leu Asp Val Lys Asn Ile Ile Val Ala Thr Gly Ser 165 170 175 Glu Val Thr Pro Phe Pro Gly Ile Glu Ile Asp Glu Glu Lys Ile Val 180 185 190 Ser Ser Thr Gly Ala Leu Ser Leu Lys Glu Ile Pro Lys Arg Leu Thr 195 200 205 Ile Ile Gly Gly Gly Ile Ile Gly Leu Glu Met Gly Ser Val Tyr Ser 210 215 220 Arg Leu Gly Ser Lys Val Thr Val Val Glu Phe Gln Pro Gln Ile Gly 225 230 235 240 Ala Ser Met Asp Gly Glu Val Ala Lys Ala Thr Gln Lys Phe Leu Lys 245 250 255 Lys Gln Gly Leu Asp Phe Lys Leu Ser Thr Lys Val Ile Ser Ala Lys 260 265 270 Arg Asn Asp Asp Lys Asn Val Val Glu Ile Val Val Glu Asp Thr Lys 275 280 285 Thr Asn Lys Gln Glu Asn Leu Glu Ala Glu Val Leu Leu Val Ala Val 290 295 300 Gly Arg Arg Pro Tyr Ile Ala Gly Leu Gly Ala Glu Lys Ile Gly Leu 305 310 315 320 Glu Val Asp Lys Arg Gly Arg Leu Val Ile Asp Asp Gln Phe Asn Ser 325 330 335 Lys Phe Pro His Ile Lys Val Val Gly Asp Val Thr Phe Gly Pro Met 340 345 350 Leu Ala His Lys Ala Glu Glu Glu Gly Ile Ala Ala Val Glu Met Leu 355 360 365 Lys Thr Gly His Gly His Val Asn Tyr Asn Asn Ile Pro Ser Val Met 370 375 380 Tyr Ser His Pro Glu Val Ala Trp Val Gly Lys Thr Glu Glu Gln Leu 385 390 395 400 Lys Glu Ala Gly Ile Asp Tyr Lys Ile Gly Lys Phe Pro Phe Ala Ala 405 410 415 Asn Ser Arg Ala Lys Thr Asn Gln Asp Thr Glu Gly Phe Val Lys Ile 420 425 430 Leu Ile Asp Ser Lys Thr Glu Arg Ile Leu Gly Ala His Ile Ile Gly 435 440 445 Pro Asn Ala Gly Glu Met Ile Ala Glu Ala Gly Leu Ala Leu Glu Tyr 450 455 460 Gly Ala Ser Ala Glu Asp Val Ala Arg Val Cys His Ala His Pro Thr 465 470 475 480 Leu Ser Glu Ala Phe Lys Glu Ala Asn Met Ala Ala Tyr Asp Lys Ala 485 490 495 Ile His Cys <210> 9 <211> 427 <212> PRT <213> Saccharomyces cerevisiae <400> 9 Met Tyr Ser Arg Lys Ser Leu Ser Leu Ile Ser Lys Cys Gly Gln Leu 1 5 10 15 Ser Arg Leu Asn Ala Gln Ala Ala Leu Gln Ala Arg Arg His Leu Ser 20 25 30 Ile His Glu Tyr Arg Ser Ala Gln Leu Leu Arg Glu Tyr Gly Ile Gly 35 40 45 Thr Pro Glu Gly Phe Pro Ala Phe Thr Pro Glu Glu Ala Phe Glu Ala 50 55 60 Ala Lys Lys Leu Asn Thr Asn Lys Leu Val Ile Lys Ala Gln Ala Leu 65 70 75 80 Thr Gly Gly Arg Gly Lys Gly His Phe Asp Thr Gly Tyr Lys Ser Gly 85 90 95 Val His Met Ile Glu Ser Pro Gln Gln Ala Glu Asp Val Ala Lys Glu 100 105 110 Met Leu Asn His Asn Leu Ile Thr Lys Gln Thr Gly Ile Ala Gly Lys 115 120 125 Pro Val Ser Ala Val Tyr Ile Val Lys Arg Val Asp Thr Lys His Glu 130 135 140 Ala Tyr Leu Ser Ile Leu Met Asp Arg Gln Thr Lys Lys Pro Met Ile 145 150 155 160 Ile Ala Ser Ser Gln Gly Gly Met Asn Ile Glu Glu Val Ala Glu Arg 165 170 175 Thr Pro Asp Ala Ile Lys Lys Phe Ser Ile Glu Thr Ser Lys Gly Leu 180 185 190 Ser Pro Gln Met Ala Lys Asp Val Ala Lys Ser Leu Gly Phe Ser Pro 195 200 205 Asp Ala Gln Asp Glu Ala Ala Lys Ala Val Ser Asn Leu Tyr Lys Ile 210 215 220 Phe Met Glu Arg Asp Ala Thr Gln Val Glu Ile Asn Pro Leu Ser Glu 225 230 235 240 Ile Glu His Asp Pro Thr His Lys Ile Met Cys Thr Asp Ala Lys Phe 245 250 255 Gly Phe Asp Asp Asn Ala Ser Phe Arg Gln Glu Lys Ile Tyr Ser Trp 260 265 270 Arg Asp Leu Ser Gln Glu Asp Pro Asp Glu Val Lys Ala Lys Lys Tyr 275 280 285 Asp Leu Asn Phe Val Lys Leu Lys Gly Asn Ile Gly Cys Leu Val Asn 290 295 300 Gly Ala Gly Leu Ala Met Ala Thr Met Asp Val Ile Lys Leu Asn Gly 305 310 315 320 Gly Asp Pro Ala Asn Phe Leu Asp Cys Gly Gly Gly Ala Thr Pro Glu 325 330 335 Thr Ile Lys Gln Gly Phe Glu Leu Ile Leu Ser Asn Lys Asn Val Asp 340 345 350 Ala Ile Phe Val Asn Ile Phe Gly Gly Ile Val Arg Cys Asp Tyr Val 355 360 365 Ala Leu Gly Leu Val Glu Ala Ala Arg Glu Leu Glu Val Arg Val Pro 370 375 380 Ile Val Ala Arg Leu Gln Gly Thr Lys Val Glu Glu Gly Arg Asp Ile 385 390 395 400 Ile Asn Lys Ser Gly Val Lys Ile Tyr Ser Phe Asp Glu Leu Asp Pro 405 410 415 Ala Ala Lys Lys Val Val Glu Leu Thr Gln Asn 420 425 <210> 10 <211> 329 <212> PRT <213> Saccharomyces cerevisiae <400> 10 Met Leu Arg Ser Thr Val Ser Lys Ala Ser Leu Lys Ile Cys Arg His 1 5 10 15 Phe His Arg Glu Ser Ile Pro Tyr Asp Lys Thr Ile Lys Asn Leu Leu 20 25 30 Leu Pro Lys Asp Thr Lys Val Ile Phe Gln Gly Phe Thr Gly Lys Gln 35 40 45 Gly Thr Phe His Ala Ser Ile Ser Gln Glu Tyr Gly Thr Asn Val Val 50 55 60 Gly Gly Thr Asn Pro Lys Lys Ala Gly Gln Thr His Leu Gly Gln Pro 65 70 75 80 Val Phe Ala Ser Val Lys Asp Ala Ile Lys Glu Thr Gly Ala Thr Ala 85 90 95 Ser Ala Ile Phe Val Pro Pro Pro Ile Ala Ala Ala Ala Ile Lys Glu 100 105 110 Ser Ile Glu Ala Glu Ile Pro Leu Ala Val Cys Ile Thr Glu Gly Ile 115 120 125 Pro Gln His Asp Met Leu Tyr Ile Ala Glu Met Leu Gln Thr Gln Asp 130 135 140 Lys Thr Arg Leu Val Gly Pro Asn Cys Pro Gly Ile Ile Asn Pro Ala 145 150 155 160 Thr Lys Val Arg Ile Gly Ile Gln Pro Pro Lys Ile Phe Gln Ala Gly 165 170 175 Lys Ile Gly Ile Ile Ser Arg Ser Gly Thr Leu Thr Tyr Glu Ala Val 180 185 190 Gln Gln Thr Thr Lys Thr Asp Leu Gly Gln Ser Leu Val Ile Gly Met 195 200 205 Gly Gly Asp Ala Phe Pro Gly Thr Asp Phe Ile Asp Ala Leu Lys Leu 210 215 220 Phe Leu Glu Asp Glu Thr Thr Glu Gly Ile Ile Met Leu Gly Glu Ile 225 230 235 240 Gly Gly Lys Ala Glu Ile Glu Ala Ala Gln Phe Leu Lys Glu Tyr Asn 245 250 