Recombinant yeast host cell for acetone and ethanol production

Abstract

The present disclosure provides a recombinant yeast host cell for converting a carbohydrate to ethanol and acetone. The recombinant yeast host cell comprises an engineered metabolic pathway to convert acetyl-CoA to acetone, a downregulated native alcohol dehydrogenase (ADH) gene, and a heterologous alcohol dehydrogenase.

Description

[0001] RECOMBINANT YEAST HOST CELL FOR ACETONE AND ETHANOL PRODUCTION

[0002] TECHNOLOGICAL FIELD

[0003] [1] The present disclosure concerns a recombinant yeast host cell for producing acetone and method thereof.

[0004] BACKGROUND

[0005] [2] Yeast, more particularly Saccharomyces cerevisiae, is utilized as the primary biocatalyst in commercial bioethanol production. Yeast can also be engineered to generate higher valued chemical products within corn ethanol fermentations either in place of ethanol or coproduced with ethanol.

[0006] [3] Production of non-ethanol chemicals during fermentation of biomass (e.g., corn, sugarcane, or lignocellulosic) derived sugars has been pursued for many years to achieve several goals, including 1) establishing their sourcing from renewable as opposed to fossil resources, 2) reducing the carbon intensity of these chemicals, 3) reducing the cost of production of these chemicals, and 4) increasing the diversification and economic performance of fuel and chemical ethanol fermentation facilities. Several companies are working to commercialize the fermentative production of non-ethanol chemicals. Yet, large scale production of non-ethanol chemicals from biomass remains rare.

[0007] [4] One promising set of chemicals to produce during fermentation is acetone and isopropanol (2-propanol). These chemicals are used in very large quantities in a variety of applications as solvents and as precursors to produce polypropylene. The scale of the markets for these chemicals makes them compatible for production with the infrastructure currently used to produce fuel ethanol from corn and sugarcane. Being able to produce these compounds using this infrastructure would meet the four goals discussed above.

[0008] [5] However, yeast also convert endogenously acetone to isopropanol (IPA), and it is therefore difficult to produce high yield of acetone using this strategy. Conversion of acetone to isopropanol is problematic for two reasons: 1) Conversion of acetone to isopropanol decreases the yields of high value acetone and 2) isopropanol would be distilled with the ethanol thus decreasing its purity.

[0009] [6] There is therefore a need to develop yeast strain engineered to co-produce acetone and ethanol while decreasing the yield of isopropanol.

[0010] SUMMARY

[0011] [7] The present disclosure concerns a recombinant yeast host cell capable of producing acetone and ethanol while producing a lower amount of isopropanol. [8] The present disclosure concerns a recombinant yeast host cell capable of producing a higher amount of acetone while producing a lower amount of isopropanol in combination with production of ethanol. The present disclosure also concerns processes for making higher amount of acetone while producing a lower amount of isopropanol in combination with production of ethanol in presence of carbohydrates.

[0012] [9] According to a first aspect, the present disclosure provides a recombinant yeast host cell for converting a carbohydrate to ethanol and acetone comprising an engineered metabolic pathway to convert acetyl-CoA to acetone; a downregulated native alcohol dehydrogenase (ADH) gene; wherein the native ADH is capable of converting acetone to isopropanol; and an heterologous alcohol dehydrogenase. The engineered metabolic pathway to convert acetyl- CoA to acetone comprises a thiolase; a CoA transferase, a HMG-CoA synthase and lyase, or an acetoacetyl-CoA hydrolase; and an acetoacetate decarboxylase. In one embodiment, the heterologous ADH has a secondary alcohol activity inferior to the secondary alcohol activity of the native ADH. In some embodiment, the heterologous ADH allow the recombinant yeast host cell to produce a yield of isopropanol inferior to 0.5 g / L in a YPD media containing 120 g / L glucose at pH 6.0 following 48 hours of fermentation. In some other embodiment, the heterologous ADH is a bacterial ADH; is an ADHA, ADHP, or ADH4; is derived from a Z. mobilis ADHA, Z. mobilis ADHP, or Saccharomyces cerevisiae ADH4; and / or has at least 75% identity with the amino acid sequence of SEQ ID NO: 238, 240, or 241 . In another embodiment, the native ADH gene is an ADH1 gene. In a further embodiment, the native ADH1 has at least 70 % identity with the amino acid sequence of SEQ ID NO: 239. In a yet additional embodiment, the downregulated native ADH1 gene is a deleted native ADH1 gene. In some embodiment, the thiolase is heterologous; of prokaryotic or eukaryotic origin; encoded by a thl gene, an erg10 gene or a phaA gene; derived from Clostridium, Saccharomyces, Cupriavidus, Clostridium, Yarrowia, Thermoanaerobacterium, Saccoglossus, Strongylocentrotus, Zygosaccharomyces or Paenibacillus species; derived from Clostridium acetobutylicum, Saccharomyces cerevisiae, Cupriavidus necator, Clostridium acetobutylicum, Clostridium kluyveri, Yarrowia lipolytica, Thermoanaerobacterium thermosaccharolyticum, Saccoglossus kowalevskii, Strongylocentrotus purpuratus, Zygosaccharomyces bailii or Paenibacillus polymyxa; and / or has the amino acid sequence of SEQ ID NO: 101 , 103, 105, 107, 109, 11 1 , 113, 115, 117, 119 or 121. In some embodiment, the CoA transferase is heterologous; of prokaryotic or eukaryotic origin; encoded by a ctfA gene, a ctfB gene, an atoD and / or an atoA gene; derived from Clostridium, Thermosipho, Escherichia, Paenibacillus, Alkaliphilus or Brevibacillus species; derived from Clostridium acetobutylicum, Thermosipho melanesiensis, Escherichia coll, Paenibacillus polymyxa, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium sticklandii, Alkaliphilus metalliredigens, or Clostridium bovifaecis or Brevibacillus laterosporus; and / or has the amino acid sequence of SEQ ID NO:123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143,145, 242, 244, 246, 248, 250, 252, 254, or 256. In some embodiment, the HMG-CoA synthase is heterologous; of prokaryotic or eukaryotic origin; encoded by a hgms gene or an erg13 gene; derived from Saccharomyces, Lacticaseibacillus, Enterococcus, Haloferax, Alloscardovia or Listeria; derived from Saccharomyces cerevisiae, Lacticaseibacillus casei UW4, Enterococcus faecalis, Haloferax volcanii DS2, Alloscardovia theropitheci, or Listeria monocytogenes; and / or has the amino acid sequence of SEQ ID NO: 165, 167, 169, 171 , 173 or 175. In some embodiment, the HMG-CoA lyase is heterologous; of prokaryotic or eukaryotic origin; encoded by an hmgcl gene; derived from Pseudomonas, Azotobacter, Bacillus, Desulfotomaculum, Acinetobacter, Moraxella, Alcaligenaceae, Macaca, Arabidopsis, Gallus, or Danio species; derived from Pseudomonas monteilii, Pseudomonas wayambapalatensis, Azotobacter vinelandii, Pseudomonas citronellolis, Pseudomonas cremoris, Pseudomonas chengduensis, Pseudomonas aeruginosa PAO1, Bacillus subtilis 168, Desulfotomaculum arcticum DSM 17038, Desulfoscipio geothermicus DSM 3669, Acinetobacter baumannii, Acinetobacter Iwoffii, Moraxella caviae, Alcaligenaceae bacterium 429, Macaca fascicularis, Arabidopsis thaliana, Gallus gallus, or Danio rerio; and / or has the amino acid sequence of SEQ ID NO: 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209 or 211.

[0013] In some embodiment, the acetoacetyl-CoA hydrolase is heterologous; of prokaryotic or eukaryotic origin; encoded by a tesB gene, | yciA gene, a yigl gene, a ydil gene, a fadM1 gene, a fadM2 gene or a fadM gene; derived from Escherichia, Methylorubrum, Pseudomonas, Campylobacter, Mycobacterium, Fibrobacter, Alcanivorax, Haemophilus, Zymomonas, or Prevotella; derived from Escherichia coll, Methylorubrum extorquens AM1, Pseudomonas aeruginosa, Campylobacter jejuni, Mycobacterium tuberculosis, Pseudomonas putida, Fibrobacter succinogenes, Alcanivorax borkumensis SK2, Haemophilus influenzae, Zymomonas mobilis subsp. Mobilis ZM4, Campylobacter jejuni, Prevotella ruminicola 23 or Providencia sneebia DSM 19967 and / or has the amino acid sequence of SEQ ID NO: 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, or 229. In some embodiment, the acetoacetate decarboxylase is heterologous; of prokaryotic or eukaryotic origin; encoded by an adc gene; derived from Clostridium, Bacillus, Lactobacillus,

[0014] Lacticaseibacillus, Rhizobium, Bradyrhizobium, Tetrahymena, Aspergillus or Paenibacillus species; derived from Clostridium acetobutylicum, Clostridium beijerinckii, Bacillus amyloliquefaciens, Lactobacillus casei, Lacticaseibacillus rhamnosus, Rhizobium leguminosarum, Bradyrhizobium japonicum, Tetrahymena thermophile, Aspergillus bertholletiae, Aspergillus niger or Paenibacillus polymyxa; and / or has the amino acid sequence of SEQ ID NO: 147, 149, 151 , 153, 155, 157, 159, 161 , or 163. In some embodiment, the recombinant yeast host cell produces at least 5% less in isopropanol yield (W / V) than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a), but lacking b) and c). In another embodiment, the recombinant yeast host cell produces 5% more in yield of acetone than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a), but lacking b) and c). In yet another embodiment, the recombinant yeast host cell produces more ethanol yield than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a) and the downregulated native ADH gene as defined in b), but lacking c). In yet another embodiment, the recombinant yeast host cell growth rate is increased when compared to a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA and acetate to acetone as defined in a) and the downregulated native ADH gene as defined in b), but lacking c).

[0015]

[0010] In a second aspect, the present disclosure provides a process for making ethanol and acetone. The process comprises contacting the recombinant yeast host cell with a carbohydrate under a condition allowing the conversion of at least a part of the carbohydrate to ethanol and acetone. In an embodiment, the carbohydrate is a corn mash.

[0016] DETAILED DESCRIPTION OF THE DRAWINGS

[0017]

[0011] Having thus generally described the nature of the invention, reference will now be made to the accompanying drawings, showing by way of illustration, a preferred embodiment thereof, and in which:

[0018]

[0012] Figure 1 provides growth rate results OD60o in YPD10 media of different heterologous ADH expression within an ADH1 KO host strain.

[0019]

[0013] Figure 2 provides the metabolites profiles following fermentation by strains SC-1 , SC- 3, SC-14, and SC-15 of YPD120 media with 5g / L acetone addition. All values are shown as g / L of isopropanol, ethanol, and reduction of acetone in the media in function of the strain / isolate tested.

[0020]

[0014] Figure 3 provides CO2production profiles from corn mash fermentation of non genetically modified S. cerevisiae strain (SC-1), an acetone producer engineered S. cerevisiae strain (SC-16), and an acetone producer engineered S. cerevisiae strain further modified by replacing the native ADH1 gene by a heterologous Z. mobilis ADH1 (SC-17).

[0021]

[0015] Figure 4 provides the metabolites profiles following fermentation by strains SC-1 , SC- 16, and SC-17 of corn mash medium. All values are shown as g / L of glycerol, acetate, acetone, isopropanol, and ethanol in function of the strain / isolate tested.

[0022]

[0016] Figure 5 provides CO2production profiles of different heterologous ADH expression within an ADH1 KO host strain in YPD120 media supplemented with 5g / L acetone. DETAILED DESCRIPTION

[0023]

[0017] The present disclosure concerns a recombinant yeast host cell capable of producing a higher amount of acetone while producing a lower amount of isopropanol. Amongst other things, the recombinant yeast host cell comprises at least (a) an engineered pathway to produce acetone, (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol, and (c) a heterologous alcohol dehydrogenase gene. The amount of acetone observed when culturing the recombinant yeast host cell is higher than a control yeast host cell comprising (a) an engineered pathways to produce acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol, and (c) a heterologous alcohol dehydrogenase gene. The amount of isopropanol observed, when culturing the recombinant yeast host cell, is lower than a control yeast host cell comprising (a) an engineered pathways to produce acetone, but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol, and (c) a heterologous alcohol dehydrogenase gene.

