Method for producing ethanol and one or more co-products in yeast

Abstract

The present invention provides a method for producing ethanol and one or more co-products from a fermentable carbon source. [Solution] Ethanol and one or more co-products are produced in an ethanol-producing yeast modified to further produce the one or more co-products. The method comprises the steps of contacting a fermentable carbon source with the modified yeast in a fermentation medium, fermenting the yeast in the fermentation medium so that the yeast produces ethanol and one or more co-products from the fermentable carbon source, and isolating the ethanol and one or more co-products. The modified yeast is an ethanol-producing yeast that produces ethanol at a higher concentration than the one or more co-products. Furthermore, this disclosure provides the modified yeast disclosed herein.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 040,445, filed Jun. 17, 2020, and U.S. Provisional Patent Application No. 62 / 979,905, filed Feb. 21, 2020, each of which is hereby incorporated by reference in its entirety.

Background Art

[0002] Background Industrial production of ethanol can be carried out by fermentation methods using various microorganisms. Process improvements to achieve higher yields and productivity include the use of different raw material sources and / or reduction of by-product production. Exemplary ethanol fermentation processes are described, for example, in U.S. Patent Application Publication 2010 / 0196978 (Patent Document 1), U.S. Patent Application Publication 2018 / 0030483 (Patent Document 2), and Chinese Patent Application Publication 101875912 A (Patent Document 3). Certain Clostridium species can carry out fermentation processes that produce ethanol, butanol, and acetone (ABE). Exemplary processes involving Clostridium are described, for example, in U.S. Patent Application Publication 2015 / 0093796 (Patent Document 4) and U.S. Patent No. 9,074,173 (Patent Document 5). Ethanol and other products can also be produced in a manner in which ethanol and other products are not produced via fermentation of a single raw material by the same microorganism. U.S. Patent Application Publication 2019 / 0106720 (Patent Document 6) describes the production of ethanol and xylitol, in which xylitol is produced from xylose present in fermentation broth and ethanol is produced from starch. U.S. Patent 5,070,016 (Patent Document 7) describes the production of methanol from carbon dioxide byproducts of anaerobic ethanol fermentation. Other byproducts of ethanol fermentation include animal feed (see, e.g., U.S. Patent 8,603,786 (Patent Document 8)), yeast (see, e.g., European Patent 1943346 B1 (Patent Document 9)), mycoproteins (see, e.g., U.S. Patent Application Publication 2017 / 0226551 (Patent Document 10)), and corn oil (see, e.g., U.S. Patent Application Publication 2006 / 0019360 (Patent Document 11)).

[0003] Therefore, there is a need in this field for improved methods to produce ethanol and one or more co-products from a single raw material using the same microorganism. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] U.S. Patent Application Publication No. 2010 / 0196978 [Patent Document 2] U.S. Patent Application Publication No. 2018 / 0030483 [Patent Document 3] Chinese Patent Application Publication No. 101875912 A [Patent Document 4] U.S. Patent Application Publication No. 2015 / 0093796 [Patent Document 5] U.S. Patent No. 9,074,173 [Patent Document 6] U.S. Patent Application Publication No. 2019 / 0106720 [Patent Document 7] U.S. Patent No. 5,070,016 [Patent Document 8] U.S. Patent No. 8,603,786 [Patent Document 9] European Patent No. 1943346 B1 [Patent Document 10] U.S. Patent Application Publication No. 2017 / 0226551 [Patent Document 11] U.S. Patent Application Publication No. 2006 / 0019360 [Overview of the project]

[0005] overview This disclosure provides a method for producing an industrially important product using ethanol-producing yeast modified to produce the product while continuously producing ethanol using a portion of a fermentable carbon source. This disclosure also provides the modified yeast.

[0006] In each or any of the embodiments described above or below, a method for producing ethanol and one or more co-products comprises the following steps: (a) contacting a fermentable carbon source with ethanol-producing yeast in a fermentation medium; (b) fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more co-products from the fermentable carbon source, and the produced ethanol is present at a higher mg / mL concentration than the produced co-products; and (c) isolating the ethanol and one or more co-products, wherein the yeast is recombinant yeast genetically modified to produce one or more co-products.

[0007] In each or any of the embodiments described above or below, the carbon source is glucose or dextrose.

[0008] In each or any of the embodiments described above or below, the carbon source is derived from a renewable cereal source obtained by saccharification of starch-based raw materials, such as corn, wheat, rye, barley, oats, rice, or mixtures thereof.

[0009] In each or any of the embodiments described above or below, the carbon source is derived from renewable sugars, such as sugarcane, sugar beet, cassava, sweet sorghum, or a mixture thereof.

[0010] In each or any of the embodiments described above or below, the ethanol-producing yeast is Saccharomyces cerevisiae.

[0011] In each or any of the embodiments described above or below, Saccharomyces cerevisiae is an industrial strain. Suitable industrial ethanol-producing strains include, but are not limited to, S. cerevisiae PE-2, CAT-1, and Red. In each or any of the embodiments described above or below, Saccharomyces cerevisiae is any common strain used in the ethanol industry, a typical laboratory strain, or any strain resulting from a typical cross between strains.

[0012] In each or any of the embodiments described above or below, Saccharomyces cerevisiae is an industrial strain already used in existing industrial ethanol processes, where such processes are based on sugarcane, sugar beet, or most preferably maize as raw materials.

[0013] In each or any of the embodiments described above or below, the ethanol-producing yeast is modified to downregulate one of the endogenous enzymes associated with the natural ethanol production metabolic pathway, such as PYK1 and / or PDC1 (pyruvate decarboxylase 1). In each or any of the embodiments described above or below, the ethanol-producing yeast is modified to downregulate or delete other endogenous enzymes not directly associated with or involved in the natural ethanol production metabolic pathway, such as glycerol pathway enzymes and / or acetate pathway enzymes. In each or any of the embodiments described above or below, the ethanol-producing yeast is modified to downregulate the endogenous pyruvate kinase that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. In each or any of the embodiments described above or below, pyruvate kinase expression is downregulated by at least 10%, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of wild-type pyruvate kinase expression. In each or any of the embodiments described above or below, pyruvate kinase activity is downregulated by at least 10%, for example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of wild-type pyruvate kinase activity. In each or any of the embodiments described above or below, the downregulation of the endogenous gene is carried out by a weak promoter (either natural or synthetic), a natural or synthetic terminator, a natural or synthetic transcription factor, a degron peptide, iCRISPR, or any other technique known in the art for gene downregulation in yeast.In each or any of the embodiments described above or below, endogenous pyruvate kinase under the control of a weak promoter is expressed at a level of 90% or less of the wild-type pyruvate kinase expression level, e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In each or any of the embodiments described above or below, the activity of endogenous pyruvate kinase under the control of a weak promoter is at a level of 90% or less of the wild-type pyruvate kinase activity level, e.g., 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In each or any of the embodiments described above or below, the weak promoter is pADH1, pCYC1, pSTE5, pREV1, pURA3, pRPLAl, pGAPl, pNUP57, or pMET25. In each or any of the embodiments described above or below, ethanol-producing yeast is modified to lack an endogenous pyruvate kinase that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate. In each or any of the embodiments described above or below, ethanol-producing yeast is modified to express an exogenous pyruvate kinase that catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate under the control of a weak promoter. In each or any of the embodiments described above or below, downregulation of the exogenous gene is carried out by a weak promoter (either natural or synthetic), a natural or synthetic terminator, a natural or synthetic transcription factor, a deglon peptide, or any other technique known in the art for downregulation of genes in yeast. In each or any of the embodiments described above or below, exogenous pyruvate kinase under the control of a weak promoter is expressed at a level of 90% or less of the wild-type pyruvate kinase expression level, for example, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less.In each or any of the embodiments described above or below, the activity of the exogenous pyruvate kinase under the control of a weak promoter is 90% or less of the level of wild-type pyruvate kinase activity, for example, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less. In each or any of the embodiments described above or below, the weak promoter is pADH1, pCYC1, pSTE5, pREV1, pURA3, pRPLAl, pGAPl, pNUP57, or pMET25.