255 Phe Ser Arg Ser Lys Pro Met Pro Val Ala Ser Phe Ile Ala Gly Thr 260 265 270 Val Ala Gly Gln Met Lys Gly Val Arg Met Gly His Ser Gly Ala Ile 275 280 285 Val Glu Gly Ser Gly Thr Asp Ala Glu Ser Lys Lys Gln Ala Leu Arg 290 295 300 Asp Val Gly Val Ala Val Val Glu Ser Pro Gly Tyr Leu Gly Gln Ala 305 310 315 320 Leu Leu Asp Gln Phe Ala Lys Phe Lys 325 <210> 11 <211> 640 <212> PRT <213> Saccharomyces cerevisiae <400> 11 Met Leu Ser Leu Lys Lys Ser Ala Leu Ser Lys Leu Thr Leu Leu Arg 1 5 10 15 Asn Thr Arg Thr Phe Thr Ser Ser Ala Leu Val Arg Gln Thr Gln Gly 20 25 30 Ser Val Asn Gly Ser Ala Ser Arg Ser Ala Asp Gly Lys Tyr His Ile 35 40 45 Ile Asp His Glu Tyr Asp Cys Val Val Ile Gly Ala Gly Gly Ala Gly 50 55 60 Leu Arg Ala Ala Phe Gly Leu Ala Glu Ala Gly Tyr Lys Thr Ala Cys 65 70 75 80 Ile Ser Lys Leu Phe Pro Thr Arg Ser His Thr Val Ala Ala Gln Gly 85 90 95 Gly Ile Asn Ala Ala Leu Gly Asn Met His Lys Asp Asn Trp Lys Trp 100 105 110 His Met Tyr Asp Thr Val Lys Gly Ser Asp Trp Leu Gly Asp Gln Asp 115 120 125 Ser Ile His Tyr Met Thr Arg Glu Ala Pro Lys Ser Ile Ile Glu Leu 130 135 140 Glu His Tyr Gly Val Pro Phe Ser Arg Thr Glu Asn Gly Lys Ile Tyr 145 150 155 160 Gln Arg Ala Phe Gly Gly Gln Thr Lys Glu Tyr Gly Lys Gly Ala Gln 165 170 175 Ala Tyr Arg Thr Cys Ala Val Ala Asp Arg Thr Gly His Ala Leu Leu 180 185 190 His Thr Leu Tyr Gly Gln Ala Leu Arg His Asp Thr His Phe Phe Ile 195 200 205 Glu Tyr Phe Ala Leu Asp Leu Leu Thr His Asn Gly Glu Val Val Gly 210 215 220 Val Ile Ala Tyr Asn Gln Glu Asp Gly Thr Ile His Arg Phe Arg Ala 225 230 235 240 His Lys Thr Ile Ile Ala Thr Gly Gly Tyr Gly Arg Ala Tyr Phe Ser 245 250 255 Cys Thr Ser Ala His Thr Cys Thr Gly Asp Gly Asn Ala Met Val Ser 260 265 270 Arg Ala Gly Phe Pro Leu Gln Asp Leu Glu Phe Val Gln Phe His Pro 275 280 285 Ser Gly Ile Tyr Gly Ser Gly Cys Leu Ile Thr Glu Gly Ala Arg Gly 290 295 300 Glu Gly Gly Phe Leu Val Asn Ser Glu Gly Glu Arg Phe Met Glu Arg 305 310 315 320 Tyr Ala Pro Thr Ala Lys Asp Leu Ala Cys Arg Asp Val Val Ser Arg 325 330 335 Ala Ile Thr Met Glu Ile Arg Glu Gly Arg Gly Val Gly Lys Lys Lys 340 345 350 Asp His Met Tyr Leu Gln Leu Ser His Leu Pro Pro Glu Val Leu Lys 355 360 365 Glu Arg Leu Pro Gly Ile Ser Glu Thr Ala Ala Ile Phe Ala Gly Val 370 375 380 Asp Val Thr Lys Glu Pro Ile Pro Ile Ile Pro Thr Val His Tyr Asn 385 390 395 400 Met Gly Gly Ile Pro Thr Lys Trp Asn Gly Glu Ala Leu Thr Ile Asp 405 410 415 Glu Glu Thr Gly Glu Asp Lys Val Ile Pro Gly Leu Met Ala Cys Gly 420 425 430 Glu Ala Ala Cys Val Ser Val His Gly Ala Asn Arg Leu Gly Ala Asn 435 440 445 Ser Leu Leu Asp Leu Val Val Phe Gly Arg Ala Val Ala His Thr Val 450 455 460 Ala Asp Thr Leu Gln Pro Gly Leu Pro His Lys Pro Leu Pro Ser Asp 465 470 475 480 Leu Gly Lys Glu Ser Ile Ala Asn Leu Asp Lys Leu Arg Asn Ala Asn 485 490 495 Gly Ser Arg Ser Thr Ala Glu Ile Arg Met Asn Met Lys Gln Thr Met 500 505 510 Gln Lys Asp Val Ser Val Phe Arg Thr Gln Ser Ser Leu Asp Glu Gly 515 520 525 Val Arg Asn Ile Thr Ala Val Glu Lys Thr Phe Asp Asp Val Lys Thr 530 535 540 Thr Asp Arg Ser Met Ile Trp Asn Ser Asp Leu Val Glu Thr Leu Glu 545 550 555 560 Leu Gln Asn Leu Leu Thr Cys Ala Ser Gln Thr Ala Val Ser Ala Ala 565 570 575 Asn Arg Lys Glu Ser Arg Gly Ala His Ala Arg Glu Asp Tyr Pro Asn 580 585 590 Arg Asp Asp Glu His Trp Met Lys His Thr Leu Ser Trp Gln Lys Asp 595 600 605 Val Ala Ala Pro Val Thr Leu Lys Tyr Arg Arg Val Ile Asp His Thr 610 615 620 Leu Asp Glu Lys Glu Cys Pro Ser Val Pro Pro Thr Val Arg Ala Tyr 625 630 635 640 <210> 12 <211> 266 <212> PRT <213> Saccharomyces cerevisiae <400> 12 Met Leu Asn Val Leu Leu Arg Arg Lys Ala Phe Cys Leu Val Thr Lys 1 5 10 15 Lys Gly Met Ala Thr Ala Thr Thr Ala Ala Ala Thr His Thr Pro Arg 20 25 30 Leu Lys Thr Phe Lys Val Tyr Arg Trp Asn Pro Asp Glu Pro Ser Ala 35 40 45 Lys Pro His Leu Gln Ser Tyr Gln Val Asp Leu Asn Asp Cys Gly Pro 50 55 60 Met Val Leu Asp Ala Leu Leu Lys Ile Lys Asp Glu Gln Asp Ser Thr 65 70 75 80 Leu Thr Phe Arg Arg Ser Cys Arg Glu Gly Ile Cys Gly Ser Cys Ala 85 90 95 Met Asn Ile Gly Gly Arg Asn Thr Leu Ala Cys Ile Cys Lys Ile Asp 100 105 110 Gln Asn Glu Ser Lys Gln Leu Lys Ile Tyr Pro Leu Pro His Met Phe 115 120 125 Ile Val Lys Asp Leu Val Pro Asp Leu Thr Asn Phe Tyr Gln Gln Tyr 130 135 140 Lys Ser Ile Gln Pro Tyr Leu Gln Arg Ser Ser Phe Pro Lys Asp Gly 145 150 155 160 Thr Glu Val Leu Gln Ser Ile Glu Asp Arg Lys Lys Leu Asp Gly Leu 165 170 175 Tyr Glu Cys Ile Leu Cys Ala Cys Cys Ser Thr Ser Cys Pro Ser Tyr 180 185 190 Trp Trp Asn Gln Glu Gln Tyr Leu Gly Pro Ala Val Leu Met Gln Ala 195 200 205 Tyr Arg Trp Leu Ile Asp Ser Arg Asp Gln Ala Thr Lys Thr Arg Lys 210 215 220 Ala Met Leu Asn Asn Ser Met Ser Leu Tyr Arg Cys His Thr Ile Met 225 230 235 240 Asn Cys Thr Arg Thr Cys Pro Lys Gly Leu Asn Pro Gly Leu Ala Ile 245 250 255 Ala Glu Ile Lys Lys Ser Leu Ala Phe Ala 260 265 <210> 13 <211> 198 <212> PRT <213> Saccharomyces cerevisiae <400> 13 Met Ser Ala Met Met Val Lys Leu Gly Leu Asn Lys Ser Ala Leu Leu 1 5 10 15 Leu Lys Pro Ser Ala Phe Ser Arg Ala