[0024] Recombinant yeast host cell

[0025]

[0018] The present disclosure provides a recombinant yeast host cell. These recombinant yeast host cells can be obtained by introducing one or more genetic modifications in a corresponding native (parental) yeast host cell. When the genetic modification is aimed at reducing or inhibiting the expression of a specific targeted gene (which is endogenous to the host cell), the genetic modifications can be made in one or all copies of the targeted gene(s). When the genetic modification is aimed at increasing the expression of a specific targeted gene, the genetic modification can be made in one or multiple genetic locations. In the context of the present disclosure, when a yeast host cell is qualified as being “genetically engineered”, it is understood to mean that it has been manipulated to either add at least one or more heterologous or exogenous nucleic acid residue and / or removed at least one endogenous (or native) nucleic acid residue. In some embodiments, the one or more nucleic acid residues that are added can be derived from a heterologous cell or the recombinant host cell itself. In the latter scenario, the nucleic acid residue(s) is (are) added at a genomic location which is different than the native genomic location. The genetic manipulations did not occur in nature and are the results of in vitro manipulations of the native yeast host cell.

[0026]

[0019] When expressed in recombinant yeast host cells, the polypeptides (including the enzymes) described herein are encoded on one or more heterologous nucleic acid molecule. The term “heterologous” when used in reference to a nucleic acid molecule (such as a promoter or a coding sequence) refers to a nucleic acid molecule that is not natively found in the recombinant host cell. “Heterologous” also includes a native coding region, or portion thereof, that is removed from the source organism and subsequently reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome or as additional copies at its natural location. The heterologous nucleic acid molecule is purposively introduced into the recombinant yeast host cell. In some embodiments, the term “heterologous” as used herein also refers to an element (nucleic acid or protein) that is derived from a source other than the endogenous source. Thus, for example, a heterologous element could be derived from a different strain of host cell, or from an organism of a different taxonomic group (e.g., different kingdom, phylum, class, order, family genus, or species, or any subgroup within one of these classifications).

[0027]

[0020] When a heterologous nucleic acid molecule is present in the recombinant yeast host cell, it can be integrated in the host cell’s genome. The term “integrated” as used herein refers to genetic elements that are placed, through molecular biology techniques, into the chromosome of a yeast host cell. For example, genetic elements can be placed into the chromosome(s) of the host cell as opposed to in a vector such as a plasmid carried by the host cell. Methods for integrating genetic elements into the genome of a host cell are well known in the art and include homologous recombination. The heterologous nucleic acid molecule can be present in one or more copies in the yeast host cell’s chromosome. Alternatively, the heterologous nucleic acid molecule can be independently replicating from the yeast host cell’s chromosome. In such embodiment, the nucleic acid molecule can be stable and selfreplicating.

[0028]

[0021] In some embodiments, heterologous nucleic acid molecules which can be introduced into the recombinant yeast host cells are codon-optimized with respect to the intended recipient recombinant yeast host cell. As used herein the term “codon-optimized coding region” means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, codons with one or more codons that are more frequently used in the genes of that organism. In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the “codon adaptation index” or “CAI,” which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism. The CAI of codon optimized heterologous nucleic acid molecule described herein corresponds to between about 0.8 and 1 .0, between about 0.8 and 0.9, or about 1 .0. In some embodiments, heterologous nucleic acid molecules which can be introduced into the recombinant host cells are codon-optimized with respect to the intended recipient recombinant host cell so as to limit or prevent homologous recombination with the corresponding native gene.

[0022] The heterologous nucleic acid molecules of the present disclosure can comprise a coding region for the one or more polypeptides (such as enzymes) to be expressed by the host cell. A DNA or RNA “coding region” is a DNA or RNA molecule which is transcribed and / or translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. “Suitable regulatory regions” refer to nucleic acid regions located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding region, and which influence the transcription, RNA processing or stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, RNA processing sites, effector binding sites and stem-loop structures. The boundaries of the coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding region can include, but is not limited to, prokaryotic regions, cDNA from mRNA, genomic DNA molecules, synthetic DNA molecules, or RNA molecules. If the coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding region. In an embodiment, the coding region can be referred to as an open reading frame. “Open reading frame" is abbreviated ORF and means a length of nucleic acid, either DNA, cDNA or RNA, that comprises a translation start signal or initiation codon, such as an ATG or AUG, and a termination codon and can be potentially translated into a polypeptide sequence.

[0029]

[0023] The nucleic acid molecules described herein can comprise a non-coding region, for example a transcriptional and / or translational control regions. “Transcriptional and translational control regions” are DNA regulatory regions, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding region in a host cell. In eukaryotic cells, polyadenylation signals are control regions.

[0030]

[0024] The heterologous nucleic acid molecule can be introduced in the host cell using a vector. A “vector,” e.g., a “plasmid”, “cosmid” or “artificial chromosome” (such as, for example, a yeast artificial chromosome) refers to an extra chromosomal element and is usually in the form of a circular double-stranded DNA molecule. Such vectors may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a host cell.

[0031]

[0025] In the heterologous nucleic acid molecules described herein, the promoter and the nucleic acid molecule coding for the one or more polypeptides (such as the one or more enzymes) can be operatively linked to one another. In the context of the present disclosure, the expressions “operatively linked” or “operatively associated” refers to fact that the promoter is physically associated to the nucleic acid molecule coding for the one or more enzyme in a manner that allows, under certain conditions, for expression of the one or more polypeptides from the heterologous nucleic acid molecule. In an embodiment, the promoter can be located upstream (5’) of the nucleic acid sequence coding for the one or more polypeptide. In still another embodiment, the promoter can be located downstream (3’) of the nucleic acid sequence coding for the one or more polypeptide. In the context of the present disclosure, one or more than one promoter can be included in the heterologous nucleic acid molecule. When more than one promoters are included in the heterologous nucleic acid molecule, each of the promoters is operatively linked to the nucleic acid sequence coding for the one or more polypeptide. The promoters can be located, in view of the nucleic acid molecule coding for the one or more protein, upstream, downstream as well as both upstream and downstream.

[0032]

[0026] “Promoter” refers to a DNA fragment capable of controlling the expression of a coding sequence or functional RNA. The term “expression,” as used herein, refers to the transcription and stable accumulation of sense (mRNA) from the heterologous nucleic acid molecule described herein. Expression may also refer to translation of mRNA into a polypeptide. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cells at most times at a substantial similar level are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity. A promoter is generally bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease S1), as well as protein binding domains (consensus sequences) responsible for the binding of the polymerase.

[0033]

[0027] The promoter can be heterologous to the nucleic acid molecule encoding the one or more polypeptides. The promoter can be heterologous or derived from a strain being from the same genus or species as the yeast host cell. In an embodiment, the promoter is derived from the same genus or species of the yeast host cell and the heterologous polypeptide is derived from different genus than the yeast host cell.

[0034]

[0028] In some embodiments, the present disclosure concerns the expression of a heterologous polypeptide (such as a heterologous enzyme), a variant thereof or a fragment thereof in a host cell. A variant comprises at least one amino acid difference when compared to the amino acid sequence of the wild-type polypeptide. The polypeptide “variants” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the heterologous polypeptides described herein. The term “percent identity”, as known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing the sequences. The level of identity can be determined conventionally using known computer programs. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome Projects (Smith, D. W., ed.) Academic Press, NY (1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, NY (1991). Preferred methods to determine identity are designed to give the best match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignments of the sequences disclosed herein were performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151 - 153) with the default parameters (GAP PENALTY=10, GAP LENGTH PEN ALT Y= 10). Default parameters for pairwise alignments using the Clustal method were KTUPLB 1 , GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.

[0035]

[0029] The heterologous polypeptide variants exhibit the biological activity associated with the wild-type heterologous polypeptide. In an embodiment, the variant polypeptide exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity (which can be, in some embodiments, the enzymatic activity) of the wild-type heterologous polypeptide. The biological activity of the polypeptides can be determined by methods and assays known in the art.

[0036]

[0030] The variant heterologous polypeptides described herein may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide for purification of the polypeptide.

[0037]

[0031] A “variant” of the polypeptide can be a conservative variant or an allelic variant. As used herein, a conservative variant refers to alterations in the amino acid sequence that do not adversely affect the biological functions of the enzyme. A substitution, insertion or deletion is said to adversely affect the protein when the altered sequence prevents or disrupts a biological function associated with the enzyme. For example, the overall charge, structure, or hydrophobic-hydrophilic properties of the polypeptide can be altered without adversely affecting a biological activity. Accordingly, the amino acid sequence can be altered, for example to renderthe polypeptide more hydrophobic or hydrophilic, without adversely affecting the biological activity of the polypeptide.

[0038]

[0032] The heterologous polypeptide can be a fragment of a heterologous wild-type polypeptide or fragment of a variant polypeptide. Polypeptide “fragments” have at least at least 50, 100, 200, 300, 400, 500 or more consecutive amino acids of the polypeptide or the enzyme variant. A fragment comprises at least one less amino acid residue when compared to the amino acid sequence of the wild-type heterologous polypeptide or of the variant polypeptide. In some embodiments, the fragments corresponding to the wild-type polypeptide or variant polypeptide to which the signal sequence was removed. In some embodiments, the “fragments” have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the wild-type polypeptides described herein. In some embodiments, fragments of the polypeptides can be employed for producing the corresponding full-length enzyme by peptide synthesis. Therefore, the fragments can be employed as intermediates for producing the full-length polypeptide.

[0039]

[0033] The fragments of heterologous wild-type polypeptides or of variant polypeptides exhibit the biological activity of the heterologous wild-type polypeptide or of the variant polypeptide. In an embodiment, the fragment polypeptide exhibits at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the biological activity of the heterologous wild-type polypeptide or of the variant polypeptide. The biological activity of fragment polypeptides can be determined by methods and assays known in the art.

[0040]

[0034] In some additional embodiments, the present disclosure also provides reducing the expression of or inactivating a gene ortholog of a gene known to encode a native enzyme. A “gene ortholog” is understood to be a gene in a different species that evolved from a common ancestral gene by speciation. In the context of the present invention, a gene ortholog encodes a polypeptide exhibiting the same biological function than the wild-type polypeptide.

[0041]

[0035] In some further embodiments, the present disclosure also provides reducing the expression or inactivating a gene paralog of a gene known to encode an enzyme. A “gene paralog” is understood to be a gene related by duplication within the genome. In the context of the present invention, a gene paralog encodes a polypeptide that could exhibit the same biological function than the wild-type polypeptide.

[0036] The recombinant yeast host cell of the present disclosure has the ability to convert a carbohydrate into one or more fermentation products (e.g., ethanol, in combination with acetone and isopropanol). In the context of the present disclosure, the recombinant yeast host cell is a fermenting yeast cell because it is capable of converting the carbohydrate into the one or more fermentation products. Suitable fermenting yeasts and recombinant yeast host cells can be, for example, from the genus Saccharomyces, Kluyveromyces, Arxula, Debaryomyces, Candida, Pichia, Phaffia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, Komagataella or Yarrowia. Suitable yeast species can include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, K. phaffii, or K. fragilis. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis. In some embodiments, the host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genus Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. In some alternative embodiments, the host cell can be an oleaginous microalgae host cell (e.g., for example, from the genus Thraustochytrium or Schizochytriurri). In an embodiment, the fermenting yeast or recombinant yeast host cell is from the genus Saccharomyces and, in some embodiments, from the species Saccharomyces cerevisiae.

[0042]

[0037] In some embodiment, the recombinant yeast host cell of the present disclosure provides an increased production of acetone yield when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol and (c) a heterologous alcohol dehydrogenase gene. In some embodiment, the recombinant yeast host cell produces at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% more in acetone yield (W / V) than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl- CoA to acetone as defined in a), but lacking b) and c).

[0043]

[0038] In some embodiment, the recombinant yeast host cell of the present disclosure provides a decreased production in isopropanol yield by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol, and (c) a heterologous alcohol dehydrogenase gene. In a further embodiment, the recombinant yeast host cell produces at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or 60% less in isopropanol yield (W / V) than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a), but lacking b) and c).

[0044]

[0039] In some embodiment, the recombinant yeast host cell of the present disclosure provides at least 70%, 80%, 85%, 90%, 95%, 98%, 99% or 100% in ethanol yield (W / V) when compared to a control yeast host cell comprising at least (a) an engineered metabolic pathway to convert acetyl-CoA and acetate to acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol and (c) a heterologous alcohol dehydrogenase gene.