[0014] In each or any of the embodiments described above or below, the ethanol-producing yeast is modified to express exogenous phosphoenolpyruvate carboxykinase (PEPCK) kinase to switch the carbon flow from PEP to oxaloacetate.

[0015] In each or any of the embodiments described above or below, the co-product is produced at a concentration that is non-toxic to ethanol-producing yeast.

[0016] In each or any of the embodiments described above or below, the recombinant yeast possesses a significant degree of the robustness and performance of stored ethanol fermentation compared to its parent industrial ethanol-producing yeast, enabling its use in existing industrial ethanol processes.

[0017] In each or any of the embodiments described above or below, the produced ethanol is present in an amount of at least 70% by weight, for example, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, or at least 95% by weight, based on the total weight of the produced ethanol and co-products.

[0018] In each or any of the embodiments described above or below, fermentation is carried out as a batch process, a fed batch process, or a continuous process.

[0019] In some or any of the above or below embodiments, the fermentation is carried out under anaerobic conditions at a temperature of about 15°C to about 60°C for about 24 hours to about 96 hours.

[0020] In some or any of the above or below embodiments, the fermentation is carried out under microaerobic conditions at a temperature of about 15°C to about 60°C for about 24 hours to about 96 hours.

[0021] In some or any of the above or below embodiments, the fermentation is carried out under aerobic conditions at a temperature of about 15°C to about 60°C for about 24 hours to about 96 hours.

[0022] In some or any of the above or below embodiments, the fermentation is carried out in an industrial ethanol plant, preferably an existing industrial ethanol plant.

[0023] In some or any of the above or below embodiments, one or more co-products are selected from the group consisting of alcohols other than ethanol; ketones; glycols; ethers; esters; diamines; carboxylic acids; amino acids; dienes, and alkenes.

[0024] In each or any of the embodiments described above or below, one or more co-products are selected from the group consisting of 1-butanol, 2-butanol, isobutanol, methanol, n-propanol, isopropanol, isoamyl alcohol, acetone, methyl ethyl ketone, methyl propionate, 1,3-propanediol, monoethylene glycol, propylene glycol, citric acid, lactic acid, succinic acid, adipic acid, acetic acid, glutamic acid, propionic acid, franzicarboxylic acid, 2,4-franzicarboxylic acid, 2,5-franzicarboxylic acid, 3-hydroxypropionic acid, acrylic acid, itaconic acid, glutamic acid, ethyl acetate, isopropyl acetate, propyl acetate, isoprenol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, diethanolamine, tryptophan, threonine, methionine, lysine, serine, tyrosine, butadiene, isoprene, ethane, and propene. In each or any of the embodiments described above or below, the co-products have low solubility in water and may aggregate or settle at the bottom of the fermentation broth tank, facilitating their separation and purification from the fermentation broth during downstream processing.

[0025] In each or any of the embodiments described above or below, the step of isolating ethanol and one or more co-products includes a process selected from distillation, adsorption, crystallization, absorption, electrodialysis, solvent extraction, ion exchange resin chromatography, or a combination thereof.

[0026] In each or any of the embodiments described above or below, a method for producing ethanol and one or more co-products comprises the following steps: (a) contacting a fermentable carbon source with ethanol-producing yeast in a fermentation medium; (b) fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more low-boiling point co-products from the fermentable carbon source, and the produced ethanol is present at a higher mg / mL concentration than the produced co-products; and (c) isolating the ethanol and one or more low-boiling point co-products, wherein the yeast is recombinant yeast genetically modified to produce one or more co-products.

[0027] In each or any of the embodiments described above or below, the low boiling point co-product has a boiling point of 100°C or less at a standard pressure of 100 kPa (1 bar), for example, 99°C or less, 98°C or less, 97°C or less, 95°C or less, 90°C or less, 85°C or less, 80°C or less, 75°C or less, 70°C or less, 65°C or less, or 60°C or less. Examples of low-boiling point products include, but are not limited to, 1-propanol (boiling point: 97°C), 2-propanol (boiling point: 82°C), acetone (boiling point: 56°C), methyl ethyl ketone (boiling point: 80°C), ethyl acetate (boiling point: 77°C), isopropyl acetate (boiling point: 88°C), ethane (boiling point: -90°C), propene (boiling point: -48°C), and ethanol (boiling point: 78.3°C).

[0028] In each or any of the embodiments described above or below, one or more low-boiling point coproducts are selected from acetone, 1-propanol, 2-propanol, or a combination thereof.

[0029] In each or any of the embodiments described above or below, the isolation of ethanol and one or more low-boiling point co-products is carried out by a continuous distillation unit.

[0030] In each or any of the embodiments described above or below, a method for producing ethanol and one or more co-products comprises the following steps: (a) contacting a fermentable carbon source with ethanol-producing yeast in a fermentation medium; (b) fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more high-boiling point co-products from the fermentable carbon source, and the produced ethanol is present at a higher mg / mL concentration than the produced co-products; and (c) isolating the ethanol and one or more high-boiling point co-products, wherein the yeast is recombinant yeast genetically modified to produce one or more high-boiling point co-products.

[0031] In each or any of the embodiments described above or below, the high-boiling-point coproducts have a boiling point above 100°C at a standard pressure of 100 kPa (1 bar), for example, above 105°C, above 110°C, above 120°C, above 130°C, above 140°C, above 150°C, above 160°C, above 170°C, above 180°C, above 190°C, above 200°C, above 210°C, above 220°C, above 230°C, above 240°C, or above 250°C. Examples of high-boiling point co-products include, but are not limited to, monoethylene glycol (boiling point: 197°C), n-butanol (boiling point: 118°C), 3-hydroxypropionic acid (boiling point: 280°C), adipic acid (boiling point: 338°C), diethanolamine (boiling point: 268°C), and 1,3-propanediol (boiling point: 214°C).

[0032] In each or any of the embodiments described above or below, one or more high-boiling point coproducts are selected from 1-butanol, isobutanol, isoamyl alcohol, or a combination thereof.

[0033] In each or any of the embodiments described above or below, the step of isolating ethanol and one or more high-boiling point co-products is carried out by distillation and a subsequent process selected from crystallization, solvent extraction, chromatographic separation, salt decomposition, precipitation, acidification, ion exchange, evaporation, or a combination thereof. [Brief explanation of the drawing]

[0034] The above summary of this disclosure and the following detailed description will be better understood when read in conjunction with the attached figures. For the purpose of illustrating the present disclosure, preferred embodiments are shown in the figures. However, it should be understood that this disclosure is not limited to the exact arrangements, embodiments and means shown.

[0035] [Figure 1] An exemplary metabolic pathway for the production of 1-propanol by fermentation is depicted. [Figure 2] This describes exemplary metabolic pathways for the production of acetone, 2-propanol, propene, and 1-butanol by fermentation. [Figure 3] Diagram an exemplary metabolic pathway for the simultaneous production of 1-propanol and acetone, or 1-propanol and 2-propanol. [Figure 4] An exemplary metabolic pathway for the production of butanone and / or 2-butanol is depicted. [Figure 5] An exemplary metabolic pathway for the co-production of 1-propanol and butanone is depicted. [Figure 6] This graph shows the inhibition of sugar consumption at various alcohol concentrations (g / L). Dotted line: Linear regression. Square: 2-butanol. Triangle: n-propanol. Circle: 2-propanol. Diamond: Ethanol. [Figure 7]This graph shows glucose and alcohol concentrations at different time points during fermentation. Solid line: Condition 1 (ethanol addition). Dotted line: Condition 2 (n-propanol and 2-propanol addition). Black circles: Glucose consumption under Condition 1. Black squares: Alcohol production / addition under Condition 1. White circles: Glucose consumption under Condition 2. White squares: Alcohol production / addition under Condition 2. [Modes for carrying out the invention]

[0036] Detailed explanation This disclosure provides modified yeast (e.g., recombinant yeast) and methods for producing industrially important products using modified yeast. The modified yeast is ethanol-producing yeast modified to produce products while continuing to produce ethanol using a portion of the fermentable carbon source. The advantage of this disclosure is that only a small portion of the carbon source can be diverted from ethanol production to the production of industrially relevant products, thereby facilitating the production of target products that would be toxic to yeast cells in large quantities. A related advantage is that diverting only a small portion of the carbon source to co-products produces potentially toxic compounds at low concentrations and below the toxic concentration range that could impair the fermentation process, thus having little to no effect on yeast cell growth and the yeast's performance in producing ethanol. A further advantage of retaining at least partially the yeast's ethanol performance while utilizing production conditions similar to those required for industrial production is that the modified yeast can be used in existing ethanol production plants. A further advantage of this disclosure is that it is possible to have modified yeast that is robust to industrial requirements and has sufficient ethanol production performance.