Ala Ala Leu Ser Ser Ser Arg 20 25 30 Arg Leu Leu Phe Asn Thr Ala Arg Thr Asn Phe Leu Ser Thr Ser Pro 35 40 45 Leu Lys Asn Val Ala Ser Glu Met Asn Thr Lys Ala Ala Ile Ala Glu 50 55 60 Glu Gln Ile Leu Asn Lys Gln Arg Ala Lys Arg Pro Ile Ser Pro His 65 70 75 80 Leu Thr Ile Tyr Gln Pro Gln Leu Thr Trp Tyr Leu Ser Ser Leu His 85 90 95 Arg Ile Ser Leu Val Leu Met Gly Leu Gly Phe Tyr Leu Phe Thr Ile 100 105 110 Leu Phe Gly Val Ser Gly Leu Leu Gly Leu Gly Leu Thr Thr Glu Lys 115 120 125 Val Ser Asn Trp Tyr His Gln Lys Phe Ser Lys Ile Thr Glu Trp Ser 130 135 140 Ile Lys Gly Ser Phe Ala Tyr Leu Phe Ala Ile His Tyr Gly Gly Ala 145 150 155 160 Ile Arg His Leu Ile Trp Asp Thr Ala Lys Glu Leu Thr Leu Lys Gly 165 170 175 Val Tyr Arg Thr Gly Tyr Ala Leu Ile Gly Phe Thr Ala Val Leu Gly 180 185 190 Thr Tyr Leu Leu Thr Leu 195 <210> 14 <211> 488 <212> PRT <213> Saccharomyces cerevisiae <400> 14 Met Leu Arg Phe Thr Asn Cys Ser Cys Lys Thr Phe Val Lys Ser Ser 1 5 10 15 Tyr Lys Leu Asn Ile Arg Arg Met Asn Ser Ser Phe Arg Thr Glu Thr 20 25 30 Asp Ala Phe Gly Glu Ile His Val Pro Ala Asp Lys Tyr Trp Gly Ala 35 40 45 Gln Thr Gln Arg Ser Phe Gln Asn Phe Lys Ile Gly Gly Ala Arg Glu 50 55 60 Arg Met Pro Leu Pro Leu Val His Ala Phe Gly Val Leu Lys Lys Ser 65 70 75 80 Ala Ala Ile Val Asn Glu Ser Leu Gly Gly Leu Asp Pro Lys Ile Ser 85 90 95 Lys Ala Ile Gln Gln Ala Ala Asp Glu Val Ala Ser Gly Lys Leu Asp 100 105 110 Asp His Phe Pro Leu Val Val Phe Gln Thr Gly Ser Gly Thr Gln Ser 115 120 125 Asn Met Asn Ala Asn Glu Val Ile Ser Asn Arg Ala Ile Glu Ile Leu 130 135 140 Gly Gly Lys Ile Gly Ser Lys Gln Val His Pro Asn Asn His Cys Asn 145 150 155 160 Gln Ser Gln Ser Ser Asn Asp Thr Phe Pro Thr Val Met His Ile Ala 165 170 175 Ala Ser Leu Gln Ile Gln Asn Glu Leu Ile Pro Glu Leu Thr Asn Leu 180 185 190 Lys Asn Ala Leu Glu Ala Lys Ser Lys Glu Phe Asp His Ile Val Lys 195 200 205 Ile Gly Arg Thr His Leu Gln Asp Ala Thr Pro Leu Thr Leu Gly Gln 210 215 220 Glu Phe Ser Gly Tyr Val Gln Gln Val Glu Asn Gly Ile Gln Arg Val 225 230 235 240 Ala His Ser Leu Lys Thr Leu Ser Phe Leu Ala Gln Gly Gly Thr Ala 245 250 255 Val Gly Thr Gly Leu Asn Thr Lys Pro Gly Phe Asp Val Lys Ile Ala 260 265 270 Glu Gln Ile Ser Lys Glu Thr Gly Leu Lys Phe Gln Thr Ala Pro Asn 275 280 285 Lys Phe Glu Ala Leu Ala Ala His Asp Ala Ile Val Glu Cys Ser Gly 290 295 300 Ala Leu Asn Thr Leu Ala Cys Ser Leu Phe Lys Ile Ala Gln Asp Ile 305 310 315 320 Arg Tyr Leu Gly Ser Gly Pro Arg Cys Gly Tyr His Glu Leu Met Leu 325 330 335 Pro Glu Asn Glu Pro Gly Ser Ser Ile Met Pro Gly Lys Val Asn Pro 340 345 350 Thr Gln Asn Glu Ala Leu Thr Gln Val Cys Val Gln Val Met Gly Asn 355 360 365 Asn Ala Ala Ile Thr Phe Ala Gly Ser Gln Gly Gln Phe Glu Leu Asn 370 375 380 Val Phe Lys Pro Val Met Ile Ala Asn Leu Leu Asn Ser Ile Arg Leu 385 390 395 400 Ile Thr Asp Ala Ala Tyr Ser Phe Arg Val His Cys Val Glu Gly Ile 405 410 415 Lys Ala Asn Glu Pro Arg Ile His Glu Leu Leu Thr Lys Ser Leu Met 420 425 430 Leu Val Thr Ala Leu Asn Pro Lys Ile Gly Tyr Asp Ala Ala Ser Lys 435 440 445 Val Ala Lys Asn Ala His Lys Lys Gly Ile Thr Leu Lys Glu Ser Ala 450 455 460 Leu Glu Leu Gly Val Leu Thr Glu Lys Glu Phe Asp Glu Trp Val Val 465 470 475 480 Pro Glu His Met Leu Gly Pro Lys 485 <210> 15 <211> 343 <212> PRT <213> Saccharomyces cerevisiae <400> 15 Met Val Lys Val Ala Ile Leu Gly Ala Ser Gly Gly Val Gly Gln Pro 1 5 10 15 Leucine, Serine, Leucine, Leucine, Leucine, Lysine, Leucine, Serine, Proline, Tyrosine, Valine, Serine, Glutamic acid, Leucine, Alanine, Leucine 20 25 30 Tyrosine, Aspartic acid, Isoleucine, Arginine, Alanine, Alanine, Glutamic acid, Glycine, Isoleucine, Glycine, Lysine, Aspartic acid, Leucine, Serine, Histidine, Isoleucine 35 40 45 Asparagine, Threonine, Asparagine, Serine, Serine, Cysteine, Valine, Glycine, Tyrosine, Aspartic acid, Lysine, Aspartic acid, Serine, Isoleucine, Glutamic acid, Asparagine 50 55 60 Threonine, Leucine, Serine, Asparagine, Alanine, Glutamine, Valine, Valine, Leucine, Isoleucine, Proline, Alanine, Glycine, Valine, Proline, Arginine 65 70 75 80 Lysine, Proline, Glycine, Leucine, Threonine, Arginine, Aspartic acid, Aspartic acid, Leucine, Phenylalanine, Lysine, Methionine, Asparagine, Alanine, Glycine, Isoleucine 85 90 95 Valine, Lysine, Serine, Leucine, Valine, Threonine, Alanine, Valine, Glycine, Lysine, Phenylalanine, Alanine, Proline, Asparagine, Alanine, Arginine 100 105 110 Isoleucine, Leucine, Valine, Isoleucine, Serine, Asparagine, Proline, Valine, Asparagine, Serine, Leucine, Valine, Proline, Isoleucine, Alanine, Valine 115 120 125 Glutamic acid, Threonine, Leucine, Lysine, Lysine, Methionine, Glycine, Lysine, Phenylalanine, Lysine, Proline, Glycine, Asparagine, Valine, Methionine, Glycine 130 135 140 Valine, Threonine, Asparagine, Leucine, Aspartic acid, Leucine, Valine, Arginine, Alanine, Glutamic acid, Threonine, Phenylalanine, Leucine, Valine, Aspartic acid, Tyrosine 145 150 155 160 Leucine, Methionine, Leucine, Lysine, Asparagine, Proline, Lysine, Isoleucine, Glycine, Glutamine, Glutamic