[0045]

[0040] In some embodiment, the recombinant yeast host cell of the present disclosure provides an increased production of ethanol by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone and (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol but lacking (c) a heterologous alcohol dehydrogenase gene. In some embodiment, the recombinant yeast host cell produces at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 100% more in ethanol yield (W / V) when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone and (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol but lacking (c) a heterologous alcohol dehydrogenase gene.

[0046]

[0041] In some embodiment, the recombinant yeast host cell of the present disclosure provides an increased growth rate when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone and (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol but lacking (c) a heterologous alcohol dehydrogenase gene.

[0047]

[0042] The recombinant yeast host cell of the present disclosure has native and / or heterologous enzymes that function in an engineered metabolic pathway to convert acetyl- CoA to acetone. In a further embodiment, the recombinant yeast host cell of the present disclosure comprises a further engineered metabolic pathway to produce acetyl-CoA, as for example, an engineered metabolic pathway to convert fructose-6-phosphate to acetylcoenzyme A. The recombinant yeast host cell of the present disclosure has native and / or heterologous enzymes that function in an engineered metabolic pathway to convert fructose- 6-phosphate to acetyl-coenzyme A. The engineered metabolic pathway to convert fructose-6- phosphate to acetyl-coenzyme A comprises a phosphoketolase, optionally in combination with an acetate kinase, a phosphotransacetylase, and / or an acetyl-CoA synthetase. As such, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, alone or in combination with an acetate kinase, a phosphotransacetylase, and / or an acetyl-CoA synthetase. In some embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase only. In additional embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and an acetate kinase. In further embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and a phosphotransacetylase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, an acetate kinase, and a phosphotransacetylase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase, an acetate kinase, a phosphotransacetylase, an acetyl-CoA synthetase. In yet other embodiments, the recombinant yeast host cell of the present disclosure comprises a phosphoketolase and an acetyl-CoA synthetase.

[0048]

[0043] As used herein, the terms "phosphoketolase" and "PHK" are intended to include the enzymes capable of converting D-xylulose 5-phosphate to D-glyceraldehyde 3-phosphate and acetyl-phosphate. The phosphoketolase can have a single-specificity activity (e.g., singlespecificity phosphoketolase), and be only capable of converting D-xylulose 5-phosphate to D- glyceraldehyde 3-phosphate. The phosphoketolase can have a multiple-specificity / dual- specificity (e.g., multiple-specificity or dual-specificity phosphoketolase), and be also capable of converting D-fructose 6-phosphate to D-erythrose 4-phosphate and acetyl-phosphate and / or D-sedoheptulose 7-phosphate into D-ribose 5-phosphate and acetyl-phosphate. Phosphoketolases include those enzymes that correspond to Enzyme Commission Number 4.1.2.9 and 4.1.2.22. The PHK is heterologous to the recombinant yeast host cell, and the recombinant yeast host cell of the present disclosure can comprise, in some embodiments, at least two copies of a heterologous nucleic acid encoding the PHK. In some embodiments, the PHK is of prokaryotic or eukaryotic origin. In other embodiments, the PHK can be encoded by a phk1 gene (e.g., PHK1) or a phk2 gene (e.g., PHK2). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 1 , be a variant of the amino acid sequence of SEQ ID NO: 1 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 1 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 2 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium bifidum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 3, be a variant of the amino acid sequence of SEQ ID NO: 3 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 3 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 4 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 3 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium gallicum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 5, be a variant of the amino acid sequence of SEQ ID NO: 5 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 5 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 6 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 5 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium animalis. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 7, be a variant of the amino acid sequence of SEQ ID NO: 7 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 7 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 8 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 7 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium breve. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 9, be a variant of the amino acid sequence of SEQ ID NO: 9 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 9 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 10 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 9 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Lactiplantibacillus sp. (previously referred to as Lactobacillus sp.), and in further embodiments from Lactiplantibacillus pentosus. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 11 , be a variant of the amino acid sequence of SEQ ID NO: 1 1 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 1 1 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 12 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 11 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Lactiplantibacillus acidophilus. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 13, be a variant of the amino acid sequence of SEQ ID NO: 13 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 13 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 14 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 13 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Lactiplantibacillus easel. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 15, be a variant of the amino acid sequence of SEQ ID NO: 15 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 15 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 16 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 15 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived Lactiplantibacillus plantarum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 17, be a variant of the amino acid sequence of SEQ ID NO: 17 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 17 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 18 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 17 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Aspergillus sp., and in further embodiments from Aspergillus niger. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 19, be a variant of the amino acid sequence of SEQ ID NO: 19 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 19 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 20 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 19 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Aspergillus nidulans. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 21 , be a variant of the amino acid sequence of SEQ ID NO: 21 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 21 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 22 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Aspergillus clavatus. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 23, be a variant of the amino acid sequence of SEQ ID NO: 23 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 23 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 24 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 23 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Penicillium sp., and in further embodiments from Penicillium chrysogenum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 25, be a variant of the amino acid sequence of SEQ ID NO: 25 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 25 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 26 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 25 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Neurospora sp., and in further embodiments from Neurospora crassa. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 27, be a variant of the amino acid sequence of SEQ ID NO: 39 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 27 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 28 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 27 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 29, be a variant of the amino acid sequence of SEQ ID NO: 29 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 29 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 30 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 29 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Oenococcus sp., and in further embodiments from Oenococcus oeni. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 31 , be a variant of the amino acid sequence of SEQ ID NO: 311 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 31 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 32 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 31 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium longum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 33, be a variant of the amino acid sequence of SEQ ID NO: 33 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 33 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 34 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 33 (a variant thereof or a fragment thereof). In some embodiments, the PHK is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the PHK can have the amino acid sequence of SEQ ID NO: 35, be a variant of the amino acid sequence of SEQ ID NO: 35 having PHK activity or be a fragment of the amino acid sequence of SEQ ID NO: 35 having PHK activity. The recombinant yeast host cell includes a copy (and in some embodiments at least two copies of) a heterologous nucleic acid molecule encoding the PHK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 36 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 35 (a variant thereof or a fragment thereof).

[0049]

[0044] As indicated above, the recombinant yeast host cell of the present disclosure can comprise at least two copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises two copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least three copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises three copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least four copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises four copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least five copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises five copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least six copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises six copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least seven copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises seven copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some additional embodiments, the recombinant yeast host cell of the present disclosure comprises at least eight copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome. In some embodiments, the recombinant yeast host cell comprises eight copies of the heterologous nucleic acid molecule encoding the PHK per haploid genome.

[0050]

[0045] In some embodiment, the recombinant yeast host cell of the present disclosure comprises a fusion polypeptide comprising both glucose-6-phosphate isomerase (PGI) and phosphoketolase (PHK) activities to increase the shuttling of fructose 6-P into acetyl-P. In the context of the disclosure, glucose-6-phosphate isomerases or GPIs, also known as phosphoglucose isomerases or phosphohexose isomerases, are classified under Enzyme Commission No. 5.3.1 .9 and are intended to encompass enzymes capable of converting D- glucose 6-phosphate (e.g., glucose 6-phosphate) into p-D-fructofuranose 6-phosphate (e.g., fructose 6-phosphate). The fusion polypeptides, which can be referred as PGI-PHK fusions in the present disclosure, comprises at least two moieties, each moiety having a distinct enzymatic activity. The fusion polypeptides of the present disclosure comprise at least one moiety exhibiting glucose-6-phosphate isomerase (PGI) activity. The fusion polypeptide of the present disclosure comprises at least one moiety exhibiting phosphoketolase (PHK) activity. The fusion polypeptides of the present disclosure exhibit both glucose-6-phosphate isomerase (PGI) activity and phosphoketolase (PHK) activity. The fusion polypeptides of the present disclosure can convert glucose-6-P into acetyl-P. The at least two moieties of the fusion polypeptides can be joined using one or more covalent bonds, just as, for example, one or more amine bonds. The at least two moieties of the fusion polypeptides can be joined directly to one another. Alternatively, the at least two moieties of the fusion polypeptides can be joined indirectly to one another by the presence of a linker. In some embodiments, the linker can comprise one or more amino acid residues. The fusion polypeptides can include, at their amino end, the PGI moiety. In such embodiment, the fusion polypeptide can include, at their carboxy end, the PHK moiety. Alternatively, the fusion polypeptide can include, at their amino end, the PHK moiety. In such embodiment, the fusion polypeptide can include, at their carboxy end, the PGI moiety. Embodiments of the fusion polypeptide are described in US provisional patent application 63 / 613,290 filed on December 21 , 2023 and herewith incorporated in its entirety.

[0051]

[0046] The engineered metabolic pathway to convert fructose-6-phosphate to acetylcoenzyme A can include, in some embodiments, an acetate kinase. As such, the recombinant yeast host cell of the present disclosure can include, in such embodiments, an acetate kinase. As used herein, the terms "acetate kinase" and "ACK" are intended to include the enzymes capable of converting acetate into acetyl-phosphate (acetyl-P). Acetate kinases include those enzymes that correspond to Enzyme Commission Number 2.72.1 . The ACK can be native or heterologous to the recombinant yeast host cell. In some embodiments, the ACK is of prokaryotic or eukaryotic origin. In other embodiments, the ACK can be encoded by a ack gene e.g., ACK). In some embodiments, the ACK is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 37, be a variant of the amino acid sequence of SEQ ID NO: 37 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 37 having ACK activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 38, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 37 (a variant thereof or a fragment thereof). In some embodiments, the ACK is of prokaryotic or eukaryotic origin. In other embodiments, the ACK can be encoded by a ack gene (e.g., ACK). In some embodiments, the ACK is derived from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 39, be a variant of the amino acid sequence of SEQ ID NO: 39 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 39 having ACK activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 40, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 39 (a variant thereof or a fragment thereof). In some embodiments, the ACK is of prokaryotic or eukaryotic origin. In other embodiments, the ACK can be encoded by a ack gene (e.g., ACK). In some embodiments, the ACK is derived from Oenococcus sp., and in further embodiments from Oenococcus oenii. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 41 , be a variant of the amino acid sequence of SEQ ID NO: 41 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 41 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 42, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 41 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 43, be a variant of the amino acid sequence of SEQ ID NO: 43 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 43 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 44, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 43 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 45, be a variant of the amino acid sequence of SEQ ID NO: 45 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 45 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 46, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 45 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 47, be a variant of the amino acid sequence of SEQ ID NO: 47 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 47 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 48, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 47 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 49, be a variant of the amino acid sequence of SEQ ID NO: 49 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 49 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 50, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 49 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Phytophthora sp., and in further embodiments from Phytophthora ramorum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 51 , be a variant of the amino acid sequence of SEQ ID NO: 51 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 51 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 52, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 51 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Chlamydomonas sp., and in further embodiments from Chlamydomonas reinhardtii. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 53, be a variant of the amino acid sequence of SEQ ID NO: 53 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 53 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 54, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 53 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Aspergillus sp., and in further embodiments from Aspergillus nidulans. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 55, be a variant of the amino acid sequence of SEQ ID NO: 55 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 55 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 56, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 55 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Lactiplantibacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 57, be a variant of the amino acid sequence of SEQ ID NO: 57 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 57 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 58, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 57 (a variant thereof or a fragment thereof). In some embodiments, the ACK is derived from Clostridium sp., and in further embodiments from Clostridium kluveryi. In such embodiments, the ACK can have the amino acid sequence of SEQ ID NO: 59, be a variant of the amino acid sequence of SEQ ID NO: 59 having ACK activity or be a fragment of the amino acid sequence of SEQ ID NO: 59 having ACK activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACK. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 60, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 59 (a variant thereof or a fragment thereof).