[0037] This disclosure provides modified yeast (e.g., recombinant yeast) suitable for use in existing industrial ethanol processes to produce industrial-related products beyond sugars and ethanol. The advantage of this disclosure is that ethanol producers can diversify their product portfolios and are not limited to the production of sugars and ethanol alone. Related advantages include the ability to produce target products and ethanol mixtures at varying concentrations depending on the market prices of the industrial-related target products and ethanol. A further advantage is the ability to divert some carbon sources from ethanol production to produce industrial-related products with higher market prices compared to ethanol, thereby increasing profitability. A further advantage of this disclosure is the provision of modified yeast suitable for use in existing industrial ethanol production plants, thereby reducing technical risks, industrialization time, and investments related to plant construction and scale-up processes in undeveloped regions.

[0038] This disclosure provides a modified yeast (e.g., recombinant yeast) that can divert a small portion of its carbon source from ethanol production to the production of industrial-related products. The advantage of this disclosure is that the modified yeast is minimally modified to produce products in smaller quantities compared to ethanol, without compromising the industrial robustness and ethanol performance requirements of industrial ethanol yeast strains. Related advantages include reduced investment in R&D programs and the ability to utilize the modified yeast in a shorter timeframe, as large-scale metabolic manipulation is not required and metabolic pathway enzymes that are fully optimized for producing products at such low concentrations are not needed. In contrast, utilizing modified yeast that can divert most or all of its carbon source to a desired product other than ethanol would require more time-consuming R&D work and increased overall costs.

[0039] As used herein, the term "derived from" may include the terms having origin in, obtained from, obtainable from, isolated from, and created from, and generally indicates that a particular substance has its origin in another particular substance or has characteristics that can be described in relation to the other particular substance.

[0040] As used herein, "exogenous polynucleotide" means any deoxyribonucleic acid derived from outside a microorganism.

[0041] As used herein, the term “expression vector” can mean a DNA construct containing a polynucleotide or nucleic acid sequence encoding a polypeptide or protein, such as a DNA coding sequence (e.g., a gene sequence), wherein the sequence is functionally linked to one or more suitable regulatory sequences that can influence the expression of the coding sequence in a host. Such regulatory sequences include promoters for influencing transcription, optional operator sequences for controlling such transcription, sequences encoding suitable mRNA-ribosome binding sites, and sequences for controlling the termination of transcription and translation. The vector may be a plasmid, cosmid, phage particle, bacterial artificial chromosome, or simply a potential genome insert. Upon transformation in a suitable host, the vector may replicate and function independently of the host genome (e.g., an independent vector or plasmid) or, in some cases, be integrated into the genome itself (e.g., an integrated vector). Plasmids are the most commonly used form of expression vector. However, this disclosure is intended to encompass other forms of expression vectors known or to become publicly known in the art that perform equivalent functions.

[0042] As used herein, the term “expression” may mean the process by which a polypeptide is produced based on a nucleic acid sequence (e.g., a gene) that encodes the polypeptide. This process includes both transcription and translation.

[0043] As used herein, the term “gene” may mean a DNA segment involved in the production of a polypeptide or protein (e.g., a fusion protein), and includes the regions before and after the coding region, as well as intervening sequences (introns) between individual coding segments (exons).

[0044] As used herein, the term “heterogeneous,” when relating to nucleic acids, polynucleotides, proteins, or peptides, may mean nucleic acids, polynucleotides, proteins, or peptides that are not naturally present in a particular cell, for example, a host cell. The term is intended to encompass proteins encoded by naturally occurring genes, mutant genes, and / or synthetic genes. In contrast, the term “homogeneous,” when relating to nucleic acids, polynucleotides, proteins, or peptides, means nucleic acids, polynucleotides, proteins, or peptides that are naturally present in a cell.

[0045] As used herein, the term “host cell” can mean a cell or cell line, including microorganisms, that can be transfected with a recombinant expression vector for the expression of a polypeptide or protein (e.g., a fusion protein). Host cells also include offspring of single-celled hosts, which may not necessarily be identical to the original parent cells (in morphology or whole-genome DNA complementation) due to natural, accidental, or intentional mutations. Host cells may also include cells that have been transfected or transformed in vivo with an expression vector.

[0046] As used herein, the term “introduced” may include transfection, transformation, or transduction in the context of insertion of a nucleic acid sequence or polynucleotide sequence into a cell, and means the incorporation of a nucleic acid sequence or polynucleotide sequence into a eukaryotic or prokaryotic cell, in which case the nucleic acid sequence or polynucleotide sequence may be incorporated into the cell’s genome (e.g., into a chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed.

[0047] Where used herein, the terms “unnatural” or “modified” as used in reference to the microorganisms of the present invention are intended to mean that the microorganism has at least one genetic alteration not typically found in naturally occurring strains of the species referred to, for example, the wild-type strain of the species referred to. Genetic alterations include, for example, modifications that introduce an expressible nucleic acid encoding a metabolic polypeptide, additions of other nucleic acids, deletions of nucleic acids, and / or other functional disruptions of the genetic material of the microorganism. Such modifications include, for example, coding regions and functional fragments of heterologous polypeptides, homologous polypeptides, or both heterologous and homologous polypeptides of the species referred to. Further modifications include, for example, non-coding regulatory regions in which the expression of a gene or operon is altered by the modification. The unnatural microorganisms of this disclosure may contain stable genetic alterations, meaning that the microorganism can be cultured for more than five generations without loss of the alteration. Generally, stable genetic modifications are those that persist for more than 10 generations, particularly stable modifications that persist for about 25 generations, and more specifically, stable genetic modifications that exist for more than 50 generations (e.g., indefinitely). Those skilled in the art will understand that genetic modifications (e.g., metabolic modifications exemplified herein) are described in relation to suitable host organisms and their corresponding metabolic reactions or suitable source organisms for desired genetic material (such as genes for desired metabolic pathways). However, given the complete genome sequencing of a wide variety of organisms and the high level of technology in the field of genomics, those skilled in the art will find it easy to apply the teachings and guidance provided herein to essentially all other organisms. Such genetic modifications include, for example, genetic modifications of homologous species, in particular, the substitution of orthologous, paralogous, or non-orthologous genes.

[0048] As used herein, the term “functionally linked” may mean that the juxtaposition or arrangement of certain elements causes them to function in a coordinated manner and produce an effect. For example, a promoter may be functionally linked to a coding sequence if it controls the transcription of that coding sequence.

[0049] As used herein, "1-propanol" is intended to mean n-propanol having the general formula CH3CH2CH2OH (CAS number 71-23-8).

[0050] As used herein, "2-propanol" is intended to mean isopropyl alcohol having the general formula CH3CH3CHOH (CAS number 67-63-0).

[0051] As used herein, the term "promoter" may mean a regulatory sequence that is involved in the binding of RNA polymerase and initiates the transcription of a gene. A promoter may be an inductive promoter or a constitutive promoter. An inductive promoter is a promoter that is active under environmentally regulated or developmentally regulated conditions.