acid, Glutamine, Aspartic acid, Lysine, Threonine, Threonine 165 170 175 Met His Arg Lys Val Thr Val Ile Gly Gly His Ser Gly Glu Thr Ile 180 185 190 Ile Pro Ile Ile Thr Asp Lys Ser Leu Val Phe Gln Leu Asp Lys Gln 195 200 205 Tyr Glu His Phe Ile His Arg Val Gln Phe Gly Gly Asp Glu Ile Val 210 215 220 Lys Ala Lys Gln Gly Ala Gly Ser Ala Thr Leu Ser Met Ala Phe Ala 225 230 235 240 Gly Ala Lys Phe Ala Glu Glu Val Leu Arg Ser Phe His Asn Glu Lys 245 250 255 Pro Glu Thr Glu Ser Leu Ser Ala Phe Val Tyr Leu Pro Gly Leu Lys 260 265 270 Asn Gly Lys Lys Ala Gln Gln Leu Val Gly Asp Asn Ser Ile Glu Tyr 275 280 285 Phe Ser Leu Pro Ile Val Leu Arg Asn Gly Ser Val Val Ser Ile Asp 290 295 300 Thr Ser Val Leu Glu Lys Leu Ser Pro Arg Glu Glu Gln Leu Val Asn 305 310 315 320 Thr Ala Val Lys Glu Leu Arg Lys Asn Ile Glu Lys Gly Lys Ser Phe 325 330 335 Ile Leu Asp Ser Ser Lys Leu 340 <210> 16 <211> 377 <212> PRT <213> Saccharomyces cerevisiae <400> 16 Met Pro His Ser Val Thr Pro Ser Ile Glu Gln Asp Ser Leu Lys Ile 1 5 10 15 Ala Ile Leu Gly Ala Ala Gly Gly Ile Gly Gln Ser Leu Ser Leu Leu 20 25 30 Leu Lys Ala Gln Leu Gln Tyr Gln Leu Lys Glu Ser Asn Arg Ser Val 35 40 45 Thr His Ile His Leu Ala Leu Tyr Asp Val Asn Gln Glu Ala Ile Asn 50 55 60 Gly Val Thr Ala Asp Leu Ser His Ile Asp Thr Pro Ile Ser Val Ser 65 70 75 80 Ser His Ser Pro Ala Gly Gly Ile Glu Asn Cys Leu His Asn Ala Ser 85 90 95 Ile Val Val Ile Pro Ala Gly Val Pro Arg Lys Pro Gly Met Thr Arg 100 105 110 Asp Asp Leu Phe Asn Val Asn Ala Gly Ile Ile Ser Gln Leu Gly Asp 115 120 125 Ser Ile Ala Glu Cys Cys Asp Leu Ser Lys Val Phe Val Leu Val Ile 130 135 140 Ser Asn Pro Val Asn Ser Leu Val Pro Val Met Val Ser Asn Ile Leu 145 150 155 160 Lys Asn His Pro Gln Ser Arg Asn Ser Gly Ile Glu Arg Arg Ile Met 165 170 175 Gly Val Thr Lys Leu Asp Ile Val Arg Ala Ser Thr Phe Leu Arg Glu 180 185 190 Ile Asn Ile Glu Ser Gly Leu Thr Pro Arg Val Asn Ser Met Pro Asp 195 200 205 Val Pro Val Ile Gly Gly His Ser Gly Glu Thr Ile Ile Pro Leu Phe 210 215 220 Ser Gln Ser Asn Phe Leu Ser Arg Leu Asn Glu Asp Gln Leu Lys Tyr 225 230 235 240 Leu Ile His Arg Val Gln Tyr Gly Gly Asp Glu Val Val Lys Ala Lys 245 250 255 Asn Gly Lys Gly Ser Ala Thr Leu Ser Met Ala His Ala Gly Tyr Lys 260 265 270 Cys Val Val Gln Phe Val Ser Leu Leu Leu Gly Asn Ile Glu Gln Ile 275 280 285 His Gly Thr Tyr Tyr Val Pro Leu Lys Asp Ala Asn Asn Phe Pro Ile 290 295 300 Ala Pro Gly Ala Asp Gln Leu Leu Pro Leu Val Asp Gly Ala Asp Tyr 305 310 315 320 Phe Ala Ile Pro Leu Thr Ile Thr Thr Lys Gly Val Ser Tyr Val Asp 325 330 335 Tyr Asp Ile Val Asn Arg Met Asn Asp Met Glu Arg Asn Gln Met Leu 340 345 350 Pro Ile Cys Val Ser Gln Leu Lys Lys Asn Ile Asp Lys Gly Leu Glu 355 360 365 Phe Val Ala Ser Arg Ser Ala Ser Ser 370 375 <210> 17 <211> 334 <212> PRT <213> Saccharomyces cerevisiae <400> 17 Met Leu Ser Arg Val Ala Lys Arg Ala Phe Ser Ser Thr Val Ala Asn 1 5 10 15 Pro Tyr Lys Val Thr Val Leu Gly Ala Gly Gly Gly Ile Gly Gln Pro 20 25 30 Leu Ser Leu Leu Leu Lys Leu Asn His Lys Val Thr Asp Leu Arg Leu 35 40 45 Tyr Asp Leu Lys Gly Ala Lys Gly Val Ala Thr Asp Leu Ser His Ile 50 55 60 Pro Thr Asn Ser Val Val Lys Gly Phe Thr Pro Glu Glu Pro Asp Gly 65 70 75 80 Leu Asn Asn Ala Leu Lys Asp Thr Asp Met Val Leu Ile Pro Ala Gly 85 90 95 Val Pro Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Ala Ile Asn 100 105 110 Ala Ser Ile Val Arg Asp Leu Ala Ala Ala Thr Ala Glu Ser Ala Pro 115 120 125 Asn Ala Ala Ile Leu Val Ile Ser Asn Pro Val Asn Ser Thr Val Pro 130 135 140 Ile Val Ala Gln Val Leu Lys Asn Lys Gly Val Tyr Asn Pro Lys Lys 145 150 155 160 Leu Phe Gly Val Thr Thr Leu Asp Ser Ile Arg Ala Ala Arg Phe Ile 165 170 175 Ser Glu Val Glu Asn Thr Asp Pro Thr Gln Glu Arg Val Asn Val Ile 180 185 190 Gly Gly His Ser Gly Ile Thr Ile Ile Pro Leu Ile Ser Gln Thr Asn 195 200 205 His Lys Leu Met Ser Asp Asp Lys Arg His Glu Leu Ile His Arg Ile 210 215 220 Gln Phe Gly Gly Asp Glu Val Val Lys Ala Lys Asn Gly Ala Gly Ser 225 230 235 240 Ala Thr Leu Ser Met Ala His Ala Gly Ala Lys Phe Ala Asn Ala Val 245 250 255 Leu Ser Gly Phe Lys Gly Glu Arg Asp Val Ile Glu Pro Ser Phe Val 260 265 270 Asp Ser Pro Leu Phe Lys Ser Glu Gly Ile Glu Phe Phe Ala Ser Pro 275 280 285 Val Thr Leu Gly Pro Asp Gly Ile Glu Lys Ile His Pro Ile Gly Glu 290 295 300 Leu Ser Ser Glu Glu Glu Glu Met Leu Gln Lys Cys Lys Glu Thr Leu 305 310 315 320 Lys Lys Asn Ile Glu Lys Gly Val Asn Phe Val Ala Ser Lys 325 330 <210> 18 <211> 1178 <212> PRT <213> Saccharomyces cerevisiae <400> 18 Met Ser Gln Arg Lys Phe Ala Gly Leu Arg Asp Asn Phe Asn Leu Leu 1 5 10 15 Gly Glu Lys Asn Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Pro Ile 20 25 30 Arg Ile Phe Arg Thr Ala His Glu Leu Ser Met Gln Thr Val Ala Ile 35 40 45 Tyr Ser His Glu Asp Arg Leu Ser Thr His Lys Gln Lys Ala Asp Glu 50 55 60 Ala Tyr Val Ile Gly Glu Val Gly Gln Tyr Thr Pro Val Gly Ala Tyr 65 70 75 80 Leu Ala Ile Asp Glu Ile Ile Ser Ile Ala Gln Lys His Gln Val Asp 85 90 95 Phe Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ser