[0052]

[0047] The engineered metabolic pathway to convert fructose-6-phosphate to acetylcoenzyme A can include, in some embodiments, a phosphotransacetylase. As such, the recombinant yeast host cell of the present disclosure can include, in such embodiments, a phosphotransacetylase. As used herein, the terms "phosphotransacetylase" and "PTA" are intended to include the enzymes capable of converting acetyl-phosphate into acetyl-coA. Phosphotransacetylases include those enzymes that correspond to Enzyme Commission Number 2.3.1 .8. The PTA can be native or heterologous to the recombinant yeast host cell. In some embodiments, the PTA is of prokaryotic or eukaryotic origin. In other embodiments, the PTA can be encoded by a pta gene (e.g., PTA). In some embodiments, the PTA is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium adolescentis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 61 , be a variant of the amino acid sequence of SEQ ID NO: 61 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 61 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 62, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 61 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Leuconostoc sp., and in further embodiments from Leuconostoc mesenteroides. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 63, be a variant of the amino acid sequence of SEQ ID NO: 63 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 63 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 64, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 63 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Oenococcus sp., and in further embodiments from Oenococcus oenii. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 65, be a variant of the amino acid sequence of SEQ ID NO: 65 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 65 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 66, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 65 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 67, be a variant of the amino acid sequence of SEQ ID NO: 67 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 67 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 68, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 67 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Bacillus sp., and in further embodiments from Bacillus subtilis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 69, be a variant of the amino acid sequence of SEQ ID NO: 69 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 69 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 70, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 69 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Clostridium sp., and in further embodiments from Clostridium kluveryi. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 71 , be a variant of the amino acid sequence of SEQ ID NO: 71 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 71 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 72, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 71 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Phytophthora sp., and in further embodiments from Phytophthora ramorum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 73, be a variant of the amino acid sequence of SEQ ID NO: 73 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 73 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 74, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 73 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Globisporangium sp., and in further embodiments from Globisporangium splendens. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 75, be a variant of the amino acid sequence of SEQ ID NO: 75 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 75 having PTA activity. The recombinant yeast host cell can include, a nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 76, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 75 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium bifidum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 77, be a variant of the amino acid sequence of SEQ ID NO: 77 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 77 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 78, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 77 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Bifidobacterium sp., and in further embodiments from Bifidobacterium animalis. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 79, be a variant of the amino acid sequence of SEQ ID NO: 79 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 79 having PTA activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 80, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 79 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Clostridium sp., and in further embodiments from Clostridium phytofermentans. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 81 , be a variant of the amino acid sequence of SEQ ID NO: 81 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 81 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 82, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 81 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Holophagae sp., and in further embodiments from Holophagae bacterium. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 83, be a variant of the amino acid sequence of SEQ ID NO: 83 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 83 having PTA activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 84, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 83 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Aspergillus sp., and in further embodiments from Aztobacter vinelandii. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 85, be a variant of the amino acid sequence of SEQ ID NO: 85 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 85 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 86, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 85 (a variant thereof or a fragment thereof). In some embodiments, the PTA is derived from Lactobacillus sp., and in further embodiments from Lactiplantibacillus plantarum. In such embodiments, the PTA can have the amino acid sequence of SEQ ID NO: 87, be a variant of the amino acid sequence of SEQ ID NO: 87 having PTA activity or be a fragment of the amino acid sequence of SEQ ID NO: 87 having PTA activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the PTA. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 88, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 87 (a variant thereof or a fragment thereof). Additional sources of PTA that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, Phytophthora sp., Phytophthora cactorum, Phytophthora parasitica, Phytophthora idaei, Chlamydomonas sp. (Chlamydomonas reinhardtii), Clostridium sp. (Clostridium cellulolyticum), and Microcystis sp. (Microcystis aeruginosa).

[0053]

[0048] In specific embodiments, the recombinant yeast host cell of the present disclosure comprises a native and / or heterologous enzyme that function in an engineered metabolic pathway to convert acetate into acetyl-coA. The recombinant yeast host cell of the present disclosure can include, in such embodiments, an acetyl-coenzyme A synthetase. As used herein, the term "acetyl-coA synthetase" and "ACS" are intended to include the enzymes capable of converting acetate to acetyl-coA. Acetyl-coA synthetases include those enzymes that correspond to Enzyme Commission Number 6.2.1.1. The ACS can be native or heterologous to the recombinant yeast host cell. In some embodiments, the ACS is of prokaryotic or eukaryotic origin. In other embodiments, the ACS can be encoded by a acs1 gene (e.g., ACS1) or a acs2 gene (e.g., ACS2). In some embodiments, the ACS is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 89 or 91 , be a variant of the amino acid sequence of SEQ ID NO: 89 or 91 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 89 or 91 having ACS activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 90 or 92 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 90 or 92 (a variant thereof or a fragment thereof). In some embodiments, the ACS is derived from Zygosaccharomyces sp., and in further embodiments from Zygosaccharomyces bailii. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 93, be a variant of the amino acid sequence of SEQ ID NO: 93 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 93 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 94 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 93 (a variant thereof or a fragment thereof). In some embodiments, the ACS is derived from Salmonella sp., and in further embodiments from Salmonella enterica. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 95, be a variant of the amino acid sequence of SEQ ID NO: 95 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 95 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 96 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 95 (a variant thereof or a fragment thereof). In some embodiments, the ACS is derived from Acetobacter sp., and in further embodiments from Acetobacter aceti. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 97, be a variant of the amino acid sequence of SEQ ID NO: 97 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 97 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 98 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 97 (a variant thereof or a fragment thereof). In some embodiments, the ACS is derived from Escherichia sp., and in further embodiments from Escherichia coll. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 99, be a variant of the amino acid sequence of SEQ ID NO: 99 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 99 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 100 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 99 (a variant thereof or a fragment thereof). Additional sources of ACS that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, Aedes togoi, Aliivibrio fischeri, Amaranthus sp., Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus nidulans, Aspergillus niger, Bacillus subtilis, Bos taurus, Bradyrhizobium japonicum, Cereibacter sphaeroides, Cryptosporidium parvum, Dunaliella tertiolecta, Euglena gracilis, Haloarcula marismortui, Homo sapiens, Hordeum vulgare, Ignicoccus hospitalis, Marmota monax, Methanosarcina acetivorans, Methanosarcina sp., Methanothermobacter thermautotrophicus, Methanothrix soehngenii, Methanothrix thermoacetophila (Uniprot A0B8F1 for exemple), Moorella thermoacetica, Mus musculus, Mus musculus, Oryctolagus cuniculus, Ovis aries, Pelotomaculum thermopropionicum, Penicillium chrysogenum, Phycomyces blakesleeanus, Pinusradiata, Pisum sativum, Populus trichocarpa, Pseudomonas putida (Uniprot Q6EMJ3 for example), Pyrobaculum aerophilum, Pyrococcus furiosus, Rattus norvegicus, Rhodotorula diobovata, Roseovarius sp., Saccharopolyspora erythraea, Spinacia oleracea, Streptomyces lividans, Taxus sp., and Zea mays.

[0054]

[0049] In specific embodiments, the recombinant yeast host cell of the present disclosure comprises a native or heterologous enzyme that function in an engineered metabolic pathway to convert acetate into acetyl-coA. In such embodiments, the native or heterologous enzyme comprises an activated, upregulated or overexpressed acetyl-coA synthetase 2 (ACS2). In some embodiments, the ACS2 is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the ACS can have the amino acid sequence of SEQ ID NO: 89, be a variant of the amino acid sequence of SEQ ID NO: 89 having ACS activity or be a fragment of the amino acid sequence of SEQ ID NO: 89 having ACS activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ACS. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 90 or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 89 (a variant thereof or a fragment thereof). Additional sources of ACS2 that can be included in the recombinant yeast host cell of the present disclosure include, without limitation, Aedes togoi, Aliivibrio fischeri, Amaranthus sp., Arabidopsis thaliana, Archaeoglobus fulgidus, Aspergillus nidulans, Aspergillus niger, Bacillus subtilis, Bos taurus, Bradyrhizobium japonicum, Cereibacter sphaeroides, Cryptosporidium parvum, Dunaliella tertiolecta, Escherichia coll, Euglena gracilis, Haloarcula marismortui, Homo sapiens, Hordeum vulgare, Ignicoccus hospitalis, Marmota monax, Methanosarcina acetivorans, Methanosarcina sp., Methanothermobacter thermautotrophicus, Methanothrix soehngenii, Methanothrix thermoacetophila (Uniprot A0B8F1 for exemple), Moorella thermoacetica, Mus musculus, Mus musculus, Oryctolagus cuniculus, Ovis aries, Pelotomaculum thermopropionicum, Penicillium chrysogenum, Phycomyces blakesleeanus, Pinus radiata, Pisum sativum, Populus trichocarpa, Pseudomonas putida (Uniprot Q6EMJ3 for example), Pyrobaculum aerophilum, Pyrococcus furiosus, Rattus norvegicus, Rhodotorula diobovata, Roseovarius sp., Saccharopolyspora erythraea, Spinacia oleracea, Streptomyces lividans, Taxus sp., and Zea mays.

[0055]

[0050] In some embodiments, the recombinant yeast host cell can include a native and / or heterologous enzyme that functions in an engineered metabolic pathway to convert acetylcoenzyme A to acetone. In such embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone is activated, upregulated, or overexpressed. In one embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprise at least a thiolase, a coenzyme A transferase, and an acetoacetate decarboxylase. In such embodiment, the engineered pathway also convert acetate along with acetylcoenzyme A. In another embodiment, the engineered metabolic pathway to convert acetylcoenzyme A to acetone comprise at least a thiolase, a HMG CoA synthase, a HMG CoA Lyase, and an acetoacetate decarboxylase. In another embodiment, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprise at least a thiolase, acetoacetyl- CoA hydrolase, and an acetoacetate decarboxylase.

[0056]

[0051] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate to acetone comprises a thiolase. As used herein, the terms "thiolase", “THL”, “ERG10” and "PHAA" are intended to include the enzymes capable of converting acetyl-coA into acetoacetyl-coA. Thiolases include enzymes that correspond to Enzyme Commission Number2.3.1 .9. The thiolase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native thiolase and optionally in combination a heterologous thiolase. In some embodiments, the thiolase is of prokaryotic or eukaryotic origin. In other embodiments, the thiolase can be encoded by a thl gene (e.g., THL), a erg10 gene (e.g., ERG10), or a phaA gene (e.g., PHAA). In some embodiments, the thiolase is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 101 , be a variant of the amino acid sequence of SEQ ID NO: 101 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 101 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 102, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 101 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 103, be a variant of the amino acid sequence of SEQ ID NO: 103 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 103 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 104, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 103 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is from Clostridium kluyveri. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 105, be a variant of the amino acid sequence of SEQ ID NO: 105 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 105 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 106, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 105 (a variant thereof or a fragment thereof).. In some embodiments, the thiolase is derived from Cupriavidus sp., and in further embodiments from Cupriavidus necator. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 107, be a variant of the amino acid sequence of SEQ ID NO: 107 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 107 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 108, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 107 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Yarrowia sp., and in further embodiments from Yarrowia lipolytica. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 109, be a variant of the amino acid sequence of SEQ ID NO: 109 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 109 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 110, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 109 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Thermoanaerobacterium sp., and in further embodiments from Thermoanaerobacterium thermosaccharolyticum. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 11 1 , be a variant of the amino acid sequence of SEQ ID NO: 111 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 111 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 112, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 11 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Saccoglossus sp., and in further embodiments from Saccoglossus kowalevskii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 1 13, be a variant of the amino acid sequence of SEQ ID NO: 113 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 1 13 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 114, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 113 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Strongylocentrotus sp., and in further embodiments from Strongylocentrotus purpuratus. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 1 15, be a variant of the amino acid sequence of SEQ ID NO: 115 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 1 15 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 116, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 115 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 117, be a variant of the amino acid sequence of SEQ ID NO: 117 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 117 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 1 18, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 17 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Zygosaccharomyces sp., and in further embodiments from Zygosaccharomyces bailii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 1 19, be a variant of the amino acid sequence of SEQ ID NO: 119 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 1 19 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 120, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 119 (a variant thereof or a fragment thereof). In some embodiments, the thiolase is derived from Clostridium sp., and in further embodiments from Clostridium beijerinckii. In such embodiments, the thiolase can have the amino acid sequence of SEQ ID NO: 121 , be a variant of the amino acid sequence of SEQ ID NO: 121 having thiolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 121 having thiolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the thiolase. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 122, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 121 (a variant thereof or a fragment thereof). Additional sources of the thiolase that can be included in the recombinant yeast host cell include, without limitation, Arabidopsis thaliana (Uniprot Q8S4Y1 or Q9FIK7, for example), Aspergillus fumigatus (Uniprot B0XMC1 , for example), Bacillus subtilis, Bacopa monnieri (Uniprot D9U856, for example), Bos taurus, Bradyrhizobium japonicum, Candida tropicalis, Catharanthus roseus, Caulobacter vibrioides, Clonorchis sinensis (Uniprot G7YHN5, for example), Dictyostelium discoideum (Uniprot Q86AD9, for example), Enterococcus faecalis, Escherichia coli (Uniprot P76461 , for example), Euphorbia helioscopia (Uniprot A0A0M4F9H9, for example), Gallus gallus (Uniprot F1 NT20, for example), Ginkgo biloba (Uniprot A0A1S6KJS1 , for example), Halobacterium sp., Haloferax mediterranei (Uniprot I3R3D1 , I3R3D0, 13RA72, or I3RA71 , for example), Helianthus annuus (Uniprot D2IH11 , for example), Homo sapiens (Uniprot Q9BWD1 , for example), Medicago sativa (Uniprot D0EUY6, for example), Metallosphaera sedula (Uniprot A4YEH9, for example), Methanothermococcus thermolithotrophicus (Uniprot A0A384E138, for example), Mycolicibacterium smegmatis, Ostrinia scapulalis (Uniprot B7XEI5, for example), Pyricularia oryzae, Pyrobaculum neutrophilum (Uniprot B1YB71 , for example), Rattus norvegicus, Rhizobium sp., Sanghuangporus baumii, Thermus thermophilus, Vitis vinifera x Vitis riparia, and Zoogloea ramigera (Uniprot P07256 or P07097, for example).