[0052] As used herein, the terms “polynucleotide” or “nucleic acid sequence” can mean a nucleotide in polymeric form of any length and any three-dimensional structure and single-stranded or multi-stranded (e.g., single-stranded, double-stranded, triple-helical, etc.), which includes deoxyribonucleotides, ribonucleotides, and / or analogues or modified forms of deoxyribonucleotides or ribonucleotides, e.g., modified nucleotides or bases or their analogues. Such polynucleotides or nucleic acid sequences may encode amino acids (e.g., polypeptides or proteins, e.g., fusion proteins). Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and this disclosure encompasses multiple polynucleotides encoding a particular amino acid sequence. Any type of modified nucleotide or nucleotide analogue, e.g., a modified nucleotide that enhances nuclease resistance (e.g., deoxyribonucleotides, 2'-O-Me, phosphorothioates, etc.), may be used, provided that the polynucleotide retains the desired functionality under the conditions of use. Furthermore, labels, such as radioactive or non-radioactive labels or anchors (e.g., biotin), may be incorporated for detection or capture purposes. The term polynucleotide also encompasses peptide nucleic acids (PNAs). Polynucleotides may be naturally occurring or unnatural. The terms polynucleotide, nucleic acid, and oligonucleotide are used interchangeably herein. Polynucleotides may include RNA, DNA, or both and / or modified forms and / or analogs thereof. Non-nucleotide components may be interposed in the nucleotide sequence. One or more phosphodiester bonds may be replaced by other linking groups.Other such linking groups include, but are not limited to, embodiments in which the phosphate is replaced with P(O)S (thioate), P(S)S (dithioate), (O)NR2 (amidate), P(O)R, P(O)OR', COCH2 (formacetal) (wherein each R or R' is independently H, or optionally a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl) containing an ether (-O-) bond). Not all bonds within the polynucleotide are to be identical. The polynucleotide may be linear or cyclic, or may contain a combination of linear and cyclic portions.

[0053] As used herein, the terms “protein” or “polypeptide” may mean a composition composed of amino acids and recognized as a protein by those skilled in the art. Herein, we use customary one- or three-letter codes to represent amino acid residues. The terms protein and polypeptide are used interchangeably herein to refer to polymers of amino acids of any length, including those constituting linked (e.g., fused) peptides / polypeptides (e.g., fusion proteins). Such polymers may be linear or branched, may contain modified amino acids, or may have non-amino acid intersperses. The terms also encompass amino acid polymers that are naturally occurring or modified by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification, such as conjugation with a labeling component. Furthermore, polypeptides containing, for example, one or more amino acid analogs (e.g., non-natural amino acids), as well as other modifications known in the art, are also included in this definition.

[0054] As used herein, the related protein, polypeptide, or peptide may include a variant protein, polypeptide, or peptide. A variant protein, polypeptide, or peptide differs from and / or from its parent protein, polypeptide, or peptide by a small number of amino acid residues. In some embodiments, the number of differing amino acid residues is approximately 1, 2, 3, 4, 5, 10, 20, 25, 30, 35, 40, 45, or 50. In some embodiments, the variant differs by approximately 1 to approximately 10 amino acids. Alternatively, the variant may have a certain degree of sequence identity with a reference protein or nucleic acid when examined using, for example, sequence alignment tools such as BLAST, ALIGN, and CLUSTAL (see below). For example, a variant protein or nucleic acid may have at least approximately 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or even 99.5% amino acid sequence identity with a reference sequence.

[0055] As used herein, the terms “recovered,” “isolated,” “purified,” and “separated” may mean a substance (e.g., a protein, peptide, nucleic acid, polynucleotide, or cell) from which at least one naturally bound component has been removed. For example, these terms may also mean a substance that is substantially or essentially devoid of components that are normally present in its natural state, such as an intact biological system.

[0056] As used herein, the term “recombinant” may mean a nucleic acid sequence or polynucleotide, polypeptide or protein, and the cells based on them, that are not identical to naturally occurring nucleic acids, polypeptides, and cells because they have been manipulated by humans. Recombinant may also mean genetic material (e.g., nucleic acid sequences or polynucleotides, polypeptides or proteins encoded therein, and vectors and cells containing such nucleic acid sequences or polynucleotides) that have been modified to alter the characteristics of their sequence or expression, for example by mutating a coding sequence to produce a modified polypeptide, fusing a coding sequence to another coding sequence or gene, placing a gene under the control of a different promoter, expressing a gene in a different organism, expressing a gene at a lower or higher level, or conditionally or constitutively expressing a gene in a manner different from its natural expression profile.

[0057] As used herein, the terms “transfection” or “transformation” may mean the insertion of an exogenous nucleic acid or polynucleotide into a host cell. The exogenous nucleic acid or polynucleotide may be maintained as a non-integrated vector (e.g., a plasmid) or may be integrated into the host cell’s genome. The terms “transfect” or “transfection” are intended to encompass all conventional methods for introducing a nucleic acid or polynucleotide into a host cell. Examples of transfection techniques include, but are not limited to, calcium phosphate precipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, and microinjection.

[0058] As used herein, the terms “transformed,” “stably transformed,” and “transgenic” may mean cells having a non-native (e.g., heterologous) nucleic acid sequence or polynucleotide sequence as an episomal plasmid that is incorporated into the genome or maintained over multiple generations.

[0059] As used herein, the term “vector” may mean a polynucleotide sequence designed to introduce nucleic acids into one or more cell types. Examples of vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, and single-stranded and double-stranded cassettes.

[0060] As used herein, the terms “wild-type,” “natural,” or “naturally occurring” protein may refer to proteins found in nature. The term “wild-type sequence” refers to a sequence of amino acids or nucleic acids found in nature or naturally occurring. In some embodiments, wild-type sequences are a starting point for protein engineering projects, such as the production of variant proteins.

[0061] As used herein, the term “non-toxic concentration” may mean a concentration of a co-product that has no effect, or only minimal effect, on the level of ethanol produced by yeast modified to produce a co-product compared to the level of ethanol produced by otherwise similar, unmodified yeast. For example, if a non-toxic concentration exists, the level of ethanol produced by the modified yeast may be reduced by 30% or less, 20% or less, or most preferably 10% or less, compared to the level of ethanol produced by the unmodified yeast.

[0062] Unless otherwise defined herein, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art in which this disclosure pertains. Singleton, et al., Dictionary of Microbiology and Molecular Biology, second ed., John Wiley and Sons, New York (1994), and Hale & Markham, The Harper Collins Dictionary of Biology, Harper Perennial, NY (1991) will serve as general dictionaries for many of the terms used herein by those skilled in the art. Furthermore, it will be understood that any substrate disclosed in any of the pathways herein may optionally include anions or cations of said substrate.

[0063] The numerical ranges provided herein include the numerical values ​​that define the range.

[0064] While this disclosure may be materialized in various forms, the following descriptions of some aspects are made with the understanding that they should be considered illustrative examples of this disclosure and are not intended to limit this disclosure to the specific aspects illustrated. The headings are provided for convenience only and should not be interpreted as limiting this disclosure to any particular form. Any aspect illustrated under any heading may be combined with any aspect illustrated under any other heading.

[0065] The use of numerical values ​​in the various quantitative values ​​specified in this application is intended to be approximate, as if the word “about” were placed before both the minimum and maximum values ​​within the specified range, unless otherwise explicitly stated. Furthermore, the disclosure of ranges is intended to be a continuous range including any value between the specified minimum and maximum values, as well as any range that can be formed by such values. Also disclosed herein are any and all ratios (and any range of such ratios) that can be formed by dividing the disclosed numerical values ​​into any other disclosed numerical values. Accordingly, those skilled in the art will recognize that many such ratios, ranges, and ranges of ratios can be clearly derived from the numerical values ​​shown herein, and in all cases, such ratios, ranges, and ranges of ratios represent various aspects of this disclosure.