Glu Phe Ala 100 105 110 Asp Lys Val Val Lys Ala Gly Ile Thr Trp Ile Gly Pro Pro Ala Glu 115 120 125 Val Ile Asp Ser Val Gly Asp Lys Val Ser Ala Arg Asn Leu Ala Ala 130 135 140 Lys Ala Asn Val Pro Thr Val Pro Gly Thr Pro Gly Pro Ile Glu Thr 145 150 155 160 Val Glu Glu Ala Leu Asp Phe Val Asn Glu Tyr Gly Tyr Pro Val Ile 165 170 175 Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly Met Arg Val Val Arg 180 185 190 Glu Gly Asp Asp Val Ala Asp Ala Phe Gln Arg Ala Thr Ser Glu Ala 195 200 205 Arg Thr Ala Phe Gly Asn Gly Thr Cys Phe Val Glu Arg Phe Leu Asp 210 215 220 Lys Pro Lys His Ile Glu Val Gln Leu Leu Ala Asp Asn His Gly Asn 225 230 235 240 Val Val His Leu Phe Glu Arg Asp Cys Ser Val Gln Arg Arg His Gln 245 250 255 Lys Val Val Glu Val Ala Pro Ala Lys Thr Leu Pro Arg Glu Val Arg 260 265 270 Asp Ala Ile Leu Thr Asp Ala Val Lys Leu Ala Lys Glu Cys Gly Tyr 275 280 285 Arg Asn Ala Gly Thr Ala Glu Phe Leu Val Asp Asn Gln Asn Arg His 290 295 300 Tyr Phe Ile Glu Ile Asn Pro Arg Ile Gln Val Glu His Thr Ile Thr 305 310 315 320 Glu Glu Ile Thr Gly Ile Asp Ile Val Ala Ala Gln Ile Gln Ile Ala 325 330 335 Ala Gly Ala Ser Leu Pro Gln Leu Gly Leu Phe Gln Asp Lys Ile Thr 340 345 350 Thr Arg Gly Phe Ala Ile Gln Cys Arg Ile Thr Thr Glu Asp Pro Ala 355 360 365 Lys Asn Phe Gln Pro Asp Thr Gly Arg Ile Glu Val Tyr Arg Ser Ala 370 375 380 Gly Gly Asn Gly Val Arg Leu Asp Gly Gly Asn Ala Tyr Ala Gly Thr 385 390 395 400 Ile Ile Ser Pro His Tyr Asp Ser Met Leu Val Lys Cys Ser Cys Ser 405 410 415 Gly Ser Thr Tyr Glu Ile Val Arg Arg Lys Met Ile Arg Ala Leu Ile 420 425 430 Glu Phe Arg Ile Arg Gly Val Lys Thr Asn Ile Pro Phe Leu Leu Thr 435 440 445 Leu Leu Thr Asn Pro Val Phe Ile Glu Gly Thr Tyr Trp Thr Thr Phe 450 455 460 Ile Asp Asp Thr Pro Gln Leu Phe Gln Met Val Ser Ser Gln Asn Arg 465 470 475 480 Ala Gln Lys Leu Leu His Tyr Leu Ala Asp Val Ala Val Asn Gly Ser 485 490 495 Ser Ile Lys Gly Gln Ile Gly Leu Pro Lys Leu Lys Ser Asn Pro Ser 500 505 510 Val Pro His Leu His Asp Ala Gln Gly Asn Val Ile Asn Val Thr Lys 515 520 525 Ser Ala Pro Pro Ser Gly Trp Arg Gln Val Leu Leu Glu Lys Gly Pro 530 535 540 Ala Glu Phe Ala Arg Gln Val Arg Gln Phe Asn Gly Thr Leu Leu Met 545 550 555 560 Asp Thr Thr Trp Arg Asp Ala His Gln Ser Leu Leu Ala Thr Arg Val 565 570 575 Arg Thr His Asp Leu Ala Thr Ile Ala Pro Thr Thr Ala His Ala Leu 580 585 590 Ala Gly Arg Phe Ala Leu Glu Cys Trp Gly Gly Ala Thr Phe Asp Val 595 600 605 Ala Met Arg Phe Leu His Glu Asp Pro Trp Glu Arg Leu Arg Lys Leu 610 615 620 Arg Ser Leu Val Pro Asn Ile Pro Phe Gln Met Leu Leu Arg Gly Ala 625 630 635 640 Asn Gly Val Ala Tyr Ser Ser Leu Pro Asp Asn Ala Ile Asp His Phe 645 650 655 Val Lys Gln Ala Lys Asp Asn Gly Val Asp Ile Phe Arg Val Phe Asp 660 665 670 Ala Leu Asn Asp Leu Glu Gln Leu Lys Val Gly Val Asp Ala Val Lys 675 680 685 Lys Ala Gly Gly Val Val Glu Ala Thr Val Cys Phe Ser Gly Asp Met 690 695 700 Leu Gln Pro Gly Lys Lys Tyr Asn Leu Asp Tyr Tyr Leu Glu Ile Ala 705 710 715 720 Glu Lys Ile Val Gln Met Gly Thr His Ile Leu Gly Ile Lys Asp Met 725 730 735 Ala Gly Thr Met Lys Pro Ala Ala Ala Lys Leu Leu Ile Gly Ser Leu 740 745 750 Arg Ala Lys Tyr Pro Asp Leu Pro Ile His Val His Thr His Asp Ser 755 760 765 Ala Gly Thr Ala Val Ala Ser Met Thr Ala Cys Ala Leu Ala Gly Ala 770 775 780 Asp Val Val Asp Val Ala Ile Asn Ser Met Ser Gly Leu Thr Ser Gln 785 790 795 800 Pro Ser Ile Asn Ala Leu Leu Ala Ser Leu Glu Gly Asn Ile Asp Thr 805 810 815 Gly Ile Asn Val Glu His Val Arg Glu Leu Asp Ala Tyr Trp Ala Glu 820 825 830 Met Arg Leu Leu Tyr Ser Cys Phe Glu Ala Asp Leu Lys Gly Pro Asp 835 840 845 Pro Glu Val Tyr Gln His Glu Ile Pro Gly Gly Gln Leu Thr Asn Leu 850 855 860 Leu Phe Gln Ala Gln Gln Leu Gly Leu Gly Glu Gln Trp Ala Glu Thr 865 870 875 880 Lys Arg Ala Tyr Arg Glu Ala Asn Tyr Leu Leu Gly Asp Ile Val Lys 885 890 895 Val Thr Pro Thr Ser Lys Val Val Gly Asp Leu Ala Gln Phe Met Val 900 905 910 Ser Asn Lys Leu Thr Ser Asp Asp Val Arg Arg Leu Ala Asn Ser Leu 915 920 925 Asp Phe Pro Asp Ser Val Met Asp Phe Phe Glu Gly Leu Ile Gly Gln 930 935 940 Pro Tyr Gly Gly Phe Pro Glu Pro Phe Arg Ser Asp Val Leu Arg Asn 945 950 955 960 Lys Arg Arg Lys Leu Thr Cys Arg Pro Gly Leu Glu Leu Glu Pro Phe 965 970 975 Asp Leu Glu Lys Ile Arg Glu Asp Leu Gln Asn Arg Phe Gly Asp Val 980 985 990 Asp Glu Cys Asp Val Ala Ser Tyr Asn Met Tyr Pro Arg Val Tyr Glu 995 1000 1005 Asp Phe Gln Lys Met Arg Glu Thr Tyr Gly Asp Leu Ser Val Leu 1010 1015 1020 Pro Thr Arg Ser Phe Leu Ser Pro Leu Glu Thr Asp Glu Glu Ile 1025 1030 1035 Glu Val Val Ile Glu Gln Gly Lys Thr Leu Ile Ile Lys Leu Gln 1040 1045 1050 Ala Val Gly Asp Leu Asn Lys Lys Thr Gly Glu Arg Glu Val Tyr 1055 1060 1065 Phe Asp Leu Asn Gly Glu Met Arg Lys Ile Arg Val Ala Asp Arg 1070 1075 1080 Ser Gln Lys Val Glu Thr Val Thr Lys Ser Lys Ala Asp Met His 1085 1090 1095 Asp Pro Leu His Ile Gly Ala Pro Met Ala Gly Val Ile Val Glu 1100 1105 1110 Val Lys Val His Lys Gly Ser Leu Ile Lys Lys Gly Gln Pro Val 1115 1120 1125 Ala Val Leu Ser Ala Met