[0057]

[0052] In some embodiments, the engineered metabolic pathway to convert acetylcoenzyme A and acetate to acetone comprises a coenzyme A transferase. As used herein, the terms "coenzyme A transferase", “coA transferase”, “CFTA / CTFB”, and “ATOA / ATOD” are intended to include the enzymes (or enzyme moieties) capable of converting acetoacetyl-coA and acetate into acetyl-coA and acetoacetate. Coenzyme A transferases include enzymes that correspond to Enzyme Commission Number 2.8.3.8. The coA transferase can be a monomer or a dimer (as for example CTFA / CTFB). The coA transferase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native coA transferase and optionally in combination a heterologous coA transferase. In some embodiments, the coA transferase is of prokaryotic or eukaryotic origin. In other embodiments, the coA transferase can be encoded by the ctfa and ctfb genes (e.g., CTFA / CTFB), or by the atoA and atoD genes (e.g., ATOA / ATOD). In some embodiments, the coA transferase is derived from Alkaliphilus sp., and in further embodiments from Alkaliphilus metalliredigens. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 123, being a variant of the amino acid sequence of SEQ ID NO: 123 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 123 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 124, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 123 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 125, being a variant of the amino acid sequence of SEQ ID NO: 125 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 125 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 126, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 125 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 242, being a variant of the amino acid sequence of SEQ ID NO: 242 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 242 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 243, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 242 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 244, being a variant of the amino acid sequence of SEQ ID NO: 244 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 244 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 245, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 244 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Thermosipho sp., and in further embodiments from Thermosipho melanesiensis. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 246, being a variant of the amino acid sequence of SEQ ID NO: 246 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 246 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 247, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 246 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 248, being a variant of the amino acid sequence of SEQ ID NO: 248 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 248 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 249, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 248 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 127, being a variant of the amino acid sequence of SEQ ID NO: 127 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 127 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 128, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 127 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 129, being a variant of the amino acid sequence of SEQ ID NO: 129 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 129 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 130, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 129 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 131 , being a variant of the amino acid sequence of SEQ ID NO: 131 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 131 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 132, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 131 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 133, being a variant of the amino acid sequence of SEQ ID NO: 133 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 133 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 134, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 133 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium beijerinckii. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 135, being a variant of the amino acid sequence of SEQ ID NO: 135 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 135 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 136, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 135 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 137, being a variant of the amino acid sequence of SEQ ID NO: 137 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 137 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 138, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 137 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Clostridium saccharoperbutylacetonicum. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 250, being a variant of the amino acid sequence of SEQ ID NO: 250 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 250 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 251 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 250 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 252, being a variant of the amino acid sequence of SEQ ID NO: 252 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 252 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 253, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 252 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium sticklandii. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 139, being a variant of the amino acid sequence of SEQ ID NO: 139 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 139 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 140, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 139 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 141 , being a variant of the amino acid sequence of SEQ ID NO: 141 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 141 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 142, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 141 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Brevibacillus sp., and in further embodiments from Brevibacillus laterosporus. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 254, being a variant of the amino acid sequence of SEQ ID NO: 254 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 254 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 255, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 254 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 256, being a variant of the amino acid sequence of SEQ ID NO: 256 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 256 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 257, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 256 (a variant thereof or a fragment thereof). In some embodiments, the coA transferase is derived from Clostridium sp., and in further embodiments from Clostridium bovifaecis. In such embodiments, the coA transferase can include a first polypeptide (CTFA) having the amino acid sequence of SEQ ID NO: 143, being a variant of the amino acid sequence of SEQ ID NO: 143 having coA transferase activity (in the presence of CFTB) or being a fragment of the amino acid sequence of SEQ ID NO: 143 having coA transferase activity (in the presence of CFTB). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFA. In such embodiment, the heterologous nucleic acid molecule encoding this first polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 144, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 143 (a variant thereof or a fragment thereof). Alternatively or in combination, the coA transferase can include a second polypeptide (CTFB) having the amino acid sequence of SEQ ID NO: 145, being a variant of the amino acid sequence of SEQ ID NO: 145 having coA transferase activity (in the presence of CFTA) or being a fragment of the amino acid sequence of SEQ ID NO: 145 having coA transferase activity (in the presence of CFTA). The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the CTFB. In such embodiment, the heterologous nucleic acid molecule encoding this second polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 146, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 145 (a variant thereof or a fragment thereof). Additional sources of coA transferase that can be included in the recombinant yeast host cell include, without limitation, Acetobacter aceti (Uniprot B3EY95, for example), Anaerobutyricum hallii (Uniprot Q2QIH1 or D2WEY8, for example), Anaerostipes caccae (Uniprot Q2QB27 or B0MC58, for example), Butyricicoccus porcorum, Butyrivibrio fibrisolvens (Uniprot D2WEY7, for example), Coprococcus sp., Faecalibacterium prausnitzii (Uniprot Q2QIH0, A8SFP6, C7H5K4, or D2WEZ2, for example), Megasphaera elsdenii, Roseburia hominis (Uniprot Q2TME9, for example), Roseburia intestinalis (Uniprot C7GB37, for example), Roseburia inulinivorans (Uniprot D2WEY6, for example), Thermoanaerobacterium saccharolyticum, Trypanosoma brucei, Eubacterium nodatum, and Eubacterium rectale (Uniprot D2WEY1 , for example).

[0058]

[0053] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A and acetate to acetone comprises an acetoacetate decarboxylase. As used herein, the terms “acetoacetate decarboxylase”, and “ADC” are intended to include the enzymes capable of converting acetoacetate to acetone and carbon dioxide. Acetoacetate decarboxylases include enzymes that correspond to Enzyme Commission Number 4.1.1.4. The acetoacetate decarboxylase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native acetoacetate decarboxylase and optionally in combination a heterologous acetoacetate decarboxylase. In some embodiments, acetoacetate decarboxylase is of prokaryotic or eukaryotic origin. In other embodiments, the acetoacetate decarboxylase can be encoded by an adc gene (e.g., ADC). In some embodiments, the acetoacetate decarboxylase is derived from Paenibacillus sp., and in further embodiments from Paenibacillus polymyxa. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 147, be a variant of the amino acid sequence of SEQ ID NO: 147 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 147 having ADC activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 148, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 147 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Clostridium sp., and in further embodiments from Clostridium acetobutylicum. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 149, be a variant of the amino acid sequence of SEQ ID NO: 149 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 149 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 150, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 149 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Clostridium beijerinckii. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 151 , be a variant of the amino acid sequence of SEQ ID NO: 151 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 151 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 152, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 151 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Bacillus sp., and in further embodiments from Bacillus amyloliquefaciens. In such embodiments acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 258, be a variant of the amino acid sequence of SEQ ID NO: 258 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 258 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 259, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 258 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Rhizobium sp., and in further embodiments from Rhizobium leguminosarum. In such embodiment, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 153, be a variant of the amino acid sequence of SEQ ID NO: 153 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 153 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 154, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 153 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Bradyrhizobium sp., and in further embodiments from Bradyrhizobium japonicum. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 155, be a variant of the amino acid sequence of SEQ ID NO: 155 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 155 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 156, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 155 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Tetrahymena sp., and in further embodiments from Tetrahymena thermophila. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 260, be a variant of the amino acid sequence of SEQ ID NO: 260 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 260 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 261 , or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 260 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Aspergillus sp., and in further embodiments from Aspergillus niger. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 157, be a variant of the amino acid sequence of SEQ ID NO: 157 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 157 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 158, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 157 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus casei. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 159, be a variant of the amino acid sequence of SEQ ID NO: 159 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 159 having ADC activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 160, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 159 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus rhamnosus. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 161 , be a variant of the amino acid sequence of SEQ ID NO: 161 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 161 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 162, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 161 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetate decarboxylase is derived from Aspergillus sp., and in further embodiments from Aspergillus bertholletiae. In such embodiments, the acetoacetate decarboxylase can include a polypeptide having the amino acid sequence of SEQ ID NO: 163, be a variant of the amino acid sequence of SEQ ID NO: 163 having ADC activity or be a fragment of the amino acid sequence of SEQ ID NO: 163 having ADC activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the ADC. In such embodiment, the heterologous nucleic acid molecule can comprise the nucleic acid sequence of SEQ ID NO: 164, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 163 (a variant thereof or a fragment thereof). Additional sources of ADC that can be included in the recombinant yeast host cell include, without limitation, Chromobacterium violaceum (Uniprot Q7NSA6, for example), Pseudomonas putida, and Ruminiclostridium cellulolyticum.

[0059]

[0054] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprises an HMG CoA Synthase (HGMS). As used herein, the terms “HMG CoA Synthase”, and “HMGS” are intended to include the enzymes capable of converting acetoacetyl-CoA and acetyl-coA into (S)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). HMG CoA Synthase include enzymes that correspond to Enzyme Commission Number 2.3.3.10. The HMG CoA Synthase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native HMG CoA Synthase and optionally in combination a heterologous HMG CoA Synthase. In some embodiments, HMG CoA Synthase is of prokaryotic or eukaryotic origin. In other embodiments, the HMG CoA Synthase can be encoded by an hgms gene. In some embodiments, the HMG CoA Synthase is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 165, be a variant of the amino acid sequence of SEQ ID NO: 165 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 165 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 166, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 165 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Synthase is derived from Lacticaseibacillus sp., and in further embodiments from Lacticaseibacillus casei UW4. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 167, be a variant of the amino acid sequence of SEQ ID NO: 167 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 167 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 168, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 167 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Synthase is derived from Enterococcus sp., and in further embodiments from Enterococcus faecalis. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 169, be a variant of the amino acid sequence of SEQ ID NO: 169 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 169 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 170, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 169 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Synthase is derived from Haloferax sp., and in further embodiments from Haloferax volcanii DS2. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 171 , be a variant of the amino acid sequence of SEQ ID NO: 171 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 171 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 172, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 171 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Synthase is derived from Alloscardovia sp., and in further embodiments from Alloscardovia theropitheci. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 173, be a variant of the amino acid sequence of SEQ ID NO: 173 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 173 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 174, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 173 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Synthase is derived from Listeria sp., and in further embodiments from Listeria monocytogenes. In such embodiments, the HMG CoA Synthase can include a polypeptide having the amino acid sequence of SEQ ID NO: 175 be a variant of the amino acid sequence of SEQ ID NO: 175 having HMGS activity or be a fragment of the amino acid sequence of SEQ ID NO: 175 having HMGS activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 176, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 175 (a variant thereof or a fragment thereof).