[0066] Yeast modification Yeast can be modified (e.g., genetically engineered) by any method known in the art to contain and / or express one or more polynucleotides encoding enzymes in pathways that catalyze the conversion of a fermentable carbon source into one or more products.

[0067] In some embodiments, yeast can be modified (e.g., genetically engineered) by any method known in the art to include and / or express one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of a fermentable carbon source to an intermediate in a pathway for the production of co-products such as 1-propanol, acetone, 2-propanol, propene, 1-butanol, 2-butanol, methyl ethyl ketone, and / or methyl propionate. Such enzymes may include, but are not limited to, any of the enzymes described herein. For example, yeast can be modified to include one or more polynucleotides encoding an enzyme that catalyzes the conversion of succinyl-CoA to 1-propanol.

[0068] In some embodiments, the yeast may contain one or more exogenous polynucleotides encoding one or more enzymes in the pathway for product production, such as 1-propanol, acetone, 2-propanol, propene, 1-butanol, 2-butanol, methyl ethyl ketone, and / or methyl propionate, from a fermentable carbon source under anaerobic conditions.

[0069] 1-A route for the production of propanol The metabolic pathways for the production of 1-propanol include, but are not limited to, pathways for producing 1-propanol from intermediates, including malonate semialdehyde, 3-hydroxypropionic acid, 1,2-propanediol, 2-ketobutyric acid (2-kB), succinyl-CoA, and acryl-CoA. As shown in Figure 1, the intermediates 2-kB, succinyl-CoA, and acryl-CoA converge to propionyl-CoA. Both propionyl-CoA and 1,2-propanediol are converted to propionaldehyde and 1-propanol by the action of aldehyde dehydrogenase (acetylation) by a bifunctional aldehyde / alcohol dehydrogenase or in combination with an alcohol dehydrogenase.

[0070] In one pathway, 1-propanol is produced via the succinyl-CoA pathway, where a sugar source is converted to succinyl-CoA via glycolysis and the citric acid cycle (TCA cycle), followed by isomerization of succinyl-CoA to methylmalonyl-CoA by methylmalonyl-CoA mutase and decarboxylation of methylmalonyl-CoA to propionyl-CoA by methylmalonyl-CoA decarboxylase. Aldehyde and alcohol dehydrogenases catalyze further conversions, converting propionyl-CoA to propionaldehyde and propionaldehyde to 1-propanol (see, for example, U.S. Patent Application Publication 2013 / 0280775). Alternatively, 1-propanol is produced via 1,2-propanediol, through which the sugar source undergoes multiple conversions catalyzed by methylglyoxal synthase, aldo-ketereductase, or glyoxylate reductase and aldehyde reductase. Hydrolases and dehydrogenases catalyze further conversions, converting 1,2-propanediol to propanol and propanal to 1-propanol (see, for example, U.S. Patent No. 9,957,530).

[0071] Alternatively, 1-propanol is produced from the 2-kB intermediate via conversion from threonine and / or citramaric acid. For example, 2-kB can be converted to propionyl-CoA by 2-oxobutanoic acid dehydrogenase or 2-oxobutanoic acid decarboxylase, respectively, or directly to propionaldehyde (see, for example, U.S. Patent Application Publication 2014 / 0377820).

[0072] In other pathways, 1-propanol is produced from β-alanine, oxaloacetate, lactic acid, or 3-hydroxypropionic acid (3-HP) intermediates, which converge on acrylyl-CoA, which is converted to propionyl-CoA by acrylyl-CoA reductase (see, for example, U.S. Patent Application Publication 2014 / 0377820). As described above, propionyl-CoA can be converted to 1-propanol by aldehyde and alcohol dehydrogenases.

[0073] Routes for the production of 1-propanol, acetone, 2-propanol, propene, and / or 1-butanol Metabolic pathways for the production of 1-propanol, acetone, 2-propanol, propene, and / or 1-butanol are shown in Figures 2 and 3. Acetone can be produced from several pathways, including but not limited to primary and secondary metabolic reactions such as glycolysis, terpenoid biosynthesis, atrazine degradation, and cyanoamino acid metabolism. In one pathway, acetyl-CoA can be derived from pyruvate and / or malonate semialdehyde by pyruvate dehydrogenase and malonate semialdehyde dehydrogenase, respectively. Acetyl-CoA is converted to acetoacetyl-CoA by thiolase or acetyl-CoA acetyltransferase (see, for example, U.S. Patent Application Publication 2018 / 0179558). Alternatively, acetoacetyl-CoA may be formed via malonyl-CoA by acetoacetyl-CoA synthase. Once acetoacetyl-CoA is formed, its conversion to acetoacetic acid can occur by acetoacetyl-CoA transferase, or via HMG-CoA by hydroxymethylglutaryl-CoA synthase and hydroxymethylglutaryl-CoA lyase. The conversion of acetoacetic acid to acetone is carried out by acetoacetic acid decarboxylase.

[0074] In another pathway, 2-propanol is produced from propane and / or acetone as precursors. As described above, acetone is produced from acetyl-CoA by multiple reactions and converted to isopropanol by isopropanol dehydrogenase (see, for example, U.S. Patent Application Publication 2018 / 0179558). In yet another pathway, propane is produced from a butyrate intermediate, and isopropanol is generated by propane 2-monooxygenase. The biosynthesis of propane from glucose with butyrate as an intermediate in Escherichia coli is described in Kallio et al. (2014) Nat Commun, 5 (4731).

[0075] In another pathway, alkenes (e.g., ethene and propene) are produced from alcohol intermediates (e.g., ethanol and propanol, respectively) by linalool dehydratase-isomerase, as described in U.S. Patent Application Publication No. 2019 / 0323016.

[0076] In another pathway, 1-butanol is produced from butanal by butanol dehydrogenase having butyrate and butyryl-CoA as precursors. Butyryl-ACP is generated via the fatty acid biosynthesis (FASII) pathway, followed by the release of butyrate by thioesterase and its conversion to butanal by carboxylic acid reductase assisted by maturase phosphopantetheinyltransferase, as described, for example, Kallio et al. (2014) Nat Commun, 5 (4731). Butyryl-CoA is produced from crotonyl-CoA by the reaction of butyryl-CoA dehydrogenase, where crotonyl-CoA is produced by amino acid metabolism and / or glycolysis via acetyl-CoA, as described, for example, Ferreira et al. (2019) Biotechnol Biofuels 12:230 and U.S. Patent No. 9,567,613.

[0077] Routes for the production of methyl ethyl ketone (butanone) and / or 2-butanol In another pathway, as shown in Figure 4, methyl ethyl ketone (also known as butanone) and / or 2-butanol are produced from malonate semialdehyde (MSA). The metabolic pathway for the production of butanone and / or 2-butanol includes a pathway that produces butanone and / or 2-butanol from intermediates including, but not limited to, malonate semialdehyde, 3-hydroxypropionic acid (3HP), 3-hydroxypropionyl-coenzyme A (3HP-CoA), acryl-CoA, propionyl-CoA, acetyl-CoA, 3-ketovaleryl-CoA, and 3-ketovaleric acid.

[0078] In some respects, the modified yeast comprises: (a) at least one nucleic acid molecule encoding one or more polypeptides that catalyze the production of acetyl-CoA from malonic acid semialdehyde; (b) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 3-hydroxypropionic acid from malonic acid semialdehyde; (c) at least one nucleic acid molecule encoding one or more polypeptides that catalyze the production of propionyl-CoA from 3-hydroxypropionic acid; and (d) at least one nucleic acid molecule encoding one or more polypeptides that catalyze the production of 2-butanone from propionyl-CoA and acetyl-CoA.