Lys Met Glu Met Ile Ile Ser Ser Pro 1130 1135 1140 Ser Asp Gly Gln Val Lys Glu Val Phe Val Ser Asp Gly Glu Asn 1145 1150 1155 Val Asp Ser Ser Asp Leu Leu Val Leu Leu Glu Asp Gln Val Pro 1160 1165 1170 Val Glu Thr Lys Ala 1175 <210> 19 <211> 1180 <212> PRT <213> Saccharomyces cerevisiae <400> 19 Met Ser Ser Ser Lys Lys Leu Ala Gly Leu Arg Asp Asn Phe Ser Leu 1 5 10 15 Leu Gly Glu Lys Asn Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Pro 20 25 30 Ile Arg Ile Phe Arg Ser Ala His Glu Leu Ser Met Arg Thr Ile Ala 35 40 45 Ile Tyr Ser His Glu Asp Arg Leu Ser Met His Arg Leu Lys Ala Asp 50 55 60 Glu Ala Tyr Val Ile Gly Glu Glu Gly Gln Tyr Thr Pro Val Gly Ala 65 70 75 80 Tyr Leu Ala Met Asp Glu Ile Ile Glu Ile Ala Lys Lys His Lys Val 85 90 95 Asp Phe Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ser Glu Phe 100 105 110 Ala Asp Lys Val Val Lys Ala Gly Ile Thr Trp Ile Gly Pro Pro Ala 115 120 125 Glu Val Ile Asp Ser Val Gly Asp Lys Val Ser Ala Arg His Leu Ala 130 135 140 Ala Arg Ala Asn Val Pro Thr Val Pro Gly Thr Pro Gly Pro Ile Glu 145 150 155 160 Thr Val Gln Glu Ala Leu Asp Phe Val Asn Glu Tyr Gly Tyr Pro Val 165 170 175 Ile Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly Met Arg Val Val 180 185 190 Arg Glu Gly Asp Asp Val Ala Asp Ala Phe Gln Arg Ala Thr Ser Glu 195 200 205 Ala Arg Thr Ala Phe Gly Asn Gly Thr Cys Phe Val Glu Arg Phe Leu 210 215 220 Asp Lys Pro Lys His Ile Glu Val Gln Leu Leu Ala Asp Asn His Gly 225 230 235 240 Asn Val Val His Leu Phe Glu Arg Asp Cys Ser Val Gln Arg Arg His 245 250 255 Gln Lys Val Val Glu Val Ala Pro Ala Lys Thr Leu Pro Arg Glu Val 260 265 270 Arg Asp Ala Ile Leu Thr Asp Ala Val Lys Leu Ala Lys Val Cys Gly 275 280 285 Tyr Arg Asn Ala Gly Thr Ala Glu Phe Leu Val Asp Asn Gln Asn Arg 290 295 300 His Tyr Phe Ile Glu Ile Asn Pro Arg Ile Gln Val Glu His Thr Ile 305 310 315 320 Thr Glu Glu Ile Thr Gly Ile Asp Ile Val Ser Ala Gln Ile Gln Ile 325 330 335 Ala Ala Gly Ala Thr Leu Thr Gln Leu Gly Leu Leu Gln Asp Lys Ile 340 345 350 Thr Thr Arg Gly Phe Ser Ile Gln Cys Arg Ile Thr Thr Glu Asp Pro 355 360 365 Ser Lys Asn Phe Gln Pro Asp Thr Gly Arg Leu Glu Val Tyr Arg Ser 370 375 380 Ala Gly Gly Asn Gly Val Arg Leu Asp Gly Gly Asn Ala Tyr Ala Gly 385 390 395 400 Ala Thr Ile Ser Pro His Tyr Asp Ser Met Leu Val Lys Cys Ser Cys 405 410 415 Ser Gly Ser Thr Tyr Glu Ile Val Arg Arg Lys Met Ile Arg Ala Leu 420 425 430 Ile Glu Phe Arg Ile Arg Gly Val Lys Thr Asn Ile Pro Phe Leu Leu 435 440 445 Thr Leu Leu Thr Asn Pro Val Phe Ile Glu Gly Thr Tyr Trp Thr Thr 450 455 460 Phe Ile Asp Asp Thr Pro Gln Leu Phe Gln Met Val Ser Ser Gln Asn 465 470 475 480 Arg Ala Gln Lys Leu Leu His Tyr Leu Ala Asp Leu Ala Val Asn Gly 485 490 495 Ser Ser Ile Lys Gly Gln Ile Gly Leu Pro Lys Leu Lys Ser Asn Pro 500 505 510 Ser Val Pro His Leu His Asp Ala Gln Gly Asn Val Ile Asn Val Thr 515 520 525 Lys Ser Ala Pro Pro Ser Gly Trp Arg Gln Val Leu Leu Glu Lys Gly 530 535 540 Pro Ser Glu Phe Ala Lys Gln Val Arg Gln Phe Asn Gly Thr Leu Leu 545 550 555 560 Met Asp Thr Thr Trp Arg Asp Ala His Gln Ser Leu Leu Ala Thr Arg 565 570 575 Val Arg Thr His Asp Leu Ala Thr Ile Ala Pro Thr Thr Ala His Ala 580 585 590 Leu Ala Gly Ala Phe Ala Leu Glu Cys Trp Gly Gly Ala Thr Phe Asp 595 600 605 Val Ala Met Arg Phe Leu His Glu Asp Pro Trp Glu Arg Leu Arg Lys 610 615 620 Leu Arg Ser Leu Val Pro Asn Ile Pro Phe Gln Met Leu Leu Arg Gly 625 630 635 640 Ala Asn Gly Val Ala Tyr Ser Ser Leu Pro Asp Asn Ala Ile Asp His 645 650 655 Phe Val Lys Gln Ala Lys Asp Asn Gly Val Asp Ile Phe Arg Val Phe 660 665 670 Asp Ala Leu Asn Asp Leu Glu Gln Leu Lys Val Gly Val Asn Ala Val 675 680 685 Lys Lys Ala Gly Gly Val Val Glu Ala Thr Val Cys Tyr Ser Gly Asp 690 695 700 Met Leu Gln Pro Gly Lys Lys Tyr Asn Leu Asp Tyr Tyr Leu Glu Val 705 710 715 720 Val Glu Lys Ile Val Gln Met Gly Thr His Ile Leu Gly Ile Lys Asp 725 730 735 Met Ala Gly Thr Met Lys Pro Ala Ala Ala Lys Leu Leu Ile Gly Ser 740 745 750 Leu Arg Thr Arg Tyr Pro Asp Leu Pro Ile His Val His Ser His Asp 755 760 765 Ser Ala Gly Thr Ala Val Ala Ser Met Thr Ala Cys Ala Leu Ala Gly 770 775 780 Ala Asp Val Val Asp Val Ala Ile Asn Ser Met Ser Gly Leu Thr Ser 785 790 795 800 Gln Pro Ser Ile Asn Ala Leu Leu Ala Ser Leu Glu Gly Asn Ile Asp 805 810 815 Thr Gly Ile Asn Val Glu His Val Arg Glu Leu Asp Ala Tyr Trp Ala 820 825 830 Glu Met Arg Leu Leu Tyr Ser Cys Phe Glu Ala Asp Leu Lys Gly Pro 835 840 845 Asp Pro Glu Val Tyr Gln His Glu Ile Pro Gly Gly Gln Leu Thr Asn 850 855 860 Leu Leu Phe Gln Ala Gln Gln Leu Gly Leu Gly Glu Gln Trp Ala Glu 865 870 875 880 Thr Lys Arg Ala Tyr Arg Glu Ala Asn Tyr Leu Leu Gly Asp Ile Val 885 890 895 Lys Val Thr Pro Thr Ser Lys Val Val Gly Asp Leu Ala Gln Phe Met 900 905 910 Val Ser Asn Lys Leu Thr Ser Asp Asp Ile Arg Arg Leu Ala Asn Ser 915 920 925 Leu Asp Phe Pro Asp Ser Val Met Asp Phe Phe Glu Gly Leu Ile Gly 930 935 940 Gln Pro Tyr Gly Gly Phe Pro Glu Pro Leu Arg Ser Asp Val Leu Arg 945 950 955 960 Asn Lys Arg Arg Lys Leu Thr Cys Arg Pro Gly Leu Glu Leu Glu Pro 