[0055] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprises an HMG CoA Lyase (HMGCL). As used herein, the terms “HMG CoA Lyase”, and “HMGCL” are intended to include the enzymes capable of converting HMG-CoA to acetyl-CoA and acetoacetate. HMG CoA Lyase includes enzymes that correspond to Enzyme Commission Number 4.1 .3.4. The HMG CoA Lyase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native HMG CoA Lyase and optionally in combination a heterologous HMG CoA Lyase. In some embodiments, HMG CoA Lyase is of prokaryotic or eukaryotic origin. In other embodiments, the HMG CoA Lyase can be encoded by an hmgcl gene. In some embodiments, the HMG CoA Lyase is derived from Danio sp., and in further embodiments from Danio rerio. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 177, be a variant of the amino acid sequence of SEQ ID NO: 177 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 177 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 178, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 177 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas monteilii. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 179, be a variant of the amino acid sequence of SEQ ID NO: 179 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 179 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 180, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 179 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas wayambapalatensis. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 181 , be a variant of the amino acid sequence of SEQ ID NO: 181 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 181 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 182, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 181 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Azotobacter sp., and in further embodiments from Azotobacter vinelandii. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 183, be a variant of the amino acid sequence of SEQ ID NO: 183 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 183 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 184, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 183 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas citronellolis. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 185, be a variant of the amino acid sequence of SEQ ID NO: 185 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 185 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 186, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 185 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas cremoris. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 187, be a variant of the amino acid sequence of SEQ ID NO: 187 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 187 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 188, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 187 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas chengduensis. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 189, be a variant of the amino acid sequence of SEQ ID NO: 189 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 189 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 190, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 189 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas aeruginosa PAO1. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 191 , be a variant of the amino acid sequence of SEQ ID NO: 191 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 191 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 192, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 191 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Bacillus sp., and in further embodiments from Bacillus subtilis 168. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 193, be a variant of the amino acid sequence of SEQ ID NO: 193 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 193 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 194, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 193 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Desulfotomaculum sp., and in further embodiments from Desulfotomaculum arcticum DSM 17038. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 195, be a variant of the amino acid sequence of SEQ ID NO: 195 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 195 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 196, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 195 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Desulfoscipio sp., and in further embodiments from Desulfoscipio geothermicus DSM 3669. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 197, be a variant of the amino acid sequence of SEQ ID NO: 197 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 197having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 198, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 197 (a variant thereof or a fragment thereof) In some embodiments, the HMG CoA Lyase is derived from Acinetobacter sp., and in further embodiments from Acinetobacter baumannii. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 199, be a variant of the amino acid sequence of SEQ ID NO: 199 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 199 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 200, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 199 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Acinetobacter sp., and in further embodiments from Acinetobacter Iwoffii. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 201 , be a variant of the amino acid sequence of SEQ ID NO: 201 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 201 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 202, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 201 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Moraxella sp., and in further embodiments from Moraxella caviae. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 203, be a variant of the amino acid sequence of SEQ ID NO: 203 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 203 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 204, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 203 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Alcaligenaceae sp., and in further embodiments from Alcaligenaceae bacterium 429. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 205, be a variant of the amino acid sequence of SEQ ID NO: 205 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 205 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 206, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 205 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Macaca sp., and in further embodiments from Macaca fascicularis. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 207, be a variant of the amino acid sequence of SEQ ID NO: 207 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 207 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 208, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 207 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Arabidopsis sp., and in further embodiments from Arabidopsis thaliana. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 209, be a variant of the amino acid sequence of SEQ ID NO: 209 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 209 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 210, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 209 (a variant thereof or a fragment thereof). In some embodiments, the HMG CoA Lyase is derived from Gallus sp., and in further embodiments from Gallus gallus. In such embodiments, the HMG CoA Lyase can include a polypeptide having the amino acid sequence of SEQ ID NO: 211 , be a variant of the amino acid sequence of SEQ ID NO: 211 having hmgcl activity or be a fragment of the amino acid sequence of SEQ ID NO: 21 1 having hmgcl activity. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise the nucleic acid sequence of SEQ ID NO: 212, or a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 1 (a variant thereof or a fragment thereof).

[0060]

[0056] In some embodiments, the engineered metabolic pathway to convert acetyl-coenzyme A to acetone comprises an acetoacetyl-CoA hydrolase. As used herein, the terms “acetoacetyl- CoA hydrolase”, “TESB”, “YCIA”, “FADM1 ”, “FADM2” and “FADM” are intended to include the enzymes capable of converting acetoacetyl-CoA to acetyl-coA and acetoacetate. Acetoacetyl- CoA hydrolase includes enzymes that correspond to Enzyme Commission Number EC 3.1.2.11. The acetoacetyl-CoA hydrolase can be native or heterologous to the recombinant yeast host cell. The recombinant yeast host cell can include a native acetoacetyl-CoA hydrolase and optionally in combination a heterologous acetoacetyl-CoA hydrolase. In some embodiments, acetoacetyl-CoA hydrolase is of prokaryotic or eukaryotic origin. In other embodiments, the acetoacetyl-CoA hydrolase can be encoded by an tesB”, “yciA”, “fadM1”, “fadM2” or “fadM gene. In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (TESB) can include a polypeptide having the amino acid sequence of SEQ ID NO: 213, be a variant of the amino acid sequence of SEQ ID NO: 213 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 213 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 213 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (YCIA) can include a polypeptide having the amino acid sequence of SEQ ID NO: 214, be a variant of the amino acid sequence of SEQ ID NO: 214 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 214 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include a heterologous nucleic acid molecule encoding the acetoacetyl- CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 214 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (YIGI) can include a polypeptide having the amino acid sequence of SEQ ID NO: 215, be a variant of the amino acid sequence of SEQ ID NO: 215 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 215 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 215 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Escherichia sp., and in further embodiments from Escherichia coli. In such embodiments, the acetoacetyl-CoA hydrolase (Ydil) can include a polypeptide having the amino acid sequence of SEQ ID NO: 216, be a variant of the amino acid sequence of SEQ ID NO: 216 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 216 having acetoacetyl- CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 216 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Methylorubrum sp., and in further embodiments from Methylorubrum extorquens AM1. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 217, be a variant of the amino acid sequence of SEQ ID NO: 217 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 217 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 217 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas aeruginosa. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 218, be a variant of the amino acid sequence of SEQ ID NO: 218 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 218 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 218 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Campylobacter sp., and in further embodiments from Campylobacter jejuni. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 219, be a variant of the amino acid sequence of SEQ ID NO: 219 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 219 having acetoacetyl- CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 219 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Mycobacterium sp., and in further embodiments from Mycobacterium tuberculosis. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 220, be a variant of the amino acid sequence of SEQ ID NO: 220 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 220 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 220 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Pseudomonas sp., and in further embodiments from Pseudomonas putida. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 221 , be a variant of the amino acid sequence of SEQ ID NO: 221 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 221 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl- CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 221 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Fibrobacter sp., and in further embodiments from Fibrobacter succinogenes. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 222, be a variant of the amino acid sequence of SEQ ID NO: 222 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 222 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 222 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Alcanivorax sp., and in further embodiments from Alcanivorax borkumensis SK2. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 223, be a variant of the amino acid sequence of SEQ ID NO: 223 having acetoacetyl- CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 223 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 223 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Haemophilus sp., and in further embodiments from Haemophilus influenzae. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 224, be a variant of the amino acid sequence of SEQ ID NO: 224 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 224 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 224 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis subsp. mobilis ZM4. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 225, be a variant of the amino acid sequence of SEQ ID NO: 225 having acetoacetyl- CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 225 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 225 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Campylobacter sp., and in further embodiments from Campylobacter jejuni. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 226, be a variant of the amino acid sequence of SEQ ID NO: 226 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 226 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl- CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 226 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Prevotella sp., and in further embodiments from Prevotella ruminicola 23. In such embodiments, the acetoacetyl-CoA hydrolase (FadM1) can include a polypeptide having the amino acid sequence of SEQ ID NO: 227, be a variant of the amino acid sequence of SEQ ID NO: 227 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 227 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 227 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Prevotella sp., and in further embodiments from Prevotella ruminicola 23. In such embodiments, the acetoacetyl- CoA hydrolase (FadM2) can include a polypeptide having the amino acid sequence of SEQ ID NO: 228, be a variant of the amino acid sequence of SEQ ID NO: 228 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 228 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 228 (a variant thereof or a fragment thereof). In some embodiments, the acetoacetyl-CoA hydrolase is derived from Providencia sp., and in further embodiments from Providencia sneebia DSM 19967. In such embodiments, the acetoacetyl-CoA hydrolase can include a polypeptide having the amino acid sequence of SEQ ID NO: 229, be a variant of the amino acid sequence of SEQ ID NO: 229 having acetoacetyl-CoA hydrolase activity or be a fragment of the amino acid sequence of SEQ ID NO: 229 having acetoacetyl-CoA hydrolase activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the acetoacetyl-CoA hydrolase. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 229 (a variant thereof or a fragment thereof).

[0057] The recombinant yeast host cell of the present disclosure has b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol. The term "downregulated" means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in a native host organism. As used in the context of the present disclosure, the downregulation of the native adh gene refers to a genetic modification which limits or impedes the expression of the native adh gene, when compared to a corresponding yeast strain which does not bear such genetic modification. In some instances, the additional genetic modification reduces but still allows the expression of the native adh gene and / or the expression of the native ADH. In other instances, the genetic modification inhibits the expression of the native adh gene and / or the expression of the native ADH. The downregulation could be done by the known methods in the art, as for example by deletion of the native adh gene, inactivation of the adh gene by insertion or deletion of nucleic acids residues, replacement of the native promoter by a heterologous promoter less active or that does not activate under propagation and / or fermentation conditions, or by modulation of a gene regulating the expression of the native adh gene. In one embodiment, the genetic modification for downregulated the native adh gene is a deletion of the native adh gene. In one embodiment the native ADH is the native ADH that is mainly responsible for the conversion of the acetone to isopropanol by the yeast host cell. In another embodiment, the native ADH is ADH1 (encoded by the adh1 gene). In another embodiment, the native ADH1 is encoded by the native adh1 gene from S. cerevisiae. In such embodiment, the native ADH1 is a polypeptide having an amino acid sequence of SEQ ID NO: 239, is a variant of the polypeptide having an amino acid sequence of SEQ ID NO: 239 having ADH1 activity or is a fragment polypeptide having an amino acid sequence of SEQ ID NO: 239 having ADH1 activity.

[0061]

[0058] The recombinant yeast host cell of the present disclosure has c) a heterologous alcohol dehydrogenase. The heterologous alcohol dehydrogenase has a decreased activity in the conversion of the acetone to isopropanol when compared to the native ADH intended for downregulation in the yeast host cell. In one embodiment, the heterologous alcohol dehydrogenase has a secondary alcohol activity inferior to the secondary alcohol activity of the native ADH intended for the downregulation in the yeast host cell. In another embodiment, the heterologous ADH allows the recombinant yeast host cell comprising a) an engineered metabolic pathway to convert acetyl-CoA and acetate to acetone and b) a downregulated ADH to produce a yield of isopropanol inferior to 0.5 g / L in a YPD media containing 120 g / L glucose at pH 6.0 following 48 hours of fermentation. In one embodiment, the heterologous ADH is a bacterial ADH. In other embodiments, the heterologous ADH can be encoded by an adha, adhp, oradh4, gene. In a further embodiment, the heterologous ADH is not a S. cerevisiae ADH1. In some embodiments, the heterologous ADH is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis. In such embodiments, the heterologous ADH (ADHA) can include a polypeptide having the amino acid sequence of SEQ ID NO: 238, be a variant of the amino acid sequence of SEQ ID NO: 238 having ADH activity or be a fragment of the amino acid sequence of SEQ ID NO: 238 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 238 (a variant thereof or a fragment thereof). In some embodiments, the heterologous ADH is derived from Zymomonas sp., and in further embodiments from Zymomonas mobilis. In such embodiments, the heterologous ADH (ADHP) can include a polypeptide having the amino acid sequence of SEQ ID NO: 241 , be a variant of the amino acid sequence of SEQ ID NO: 241 having ADH activity or be a fragment of the amino acid sequence of SEQ ID NO: 241 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 241 (a variant thereof or a fragment thereof). In some embodiments, the heterologous ADH is derived from Saccharomyces sp., and in further embodiments from Saccharomyces cerevisiae. In such embodiments, the heterologous ADH (ADH4) can include a polypeptide having the amino acid sequence of SEQ ID NO: 240, be a variant of the amino acid sequence of SEQ ID NO: 240 having ADH activity or be a fragment of the amino acid sequence of SEQ ID NO: 240 having ADH activity. The recombinant yeast host cell can include, a heterologous nucleic acid molecule encoding the heterologous ADH. In such embodiment, the heterologous nucleic acid molecule encoding this polypeptide can comprise a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 240 (a variant thereof or a fragment thereof).