[0079] In some cases, malonate semialdehyde can be converted to acetyl-CoA by malonate semialdehyde dehydrogenase. In some cases, the modified yeast contains one or more malonate semialdehyde dehydrogenases, including but not limited to those listed in Table 1, which have EC numbers 1.2.1.18 or 1.2.1.27. In some cases, malonate semialdehyde dehydrogenase (bauC) is derived from Pseudomonas aeruginosa. In some cases, malonate semialdehyde dehydrogenase (Ald6) is derived from Candida albicans. In some cases, malonate semialdehyde dehydrogenase (iolA) is derived from Lysteria monocytogenes. In some aspects, malonate semialdehyde dehydrogenase (dddC) is derived from Halomonas sp. HTNK1.

[0080] (Table 1) Candidates for the conversion of malonate semialdehyde to acetyl-CoA TIFF2026094284000002.tif52157

[0081] In some aspects, malonyl semialdehyde can be converted to acetyl-CoA by a sequential reaction of (i) malonyl-CoA reductase and / or 2-keto acid decarboxylase, and (ii) malonyl-CoA decarboxylase. In some aspects, malonyl-CoA reductase and / or 2-keto acid decarboxylase catalyze the conversion of malonyl semialdehyde to malonyl-CoA. In some aspects, malonyl-CoA decarboxylase catalyzes the production of acetyl-CoA from malonyl-CoA. In some aspects, the modified yeast contains one or more malonyl-CoA reductases and / or 2-keto acid decarboxylases, including but not limited to those listed in Table 2, which have EC number 1.1.1.298. In some aspects, the modified yeast contains one or more malonyl-CoA decarboxylases, including but not limited to those listed in Table 2, which have EC number 4.1.1.9. In some aspects, malonyl-CoA reductase (mcr) is derived from Chloroflexus aurantiacus. In some aspects, 2-keto acid decarboxylase (kivD) is derived from Lactococcus lactis. In some aspects, 2-keto acid decarboxylase (kdcA) is derived from Lactococcus lactis. In some aspects, 2-keto acid decarboxylase (ARO10) is derived from Saccharomyces cerevisiae. In some aspects, malonyl-CoA decarboxylase (MatA) is derived from Rhizobium trifolii. In some respects, malonyl-CoA decarboxylase (MLYCD) is derived from Homo sapiens.

[0082] (Table 2) Candidates for the conversion of malonate semialdehyde to acetyl-CoA via malonyl-CoA intermediates TIFF2026094284000003.tif70142

[0083] In some cases, malonic acid semialdehyde can be converted to 3HP by 3-hydroxypropionic acid dehydrogenase. In some cases, the modified yeast contains one or more 3-hydroxypropionic acid dehydrogenases, including but not limited to those listed in Table 3, which have EC numbers 1.1.1.298 or 1.1.1.381. In some cases, 3-hydroxypropionic acid dehydrogenase (ydfg) is derived from Escherichia coli. In some cases, 3-hydroxypropionic acid dehydrogenase (mcr-1) is derived from Chloroflexus aurantiacus. In some cases, 3-hydroxypropionic acid dehydrogenase (Ydf1) is derived from Saccharomyces cerevisiae. In some cases, 3-hydroxypropionic acid dehydrogenase (Hpd1) is derived from Candida albicans.

[0084] (Table 3) Candidates for the conversion of malonic acid semialdehyde to 3-hydroxypropionic acid TIFF2026094284000004.tif52142

[0085] In some cases, 3HP can be converted to propionyl-CoA by a series of reactions involving (i) 3-hydroxypropionyl-CoA transferase, 3-hydroxypropionyl-CoA ligase, or 3-hydroxypropionyl-CoA synthase; (ii) 3-hydroxypropionyl-CoA dehydratase; and (iii) acrylyl-CoA reductase.

[0086] In some aspects, the modified yeast contains, but is not limited to, one or more 3-hydroxypropionyl-CoA transferases, 3-hydroxypropionyl-CoA ligases, and / or 3-hydroxypropionyl-CoA synthases having EC numbers 2.8.3.1, 6.2.1.17, or 6.2.1.36, such as those listed in Table 4. In some aspects, the 3-hydroxypropionyl-CoA transferase (pct) is derived from Cupriavidus necator, Clostridium propionicum, or Megasphaera elsdenii. In some aspects, the 3-hydroxypropionyl-CoA ligase (prpE) is derived from Salmonella enterica or Escherichia coli. In some cases, 3-hydroxypropionyl-CoA ligase (Nmar_1309) is derived from Nitrosopumilus maritimus. In some cases, 3-hydroxypropionyl-CoA synthase (Msed_1456) is derived from Metallosphaera sedula. In some cases, 3-hydroxypropionyl-CoA synthase (Stk_07830) is derived from Sulfolobus tokodaii.

[0087] In some cases, 3-hydroxypropionyl-CoA transferase transfers coenzyme-A from acetyl-CoA to 3-hydroxypropionic acid, producing acetic acid. This coenzyme is then reused in two consecutive reactions: acetic acid is converted to acetic acid-P by acetic acid kinase, and acetic acid-P is converted to acetyl-CoA by phosphate acetyltransferase. Acetate kinase and phosphate acetyltransferase include, but are not limited to, enzymes with EC numbers 2.7.2.1 and 2.3.1.8, respectively. In some cases, acetic acid kinase is derived from Corynebacterium glutamicum or Escherichia coli. In some cases, acetic acid kinase is derived from Escherichia coli (ackA). In some cases, phosphate acetyltransferase is derived from Escherichia coli or Corynebacterium glutamicum. In some aspects, phosphate acetyltransferase is derived from Corynebacterium glutamicum (PTA). In some aspects, phosphate acetyltransferase is derived from Corynebacterium glutamicum, and acetate kinase is derived from Escherichia coli.

[0088] In some aspects, the modified yeast contains one or more 3-hydroxypropionyl-CoA dehydrats, including but not limited to those listed in Table 4, such as EC numbers 4.2.1.116, 4.2.1.55, 4.2.1.150, or 4.2.1.17. In some aspects, the 3-hydroxypropionyl-CoA dehydratase (hpcd) is derived from Metallospaella sedula, Bacillus sp., or Sporanaerobacter acetigenes. In some aspects, the 3-hydroxypropionyl-CoA dehydratase is derived from Ruegeria pomeroyi. In some cases, 3-hydroxypropionyl-CoA dehydratase (St1516) is derived from *Sulfolobus tocodanei*. In some cases, 3-hydroxypropionyl-CoA dehydratase (Nmar_1308) is derived from *Nitrosopmylus maritimus*. In some cases, 3-hydroxypropionyl-CoA dehydratase (Hpcd) is derived from *Chloroflexus aurantiacus*. In some cases, 3-hydroxypropionyl-CoA dehydratase (Crt) is derived from *Clostridium acetobutylicum* or *Clostridium pasteuranum*. In some cases, 3-hydroxypropionyl-CoA dehydratase is derived from *Clostridium pasteuranum*. In some cases, 3-hydroxypropionyl-CoA dehydratase (Mels_1449) is derived from Megasfera elsdenii. In some cases, 3-hydroxypropionyl-CoA dehydratase (Aflv_0566) is derived from Anoxybacillus flavithermus.

[0089] In some aspects, the modified yeast contains one or more acrylyl-CoA reductases, including but not limited to those listed in Table 4, which have EC numbers 1.3.1.84 or 1.3.1.95. In some aspects, acrylyl-CoA reductase (acuI) is derived from Ruegeria pomeroi, Escherichia coli, or Rhodobacter sphaeroides. In some aspects, acrylyl-CoA reductase (pcdh) is derived from Clostridium propionicum. In some aspects, acrylyl-CoA reductase (acuI) is derived from Alcaligenes faecalis. In some aspects, acrylyl-CoA reductase (Acr) is derived from Sulfolobus tokodani. In some cases, acrylyl-CoA reductase (acuI) is derived from Escherichia coli. In some cases, acrylyl-CoA reductase (Acr) is derived from Metallosphaera sedula. In some cases, acrylyl-CoA reductase (Nmar_1565) is derived from Nitrosopmylus maritimus.