965 970 975 Phe Asp Leu Glu Lys Ile Arg Glu Asp Leu Gln Asn Arg Phe Gly Asp 980 985 990 Ile Asp Glu Cys Asp Val Ala Ser Tyr Asn Met Tyr Pro Arg Val Tyr 995 1000 1005 Glu Asp Phe Gln Lys Ile Arg Glu Thr Tyr Gly Asp Leu Ser Val 1010 1015 1020 Leu Pro Thr Lys Asn Phe Leu Ala Pro Ala Glu Pro Asp Glu Glu 1025 1030 1035 Ile Glu Val Thr Ile Glu Gln Gly Lys Thr Leu Ile Ile Lys Leu 1040 1045 1050 Gln Ala Val Gly Asp Leu Asn Lys Lys Thr Gly Gln Arg Glu Val 1055 1060 1065 Tyr Phe Glu Leu Asn Gly Glu Leu Arg Lys Ile Arg Val Ala Asp 1070 1075 1080 Lys Ser Gln Asn Ile Gln Ser Val Ala Lys Pro Lys Ala Asp Val 1085 1090 1095 His Asp Thr His Gln Ile Gly Ala Pro Met Ala Gly Val Ile Ile 1100 1105 1110 Glu Val Lys Val His Lys Gly Ser Leu Val Lys Lys Gly Glu Ser 1115 1120 1125 Ile Ala Val Leu Ser Ala Met Lys Met Glu Met Val Val Ser Ser 1130 1135 1140 Pro Ala Asp Gly Gln Val Lys Asp Val Phe Ile Lys Asp Gly Glu 1145 1150 1155 Ser Val Asp Ala Ser Asp Leu Leu Val Val Leu Glu Glu Glu Thr 1160 1165 1170 Leu Pro Pro Ser Gln Lys Lys 1175 1180 <210> 20 <211> 446 <212> PRT <213> Saccharomyces cerevisiae <400> 20 Met Lys Ile Val Val Ile Gly Thr Asn His Ala Gly Ile Ala Thr Ala 1 5 10 15 Asn Thr Leu Leu Glu Gln Tyr Pro Gly His Glu Ile Val Met Ile Asp 20 25 30 Arg Asn Ser Asn Met Ser Tyr Leu Gly Cys Gly Thr Ala Ile Trp Val 35 40 45 Gly Arg Gln Ile Glu Lys Pro Asp Glu Leu Phe Tyr Ala Lys Ala Glu 50 55 60 Asp Phe Glu Ala Lys Gly Val Lys Ile Leu Thr Glu Thr Glu Val Ser 65 70 75 80 Glu Ile Asp Phe Ala Asn Lys Lys Val Tyr Ala Lys Thr Lys Ser Asp 85 90 95 Asp Glu Ile Ile Glu Ala Tyr Asp Lys Leu Val Leu Ala Thr Gly Ser 100 105 110 Arg Pro Ile Ile Pro Asn Leu Pro Gly Lys Asp Leu Lys Gly Ile His 115 120 125 Phe Leu Lys Leu Phe Gln Glu Gly Gln Ala Ile Asp Ala Glu Phe Ala 130 135 140 Lys Glu Lys Val Lys Arg Ile Ala Val Ile Gly Ala Gly Tyr Ile Gly 145 150 155 160 Thr Glu Ile Ala Glu Ala Ala Lys Arg Arg Gly Lys Glu Val Leu Leu 165 170 175 Phe Asp Ala Glu Asn Thr Ser Leu Ala Ser Tyr Tyr Asp Glu Glu Phe 180 185 190 Ala Lys Gly Met Asp Glu Asn Leu Ala Gln His Gly Ile Glu Leu His 195 200 205 Phe Gly Glu Leu Ala Lys Glu Phe Lys Ala Asn Glu Glu Gly Tyr Val 210 215 220 Ser Gln Ile Val Thr Asn Lys Ala Thr Tyr Asp Val Asp Leu Val Ile 225 230 235 240 Asn Cys Ile Gly Phe Thr Ala Asn Ser Ala Leu Ala Ser Asp Lys Leu 245 250 255 Ala Thr Phe Lys Asn Gly Ala Ile Lys Val Asp Lys His Gln Gln Ser 260 265 270 Ser Asp Pro Asp Val Tyr Ala Val Gly Asp Val Ala Thr Ile Tyr Ser 275 280 285 Asn Ala Leu Gln Asp Phe Thr Tyr Ile Ala Leu Ala Ser Asn Ala Val 290 295 300 Arg Ser Gly Ile Val Ala Gly His Asn Ile Gly Gly Lys Glu Leu Glu 305 310 315 320 Ser Val Gly Val Gln Gly Ser Asn Gly Ile Ser Ile Phe Gly Tyr Asn 325 330 335 Met Thr Ser Thr Gly Leu Ser Val Lys Ala Ala Lys Lys Leu Gly Leu 340 345 350 Glu Val Ser Phe Ser Asp Phe Glu Asp Lys Gln Lys Ala Trp Phe Leu 355 360 365 His Glu Asn Asn Asp Ser Val Lys Ile Arg Ile Val Tyr Glu Thr Lys 370 375 380 Ser Arg Arg Ile Ile Gly Ala Gln Leu Ala Ser Lys Ser Glu Ile Ile 385 390 395 400 Ala Gly Asn Ile Asn Met Phe Ser Leu Ala Ile Gln Glu Lys Lys Thr 405 410 415 Ile Asp Glu Leu Ala Leu Leu Asp Leu Phe Phe Leu Pro His Phe Asn 420 425 430 Ser Pro Tyr Asn Tyr Met Thr Val Ala Ala Leu Asn Ala Lys 435 440 445 <210> 21 <211> 356 <212> PRT <213> Saccharomyces cerevisiae <400> 21 Met Tyr Pro Thr Ser Gly Cys Ala Arg Val Leu Met Ala Cys Pro Ala 1 5 10 15 Pro Ala Met Leu Arg Gly Pro Leu Leu Arg Pro Ser Thr Thr Ala Ile 20 25 30 Arg Gly Leu Arg Gly Ser Pro Leu Leu Tyr His Tyr Ala Ala Thr Ser 35 40 45 Asn Ser Asn Met Arg Tyr Phe Ser Ser Thr Ser Arg Arg Trp Ile Lys 50 55 60 Glu Phe Phe Ala Pro Pro Lys Glu Thr Asp His Ile Val Glu Ser Val 65 70 75 80 Thr Thr Trp Lys His Pro Val Phe Thr Glu Lys Gln Met Lys Glu Ile 85 90 95 Ala Ile Ala His Arg Glu Ala Lys Asn Trp Ser Asp Trp Val Ala Leu 100 105 110 Gly Thr Val Arg Phe Leu Arg Trp Ala Thr Asp Leu Ala Thr Gly Tyr 115 120 125 Arg His Ala Ala Pro Gly Lys Gln Gly Val Glu Val Pro Glu Gln Phe 130 135 140 Gln Met Thr Glu Arg Lys Trp Val Ile Arg Phe Ile Phe Leu Glu Thr 145 150 155 160 Val Ala Gly Val Pro Gly Met Val Gly Gly Met Leu Arg His Leu Arg 165 170 175 Ser Leu Arg Arg Met Lys Arg Asp Asn Gly Trp Ile Glu Thr Leu Leu 180 185 190 Glu Glu Ala Tyr Asn Glu Arg Met His Leu Leu Ser Phe Leu Lys Leu 195 200 205 Ala Gln Pro Gly Trp Phe Met Arg Leu Met Val Leu Gly Ala Gln Gly 210 215 220 Val Phe Phe Asn Gly Phe Phe Ile Ser Tyr Leu Ile Ser Pro Arg Thr 225 230 235 240 Cys His Arg Phe Val Gly Tyr Leu Glu Glu Glu Ala Val Met Thr Tyr 245 250 255 Thr His Ala Ile Lys Asp Leu Glu Ser Gly Lys Leu Pro Asn Trp Ala 260 265 270 Asn Gln Pro Ala Pro Asp Ile Ala Val Ala Tyr Trp Gln Met Pro Glu 275 280 285 Gly Lys Arg Thr Ile Leu Asp Leu Leu Tyr Tyr Ile Arg Ala Asp Glu 290 295 300 Ala Lys His Arg Glu Val Asn His Thr Leu Ala Asn Leu Lys Gln Gly 305 310 315 320 Val Asp Pro Asn Pro Tyr Ala Ala Lys Tyr Asp Asn Pro Glu Ala Pro 325 330 335 His Pro Thr Lys Ser Ala Glu Ile Val Lys Pro Thr Gly Trp Glu Arg 340 345 350 Asp Glu Val Ile 355