[0062]

[0059] In yet additional embodiments, the recombinant yeast host cell of the present disclosure comprises a native and / or heterologous enzyme that functions in the engineered non-oxidative pentose phosphate pathway. In such embodiment the recombinant yeast host cell of the present disclosure comprises at least one of: a transaldolase, a transketolase, an epimerase, or an isomerase. In some embodiments, the transaldolase: has the ability to convert glyceraldehyde 3-phosphate into erythrose 4-phosphate; has the ability to convert sedoheptulose 7-phosphate into fructose 6-phosphate; is heterologous; is of prokaryotic or eukaryotic origin; is a TAL1 polypeptide; is encoded by a tall gene; is derived from Saccharomyces sp.; is derived from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 264 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 264 (a variant thereof or a fragment thereof). In some embodiments, the transketolase: has the ability to convert ribose 5-phosphate into glyceraldehyde 3-phosphate; has the ability to convert fructose 6-phosphate into xylulose 5- phosphate; has the ability to convert xylulose 5-phoshate into sedoheptulose 7-phosphate; has the ability to convert glyceraldehyde 3-phosphate into erythrose 4-phosphate; is heterologous; is of prokaryotic or eukaryotic origin; is a TKL1 polypeptide; is encoded by a tkl1 gene; is derived from Saccharomyces sp.; is derived from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 263 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 263 (a variant thereof or a fragment thereof). In some embodiments, the epimerase: has the ability to convert ribulose 5-phosphate into xylulose 5-phosphate; has the ability to convert xylulose 5-phosphate into ribulose 5- phosphate; is heterologous; is of prokaryotic or eukaryotic origin; is a RPE1 polypeptide; is encoded by a rpe1 gene; is derived from Saccharomyces sp.; is derived from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 266 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 266 (a variantthereof or a fragment thereof). In some embodiments, the isomerase: has the ability to convert ribulose 5-phosphate into ribose 5-phosphate; has the ability to convert ribose 5-phosphate into ribulose 5-phosphate; is heterologous; is of prokaryotic or eukaryotic origin; is a RKI1 polypeptide; is encoded by a rki1 gene; is derived from Saccharomyces sp.; is derived from Saccharomyces cere visiae; has the amino acid sequence of SEQ ID NO: 265 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 265 (a variant thereof or a fragment thereof). In further embodiments, the native and / or heterologous enzyme that functions in the engineered non-oxidative pentose phosphate pathway comprises at least two of the transaldolase, the transketolase, the epimerase, or the isomerase. In additional embodiments, the native and / or heterologous enzyme that functions in an engineered non-oxidative pentose phosphate pathway comprises at least three of the transaldolase, the transketolase, the epimerase, or the isomerase. In yet further embodiments, the native and / or heterologous enzyme that functions in an engineered non-oxidative pentose phosphate pathway comprises the transaldolase, the transketolase, the epimerase, and the isomerase.

[0063]

[0060] In yet additional embodiments, the recombinant yeast host cell of the present disclosure comprises a native and / or heterologous protein that functions in an engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A. In such embodiment the recombinant yeast host cell of the present disclosure comprises at least one of FEN2 or CABI . In some embodiments, the FEN2: is a plasma membrane proton-pantothenate symporter; is heterologous; is of prokaryotic or eukaryotic origin is encoded by a fen2 gene;is derived from Saccharomyces sp.; is derived from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 267 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 267 (a variant thereof or a fragment thereof). In some embodiments, the CAB1 : has pantothenate kinase activity; is heterologous; is of prokaryotic or eukaryotic origin; is encoded by a cab1 gene; is derived from Saccharomyces sp.; is derived from Saccharomyces cerevisiae; has the amino acid sequence of SEQ ID NO: 268 (a variant thereof or a fragment thereof); and / or is encoded by a heterologous nucleic acid molecule comprising a degenerate nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 268 (a variant thereof or a fragment thereof). In yet additional embodiments, the native and / or heterologous protein that functions in the engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A comprises FEN2. In yet further embodiments, the native and / or heterologous protein that functions in the engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A comprises CAB1. In still additional embodiments, the native and / or heterologous protein that functions in the engineered metabolic pathway to convert pantothenate into acetyl-coenzyme A comprises FEN2 and CAB1 .

[0064] Process of using the recombinant yeast host cell

[0065]

[0061] The present disclosure provides a process for increasing acetone production by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol and (c) a heterologous alcohol dehydrogenase gene. In one embodiment, the process of the present disclosure provides a decrease in isopropanol production by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol, and (c) a heterologous alcohol dehydrogenase gene. In a further embodiment, the process of the present disclosure provides a production of at least 80% w / v ethanol yield by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered metabolic pathway to convert acetyl-CoA and acetate to acetone but lacking (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol and (c) a heterologous alcohol dehydrogenase gene. In a further embodiment, the process of the present disclosure provides an increased production of ethanol by the recombinant yeast host cell when compared to a control yeast host cell comprising at least (a) an engineered pathways to produce acetone and (b) a downregulated native alcohol dehydrogenase (ADH) gene, wherein the native ADH is capable of converting acetone to isopropanol but lacking (c) a heterologous alcohol dehydrogenase gene.

[0066]

[0062] Broadly, the process of the present disclosure comprises contacting the recombinant yeast host cell with a carbohydrate under conditions to allow the conversion of at least in part of the carbohydrates into acetone and ethanol (e.g., fermenting step). In some embodiments, the contacting step occurs under conditions to allow the conversion of at least in part of the carbohydrate into acetone. Alternatively, or in combination, the contacting step occurs under conditions to allow the conversion of at least in part of the carbohydrate into isopropanol. The process can optionally include a step of isolating the acetone, ethanol, and / or the isopropanol from the fermented carbohydrate (using distillation for example).

[0067]

[0063] In some embodiments, the process of the present disclosure comprises a plurality of fermentations in which the carbohydrate is recycled between two rounds of fermentations. In some embodiments, the recombinant yeast host cells are only exogenously added in the initial fermentation cycle and are then recycled in further fermentation cycles. Each fermentation cycle of the process includes contacting a fermentation medium (comprising a fermentable carbohydrate) with a fermenting population under conditions so as to allow the conversion of the fermentable carbohydrate in a fermentation product (e.g., fermentation). At the end of the fermentation, the fermenting population present in the fermented fermentation medium is substantially isolated from the fermented fermentation medium and use to initiate another fermentation cycle. It is understood that, in such embodiments, the initial fermenting population consists essentially in the recombinant yeast host cells of the present disclosure and that, during the plurality of the fermentation cycles, the recycled fermenting population can include some contaminating wild (non-genetically modified) yeasts. The plurality offermentation cycles can include at least one continuous fermentation. The plurality of fermentation cycles can only include continuous fermentations. The plurality of fermentation cycles can include at least one batch fermentation. The plurality of fermentation cycles can only include batch fermentations. The processes of the present disclosure can include an initial fermentation cycle at least one, two, three, four, five, six, seven, eight, nine, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 or more further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 39 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 49 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 59 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 69 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 79 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 89 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 99 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 109 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 119 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 129 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 139 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 149 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 159 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 169 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 179 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 189 further fermentation cycles. In specific embodiments, the processes of the present disclosure include an initial fermentation cycle at least 199 further fermentation cycles.

[0068]

[0064] The carbohydrate that can be fermented with the recombinant yeast host cells described herein includes any type of fermentable carbohydrate known in the art and described herein. In one embodiment, the carbohydrate source is a biomass. For example, the biomass can include, but is not limited to, starch, sugar and lignocellulosic materials comprising lignocellulosic fibers. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. The starch present in the biomass can be totally or in part in a raw form or in a gelatinized form. When the biomass comprises or is derived from corn, it can include a corn mash. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, molasses or cane. The terms “lignocellulosic material”, “lignocellulosic substrate” and “cellulosic biomass” mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and / or agricultural residues, forestry residues and / or forestry wastes, paper-production sludge and / or waste paper sludge, waste -water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants and sugar-processing residues. The terms “hemicellulosics”, “hemicellulosic portions” and “hemicellulosic fractions” mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising mannan, glucomannan and galactoglucomannan), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan) and proteoglycans e.g., arabinogalactan-protein). In some embodiments, the biomass can include and / or be supplemented with citric acid (especially when acetic acid or acetate is the first metabolic product).

[0069]

[0065] In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; sugar cane must; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.

[0070]

[0066] Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

[0071]

[0067] It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and / or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, molasses, sugarcane, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.

[0068] Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making and is typically removed from process wastewater in a primary clarifier. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol. Processes provided by the present invention are widely applicable. Moreover, the saccharification and / or fermentation products may be used to produce ethanol and acetone or higher value-added chemicals, such as organic acids, aromatics, esters, and polymer intermediates.

[0072]

[0069] The fermentation step of the process can be performed at temperatures of at least about 25°C, about 28°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C. In some embodiments, the process can be conducted at temperatures above about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41 °C, about 42°C, or about 50°C.

[0073]

[0070] In some embodiments, prior to fermentation, a step of liquefying starch can be included in the process. In such embodiment, the liquefied starch is then submitted to a following fermentation step. The liquefaction of starch can be performed at a temperature of between about 70°C-105°C to allow for proper gelatinization and hydrolysis of the starch. In an embodiment, the liquefaction occurs at a temperature of at least about 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C or 105°C. Alternatively or in combination, the liquefaction occurs at a temperate of no more than about 105°C, 100°C, 95°C, 90°C, 85°C, 80°C, 75°C or 70°C. In yet another embodiment, the liquefaction occurs at a temperature between about 80°C and 85°C (which can include a thermal treatment spike at 105°C).

[0074]

[0071] During fermentation, the pH of the biomass can be equal to or below 5.5, 5.4, 5.3, 5.2, 5.1 , 5.0, 4.9, 4.8, 4.7., 4.6, 4.5, 4.4, 4.3, 4.2, 4.1 , 4.0 or lower. In an embodiment, the pH of the fermentation medium (during fermentation) is between 4.0 and 5.5.

[0075]

[0072] In the process described herein, it is possible to add an exogenous source (e.g., to dose) of an enzyme to facilitate saccharification or improve fermentation yield. As such, the process can comprise including one or more dose of one or more exogenous enzyme during the liquefaction / saccharification and / or the fermentation step. The exogenous enzyme can be provided in a purified form or in combination with other enzymes (e.g., a cocktail). In the context of the present disclosure, the term “exogenous” refers to a characteristic of the enzyme, namely that it has not been produced during the saccharification or the fermentation step, but that it was produced prior to the saccharification or the fermentation step. The exogenous enzyme that can be used during the saccharification / fermentation process can include, without limitation, an alpha-amylase, a glucoamylase, a protease, a phytase, a pullulanase, a cellulase, a xylanase, a trehalase, or any combination thereof.

[0073] In the process described herein, it is possible to add a nitrogen source (usually urea or ammonia) to facilitate liquefaction / saccharification or improve fermentation yield. As such, the process can comprise including one or more amount of the nitrogen source prior to or during the saccharification and / or the fermentation step.

[0076]

[0074] The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

[0077] EXAMPLE I

[0078]

[0075] Table 1 provides a description of the genetic modifications introduced in Saccharomyces cerevisiae strains of Example I.

[0079] Native ADH1 was identified as the likely enzyme responsible for conversion of acetone to isopropanol in S. cerevisiae. However, deletion of native ADH1 has demonstrated extremely poor growth of the strain coupled with incomplete fermentation (data not shown). It was hypothesized that replacing the deleted native ADH1 with a heterologous ADH having reduced secondary alcohol dehydrogenase activity could restore the fermentative performance and kinetics back to wild type levels while minimizing the side activity resulting in isopropanol formation. Nine heterologous ADH enzymes (see Table 1) were expressed within a S. cerevisiae ADH1 Knock Out strain (strain SC-3) using a plasmid-based expression system and screened for growth rate in YPD (Figure 1). The S. cerevisiae ADH1 was also reintroduced using the plasmid expression system as a positive control (strain SC-13).

[0080] Table 1. Genetic modifications of the strains characterized in Example I. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC-1).

[0081]

[0076] The strains were grown overnight in YPD40 media at 32°C and cells were normalized to an OD600 of 0.05 in sterilized ddH2O. Two (2) pL of each sample were inoculated in duplicate, into 98 pL of YPD10 media in a 96 well plate and incubated at 35°C for 48 hours with constant shaking. The OD600 was recorded every 10 minutes (figure 1). Of the nine enzymes tested only the adhA of Z. mobilis (strain SC-12) was found to restore growth to a similar rate as the S. cerevisiae ADH1 control.