[0090] In some aspects, 3-hydroxypropionyl-CoA transferase (pct) is derived from Clostridium propionicum, 3-hydroxypropionyl-CoA dehydratase (hpcd) is derived from Sporanaerobacter acetylenes and / or Metallospaella sedula, and acrylyl-CoA reductase (acr) is derived from Ruegeria pomeroi.

[0091] (Table 4) Candidates for the conversion of 3-hydroxypropionic acid to propionyl-CoA TIFF2026094284000005.tif120143TIFF2026094284000006.tif179143

[0092] In some cases, 3HP can be converted to propionyl-CoA by trifunctional propionyl-CoA synthase (PCS). In some cases, the modified yeast contains one or more propionyl-CoA synthases, including but not limited to those listed in Table 5, which have EC number 6.2.1.17. In some cases, the propionyl-CoA synthases (pcs) are derived from Chloroflexus aurantiacus, Chloroflexus aggregans, Roseiflexus castenholzii, Natronococcus occultus, Halioglobus japonicus, or Erythrobacter sp. NAP1.

[0093] (Table 5) Candidates for the conversion of 3-hydroxypropionic acid to propionyl-CoA TIFF2026094284000007.tif70143

[0094] In some respects, the modified yeast comprises: (i) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 3-ketovaleryl-CoA from propionyl-CoA and acetyl-CoA; (ii) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 3-oxovaleric acid from 3-ketovaleryl-CoA; and (iii) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 2-butanone from 3-oxovaleric acid.

[0095] In some aspects, propionyl-CoA and acetyl-CoA can be converted together to 3-ketovaleryl-CoA by β-ketothiolase or acetyl-CoA acetyltransferase. In some aspects, the modified yeast contains one or more β-ketothiolases or acetyl-CoA acetyltransferases, including but not limited to those listed in Table 6, which have EC numbers 2.3.1.16 or 2.3.1.9. In some aspects, β-ketothiolase (phaA) is derived from Acinetobacter sp. RA384. In some aspects, β-ketothiolase (BktB) is derived from Cupriviadus necator. In some aspects, β-ketothiolase (BktC) is derived from Cupriviadus necator. In some cases, β-ketothiolase (BktB) is derived from Cupriavidus taiwanensis. In some cases, β-ketothiolase (POT1) is derived from Saccharomyces cerevisiae. In some cases, acetyl-CoA acetyltransferase (phaA) is derived from Cupriavidus nekator. In some cases, acetyl-CoA acetyltransferase (thlA) is derived from Clostridium acetobutyricum. In some cases, acetyl-CoA acetyltransferase (thlB) is derived from Clostridium acetobutyricum. In some cases, acetyl-CoA acetyltransferase (phaA) is derived from Zoogloea ramigera. In some cases, acetyl-CoA acetyltransferase (atoB) is derived from Escherichia coli. In several aspects, acetyl-CoA acetyltransferase (ERG10) is derived from Saccharomyces cerevisiae.

[0096] (Table 6) Candidate conversions of propionyl-CoA and acetyl-CoA to 3-ketovaleryl-CoA TIFF2026094284000008.tif120143

[0097] In some cases, 3-ketovaleryl-CoA can be converted to 3-ketovaleric acid (also known as 3-oxovaleric acid) by 3-ketovaleryl-CoA transferase or 3-ketovaleryl-CoA hydrolase. In some cases, modified yeasts include, but are not limited to, enzymes having EC numbers 2.8.3.5, 2.8.3.8, or 2.8.3.9, such as those listed in Table 7, one or more 3-ketovaleryl-CoA transferases or 3-ketovaleryl-CoA hydrolases selected from succinyl-CoA:3-keto acid-CoA transferase, acetate-CoA transferase, butyrate-acetoacetate-CoA transferase, and acetoacetyl-CoA:acetyl-CoA transferase. In some aspects, succinyl-CoA:3-keto-CoA transferase (ScoA) is derived from Bacillus subtilis. In some aspects, succinyl-CoA:3-keto-CoA transferase (ScoB) is derived from Bacillus subtilis. In some aspects, acetate-CoA transferase (atoA) is derived from Escherichia coli. In some aspects, acetate-CoA transferase (atoD) is derived from Escherichia coli. In some aspects, butyrate-acetoacetate-CoA transferase (ctfA) is derived from Clostridium acetobutyricum. In some aspects, butyrate-acetoacetate-CoA transferase (ctfB) is derived from Clostridium acetobutyricum. In some aspects, butyrate-acetoacetate-CoA transferase (ctfA) is derived from Clostridium saccharoperbutylacetonium. In some aspects, butyrate-acetoacetate-CoA transferase (ctfB) is derived from Clostridium saccharoperbutylacetonium. In some aspects, acetoacetyl-CoA:acetyl-CoA transferase (ctfA) is derived from Escherichia coli.In some aspects, acetoacetyl-CoA:acetyl-CoA transferase (ctfB) is derived from E. coli. In some aspects, acetate-CoA transferase (ydiF) is derived from E. coli.

[0098] In some aspects, the transferase transfers coenzyme A from 3-ketovaleryl-CoA to acetate, producing acetyl-CoA. Acetate is reused by two consecutive reactions in which acetyl-CoA is converted to acetyl-P by phosphate acetyltransferase, and acetyl-P is converted to acetate by acetate kinase. Acetate kinase and phosphate acetyltransferase include, but are not limited to, enzymes with EC numbers 2.7.2.1 and 2.3.1.8, respectively. In some aspects, acetate kinase is derived from Corynebacterium glutamicum or Escherichia coli. In some aspects, acetate kinase is derived from Escherichia coli (ackA). In some aspects, phosphate acetyltransferase is derived from Escherichia coli or Corynebacterium glutamicum. In some aspects, phosphate acetyltransferase is derived from Corynebacterium glutamicum (pta). In some aspects, phosphate acetyltransferase is derived from Corynebacterium glutamicum, and acetate kinase is derived from Escherichia coli.

[0099] (Table 7) Candidates for the conversion of 3-ketovaleryl-CoA to 3-ketovaleric acid (3-oxovaleric acid) TIFF2026094284000009.tif120143

[0100] In some aspects, 3-ketovaleric acid (also known as 3-oxovaleric acid), which is structurally similar to acetoacetate, can be converted to butanone by acetoacetate decarboxylase. In some aspects, modified yeasts contain one or more enzymes having acetoacetate decarboxylase activity, including but not limited to enzymes having EC number 4.1.1.4, such as those listed in Table 8. In some aspects, acetoacetate decarboxylase (adc) is derived from Clostridium acetobutyricum. In some aspects, acetoacetate decarboxylase (adc) is derived from Clostridium saccharoperbutylacetonicum. In some aspects, acetoacetate decarboxylase (adc) is derived from Clostridium beijerinkii. In some aspects, acetoacetate decarboxylase (adc) is derived from Clostridium pasteuranum. In some aspects, acetoacetate decarboxylase (adc) is derived from Pseudomonas putida.

[0101] (Table 8) Candidates for the conversion of 3-ketovaleric acid (3-oxovaleric acid) to butanone TIFF2026094284000010.tif60143

[0102] In some cases, the enzymes used to convert propionyl-CoA and acetyl-CoA to butanone are (i) β-ketothiolase (BktB) from Capriavidus nekator and / or β-ketothiolase (phaA) from Acinetobacter species, (ii) CoA transferase (atoAD) from Escherichia coli and / or CoA transferase (ctfAB) from Clostridium acetobutyricum, and (iii) acetate decarboxylase (adc) from Clostridium acetobutyricum or Pseudomonas putida. Conveniently, in some cases, the enzymes convert propionyl-CoA and acetyl-CoA to butanone without the formation of significant levels of undesirable byproducts such as acetone, thereby avoiding an undesirable decrease in yield.