Claims

1. A method for decoupling the yield and productivity of isoprenoids produced in yeast cells capable of producing isoprenoids, wherein the method comprises adding one or more ATP-consuming agents to the yeast cells and reducing the ATP level during the isoprenoid production process.

2. The method according to claim 1, wherein, the one or more ATP-consuming agents are weak organic acids.

3. The method according to claim 2, wherein, the weak organic acids are selected from sorbic acid, acetic acid, benzoic acid, and propionic acid.

4. The method according to claim 3, wherein, the weak organic acid is benzoic acid.

5. The method according to claim 1, wherein, the ATP level is also reduced by overexpression of one or more ATP-dissipating enzymes.

6. The method according to claim 5, wherein, the one or more ATP-dissipating enzymes are selected from Saccharomyces cerevisiae SSB1 and ATP-diphosphohydrolase.

7. The method according to claim 1, wherein, the ATP level is also reduced by overexpression of one or more ATP-uncoupling enzymes.

8. The method according to claim 7, wherein, the one or more ATP-uncoupling enzymes are selected from NADH oxidase (NOX) and alternative oxidase (AOX).

9. The method according to claim 1, wherein, the ATP level is also reduced by expression of futile cycles in the yeast cells.

10. The method according to claim 9, wherein, the futile cycles are selected from the simultaneous overexpression of phosphofructokinase and fructose-1,6-bisphosphatase and the simultaneous overexpression of phosphoenolpyruvate carboxykinase and pyruvate carboxylase.

11. The method according to claim 1, wherein, the isoprenoids are selected from hemiterpenes, monoterpenes, diterpenes, triterpenes, tetraterpenes, sesquiterpenes, and polyterpenes.

12. The method according to claim 1, wherein, the isoprenoids are selected from rosadiene, the precursor of artemisinin, amorpha-4,11-diene, carene, α-farnesene, β-farnesene, farnesol, geraniol, geranylgeraniol, isoprene, linalool, limonene, myrcene, nerolidol, ocimene, patchouli alcohol, β-pinene, sabinene, γ-terpinene, terpinolene, and valencene.

13. The method according to claim 12, wherein, the isoprenoid is β-farnesene.

14. The method according to claim 1, wherein, the yeast cells are Saccharomyces cerevisiae.

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