[0082] EXAMPLE II

[0083]

[0077] Table 2 provides a description of the genetic modifications introduced in Saccharomyces cerevisiae strains of Example II.

[0084] Further the discovery that heterologous expression of Z.mobilis adhA could restore the growth in a S. cerevisiae ADH1 Knock Out strain, a fermentation was performed to determine the fermentative performance of the strain. S. cerevisiae ADH1 Knock Out strain SC-3 and on strain having S. cerevisiae ADH1 reintroduced (SC-15) was also used as control.

[0085] Table 2. Genetic modifications of the strains characterized in Example II. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC-1).

[0086]

[0078] Lab-scale fermentation conditions. Strains were grown overnight in YPD40 media at 32°C. Each strain was inoculated to an initial OD of 0.025 in duplicate into 30ml of YPD120 media containing 5g / L acetone at pH 5.5. Fermentations were carried out in sealed 60ml serum bottles and end point metabolites were quantified via HPLC at 66 hours of fermentation (see figure 2).

[0087] EXAMPLE III

[0088] Table 3 provides a description of the genetic modifications introduced in Saccharomyces cerevisiae strains of Example III.

[0089] Table 3. Genetic modifications of the strains characterized in Example III. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC-1).

[0090]

[0079] Lab-scale corn mash fermentation conditions. Each strain was grown overnight in YPD40 media at 32°C and then was inoculated at an OD600 of 0.12, in duplicate, into 30 g corn mash medium in sealed 60ml serum bottles. In addition to the yeast, each bottle also received 236 ppm Urea addition and 0.45 AGU / g total solids of glucoamylase. The fermentations were carried out for 24 hours at 33°C and at a temperature of 31 °C thereafter. The fermentations were monitored via CO2 off gas to determine fermentation kinetics (figure 3) and end point metabolites were quantified by HPLC following 66 hours of fermentation (figure 4).

[0091] EXAMPLE IV

[0092]

[0080] Table 4 provides a description of the genetic modifications introduced in Saccharomyces cerevisiae strains of Example IV.

[0093]

[0081] Following the identification of ZmAdhA in replacing native ADH1 for decreased IPA formation, ZmAdhA homologs as well as other native S. cerevisiae ADH isozymes were chosen for follow-up screening. The ADH enzymes were screened using the ADH1 promoter and chromosomal integration at the FCY1 loci in strain SC-2 adhlKO. Two isolates of each transformation were applied and wildtype ADH1 (SC-18) and ZmAdhA (SC-19) containing strains were included as controls.

[0082] Table 4. Genetic modifications of the strains characterized in Example IV. All the strains were derived from a non-genetically modified Saccharomyces cerevisiae strain (e.g., SC-1).

[0094]

[0083] Lab-scale fermentation conditions. Each strain was grown overnight in YPD40 media at 32°C and then was inoculated at an OD600 of 0.035, in duplicate, into YPD120 media supplemented with 5g / L acetone in sealed 60 ml serum bottles. The fermentations were carried out for 48 hours at 32°C at 150 RPM. The fermentations were monitored via CO2 off gas to determine fermentation kinetics (figure 5) and end point metabolites were quantified by HPLC following 48 hours of fermentation (Table 5).

[0095] Table 5. HPLC analysis following fermentation of different heterologous ADH expression within an ADH1 KO host strain in YPD120 media supplemented with 5g / L acetone.

[0096]

[0084] Wildtype ADH1 (strain SC-18) and ZmAdhA (strain SC-19) containing strains were included as controls. ZmAdhP from Z. mobilis (strain SC-20) was able to maintain a similar level of ethanol production (53g / L) and comparable IPA production (0.45g / L) as the ZmAdhA enzyme (strain SC-19), while ScADH4 (strain SC-21) maintained high ethanol production (53g / L) and even lower IPA production (0.15g / L) (Table 5). All ADH enzymes restored cell growth of SC-2 to a level similar to SC-1 as indicated by the CO2production profiles (Figure 5).

Claims

WHAT IS CLAIMED IS:1 . A recombinant yeast host cell for converting a carbohydrate to ethanol and acetone comprising: a) an engineered metabolic pathway to convert acetyl-CoA to acetone comprising: i. a thiolase; ii. a CoA transferase, a HMG-CoA synthase and lyase, or an acetoacetyl- CoA hydrolase; andHi. an acetoacetate decarboxylase; b) a downregulated native alcohol dehydrogenase (ADH) gene; wherein the native ADH is capable of converting acetone to isopropanol; and c) an heterologous alcohol dehydrogenase.

2. The recombinant yeast host cell of claim 1 , wherein the heterologous ADH has a secondary alcohol activity inferior to the secondary alcohol activity of the native ADH.

3. The recombinant yeast host cell of claim 1 , wherein the heterologous ADH allow the recombinant yeast host cell to produce a yield of isopropanol inferior to 0.5 g / L in a YPD media containing 120 g / L glucose at pH 6.0 following 48 hours of fermentation.

4. The recombinant yeast host cell of either claims 1 or 3, wherein the heterologous ADH is a bacterial ADH.

5. The recombinant yeast host cell of any one of claims 1 to 4, wherein the heterologous ADH is an ADHA, ADHP, or ADH4.

6. The recombinant yeast host cell of any one of claims 1 to 5, wherein the heterologous ADH is derived from a Z. mobilis ADHA, Z. mobilis ADHP, or Saccharomyces cerevisiae ADH4.

7. The recombinant yeast host cell of claim 6, wherein the heterologous ADH has at least 75% identity with the amino acid sequence of SEQ ID NO: 238, 240, or 241.

8. The recombinant yeast host cell of any one of claims 1 to 7, wherein the native ADH gene is an ADH1 gene.

9. The recombinant yeast host cell of claim 8, wherein the native ADH1 has at least 70 % identity with the amino acid sequence of SEQ ID NO: 239.

10. The recombinant yeast host cell of any one of claims 1 to 9, wherein the downregulated native ADH1 gene is a deleted native ADH1 gene.11 . The recombinant yeast host cell of any one of claims 1 to 10, wherein the thiolase is:heterologous;• of prokaryotic or eukaryotic origin;• encoded by a thl gene, an erg10 gene or a phaA gene;• derived from Clostridium, Saccharomyces, Cupriavidus, Clostridium, Yarrowia, Thermoanaerobacterium, Saccoglossus, Strongylocentrotus, Zygosaccharomyces or Paenibacillus species;• derived from Clostridium acetobutylicum, Saccharomyces cerevisiae, Cupriavidus necator, Clostridium acetobutylicum, Clostridium kluyveri, Yarrowia lipolytica, Thermoanaerobacterium thermosaccharolyticum, Saccoglossus kowalevskii, Strongylocentrotus purpuratus, Zygosaccharomyces bailii or Paenibacillus polymyxa; and / or• has the amino acid sequence of SEQ ID NO: 101 , 103, 105, 107, 109, 111 , 113, 115, 117, 119 or 121.

12. The recombinant microorganism of any one of claims 1 to 11 , wherein the CoA transferase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by a ctfA gene, a ctfB gene, an atoD and / or an atoA gene;• derived from Clostridium, Thermosipho, Escherichia, Paenibacillus, Alkaliphilus or Brevibacillus species;• derived from Clostridium acetobutylicum, Thermosipho melanesiensis, Escherichia coll, Paenibacillus polymyxa, Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum, Clostridium sticklandii, Alkaliphilus metalliredigens, or Clostridium bovifaecis or Brevibacillus laterosporus; and / or• has the amino acid sequence of SEQ ID NO:123, 125, 127, 129, 131 , 133, 135, 137, 139, 141 , 143,145, 242, 244, 246, 248, 250, 252, 254, or 256.

13. The recombinant microorganism of any one of claims 1 to 12, wherein the HMG-CoA synthase is:• heterologous; of prokaryotic or eukaryotic origin;• encoded by a hgms gene or an erg13 gene;• derived from Saccharomyces, Lacticaseibacillus, Enterococcus, Haloferax, Alloscardovia or Listeria;• derived from Saccharomyces cerevisiae, Lacticaseibacillus easel UW4, Enterococcus faecalis, Haloferax volcanii DS2, Alloscardovia theropitheci, or Listeria monocytogenes; and / or• has the amino acid sequence of SEQ ID NO: 165, 167, 169, 171 , 173 or 175.

14. The recombinant microorganism of any one of claims 1 to 13, wherein the HMG-CoA lyase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by an hmgcl gene;• derived from Pseudomonas, Azotobacter, Bacillus, Desulfotomaculum, Acinetobacter, Moraxella, Alcaligenaceae, Macaca, Arabidopsis, Gallus, or Danio species;• derived from Pseudomonas monteilii, Pseudomonas wayambapalatensis, Azotobacter vinelandii, Pseudomonas citronellolis, Pseudomonas cremoris, Pseudomonas chengduensis, Pseudomonas aeruginosa PA01, Bacillus subtilis 168, Desulfotomaculum arcticum DSM 17038, Desulfoscipio geothermicus DSM 3669, Acinetobacter baumannii, Acinetobacter Iwoffii, Moraxella caviae, Alcaligenaceae bacterium 429, Macaca fascicularis, Arabidopsis thaliana, Gallus gallus, or Danio rerio; and / or• has the amino acid sequence of SEQ ID NO: 177, 179, 181 , 183, 185, 187, 189, 191 , 193, 195, 197, 199, 201 , 203, 205, 207, 209 or 211.

15. The recombinant microorganism of any one of claims 1 to 14, wherein the acetoacetyl- CoA hydrolase is:• heterologous;• of prokaryotic or eukaryotic origin; encoded by a tesB gene, a yciA gene, a yigl gene, a ydil gene, a fadM1 gene, a fad M2 gene or a fadM gene;• derived from Escherichia, Methylorubrum, Pseudomonas, Campylobacter, Mycobacterium, Fibrobacter, Alcanivorax, Haemophilus, Zymomonas, or Prevotella;• derived from Escherichia coll, Methylorubrum extorquens AM1, Pseudomonas aeruginosa, Campylobacter jejuni, Mycobacterium tuberculosis, Pseudomonas putida, Fibrobacter succinogenes, Alcanivorax borkumensis SK2, Haemophilus influenzae, Zymomonas mobilis subsp. Mobilis ZM4, Campylobacter jejuni, Prevotella ruminicola 23 or Providencia sneebia DSM 19967; and / or• has the amino acid sequence of SEQ ID NO: 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, or 229.

16. The recombinant microorganism of any one of claims 1 to 15, wherein the acetoacetate decarboxylase is:• heterologous;• of prokaryotic or eukaryotic origin;• encoded by an adc gene;• derived from Clostridium, Bacillus, Lactobacillus, Lacticaseibacillus, Rhizobium, Bradyrhizobium, Tetrahymena, Aspergillus or Paenibacillus species;• derived from Clostridium acetobutylicum, Clostridium beijerinckii, Bacillus amyloliquefaciensi Lactobacillus casei, Lacticaseibacillus rhamnosus,Rhizobium leguminosarum, Bradyrhizobium japonicum, Tetrahymena thermophile, Aspergillus bertholletiae, Aspergillus niger or Paenibacillus polymyxa; and / or• has the amino acid sequence of SEQ ID NO: 147, 149, 151 , 153, 155, 157, 159, 161 , or 163.

17. The recombinant yeast host cell of any one of claims 1 to 16, wherein the recombinant yeast host cell produces at least 5% less in isopropanol yield (W / V) than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a), but lacking b) and c).

18. The recombinant yeast host cell of any one of claims 1 to 17, wherein the recombinant yeast host cell produces 5% more in yield of acetone than a control yeast host cellcomprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a), but lacking b) and c).

19. The recombinant yeast host cell of any one of claims 1 to 18, wherein the recombinant yeast host cell produces more ethanol yield than a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA to acetone as defined in a) and the downregulated native ADH gene as defined in b), but lacking c).

20. The recombinant yeast host cell of any one of claims 1 to 19, wherein the recombinant yeast host cell growth rate is increased when compared to a control yeast host cell comprising the engineered metabolic pathway to convert acetyl-CoA and acetate to acetone as defined in a) and the downregulated native ADH gene as defined in b), but lacking c).21 . A process for making ethanol and acetone, the method comprising contacting the recombinant yeast host cell of any one of claims 1 to 20 with a carbohydrate under a condition allowing the conversion of at least a part of the carbohydrate to ethanol and acetone.

22. The process of claim 21 , wherein the carbohydrate is a corn mash.

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