[0103] In some respects, the modified yeast comprises: (i) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 2-methylacetoacetyl-CoA from propionyl-CoA and acetyl-CoA; (ii) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 2-methylacetoacetic acid from 2-methylacetoacetyl-CoA; and (iii) at least one nucleic acid molecule encoding a polypeptide that catalyzes the production of 2-butanone from 2-methylacetoacetic acid.

[0104] In some aspects, propionyl-CoA and acetyl-CoA can be converted together to 2-methylacetoacetyl-CoA by 2-methylacetoacetyl-CoA thiolase. In some aspects, 2-methylacetoacetyl-CoA can be converted to 2-methylacetoacetate by CoA hydrolase or CoA-transferase. In some aspects, CoA hydrolase is acetyl-CoA hydrolase. In some aspects, CoA-transferase is acetyl-CoA acetyltransferase or succinyl-CoA:3-keto acid-CoA transferase. In some aspects, modified yeast contains one or more CoA hydrolases or CoA-transferases, including but not limited to those listed in Table 9, which have EC numbers 2.3.1.9, 2.8.3.5, or 3.1.2.1. In some aspects, acetyl-CoA acetyltransferase (Act1) is derived from Homo sapiens. In some aspects, succinyl-CoA:3-keto-acid-CoA transferase (ScoA) is derived from Bacillus subtilis. In some aspects, succinyl-CoA:3-keto-acid-CoA transferase (ScoB) is derived from Bacillus subtilis. In some aspects, acetyl-CoA hydrolase (Ach1) is derived from Saccharomyces cerevisiae.

[0105] In some aspects, 2-methylacetoacetate can be converted to butanone by 2-methylacetoacetate decarboxylase. In some aspects, the modified yeast contains one or more 2-methylacetoacetate decarboxylases, including but not limited to enzymes having EC number 4.1.1.5, such as those listed in Table 9. In some aspects, 2-methylacetoacetate decarboxylase is A-acetolactate decarboxylase. In some aspects, A-acetolactate decarboxylase (ALDC) is derived from Acetobacter aceti. In some aspects, A-acetolactate decarboxylase (Aldc) is derived from Enterobacter aerogenes. In some aspects, A-acetolactate decarboxylase (budA) is derived from Rauoltella terrigena.

[0106] (Table 9) Candidates for the conversion of propionyl-CoA and acetyl-CoA to butanone TIFF2026094284000011.tif80143

[0107] In some cases, butanone can be converted to 2-butanol by alcohol dehydrogenase (e.g., 2-butanol dehydrogenase) or MEK reductase. In some cases, alcohol dehydrogenase is NAD-dependent. In some cases, alcohol dehydrogenase is NADP-dependent.

[0108] In some aspects, the modified yeast contains one or more alcohol dehydrogenases, including but not limited to enzymes having EC numbers 1.1.1.1, 1.1.1.2, 1.1.1.80, or 1.1.1.-, such as those listed in Table 10. In some aspects, the NAD-dependent enzyme is known as EC number 1.1.1.1. In some aspects, the NADP-dependent enzyme is known as EC number 1.1.1.2. In some aspects, 2-butanol dehydrogenase (sadh) is derived from Rhodococcus ruber. In some aspects, 2-butanol dehydrogenase (adhA) is derived from Pyrococcus furious. In some cases, 2-butanol dehydrogenase (adh) is derived from Clostridium beijerinckii. In some cases, 2-butanol dehydrogenase (adh) is derived from Thermoanaerobacter brockii. In some cases, 2-butanol dehydrogenase (yqhD) is derived from Escherichia coli. In some cases, 2-butanol dehydrogenase (chnA) is derived from Acinetobacter species.

[0109] (Table 10) Candidates for the conversion of butanone to 2-butanol TIFF2026094284000012.tif70143

[0110] Route for the production of methyl propionate In an alternative pathway, methyl propionate is produced from butanone by Baeyer-Villiger monooxygenase, which includes, but is not limited to, the enzyme having EC number 1.14.13. In one embodiment, Baeyer-Villiger monooxygenase is derived from Acinetobacter calcoaceticus, Rhodococcus jostii, and / or Xanthobacter flavus.

[0111] A route for the simultaneous production of 1-propanol and butanone. In another pathway, 1-propanol and butanone are co-produced from malonate semialdehyde (MSA), as shown in Figure 5. The metabolic pathway for the co-production of 1-propanol and butanone includes pathways for producing 1-propanol and butanone from intermediates, including but not limited to malonate semialdehyde, 3-hydroxypropionic acid (3HP), 3-hydroxypropionyl-coenzyme A (3HP-CoA), acryl-CoA, propionyl-CoA, acetyl-CoA, 3-ketovaleryl-CoA, and 3-ketovaleric acid. In the pathway for butanone production discussed herein, a portion of the produced propionyl-CoA is used to produce butanone, and a portion is used to produce 1-propanol.

[0112] In some aspects, propionyl-CoA can be converted to 1-propanol by a difunctional alcohol / aldehyde dehydrogenase. In some aspects, the modified yeast contains one or more difunctional alcohol / aldehyde dehydrogenases, including but not limited to those listed in Table 11, which have EC numbers 1.1.1.1, 1.2.1.4, or 1.2.1.5. In some aspects, the alcohol / aldehyde dehydrogenase (adhe) is derived from Clostridium acetobutyricum. In some aspects, the alcohol / aldehyde dehydrogenase (adhe) is derived from Clostridium beigerlinky. In some aspects, the alcohol / aldehyde dehydrogenase (adhe) is derived from Clostridium typhimurium. In some aspects, alcohol / aldehyde dehydrogenase (adhe) is derived from Clostridium arbusti. In some aspects, alcohol / aldehyde dehydrogenase (adhE) is derived from Escherichia coli. In some aspects, alcohol / aldehyde dehydrogenase (adhP) is derived from Escherichia coli. In some aspects, alcohol / aldehyde dehydrogenase (bdhB) is derived from Clostridium acetobutyricum. In some aspects, alcohol / aldehyde dehydrogenase (Adh2) is derived from Saccharomyces cerevisiae. In some aspects, alcohol / aldehyde dehydrogenase (adhE) is derived from Clostridium roseum. In some aspects, alcohol / aldehyde dehydrogenase (adhA) is derived from Thermoanaerobacterium saccharolyticum. In some aspects, alcohol / aldehyde dehydrogenase (Ald6) is derived from Saccharomyces cerevisiae.In several respects, alcohol / aldehyde dehydrogenase (Aldh3A1) is of Homo sapiens origin.

[0113] (Table 11) Candidates for direct conversion of propionyl-CoA to 1-propanol TIFF2026094284000013.tif130143

[0114] In some aspects, propionyl-CoA can be converted to 1-propanol by a sequential reaction of aldehyde dehydrogenase (acetylation) and alcohol dehydrogenase. In some aspects, the modified yeast contains one or more aldehyde dehydrogenases (acetylation), including but not limited to those listed in Table 12, which have EC number 1.2.1.10. In some aspects, aldehyde dehydrogenase (acetylation) (mhpf) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylation) (Mhpf) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylation) (Mhpf) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylation) (mhpf) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylation) (Pdup) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylated) (pdup) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylated) (Pdup) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylated) (aldH) is derived from E. coli. In some aspects, aldehyde dehydrogenase (acetylated) (ald) is derived from E. coli. In some aspects, the modified yeast contains one or more alcohol dehydrogenases, including but not limited to those listed in Table 12, which have EC numbers 1.1.1.2 or 1.2.1.87. In some aspects, alcohol dehydrogenase (alrA) is derived from Acinetobacter species. In some aspects, alcohol dehydrogenase (bdhI) is derived from Clostridium acetobutylicum. In some aspects, alcohol dehydrogenase (bdhII) is derived from Clostridium acetobutyricum. In some aspects, alcohol dehydrogenase (adhA) is derived from Clostridium glutamicum.In some cases, alcohol dehydrogenase (yqhD) is derived from Escherichia coli. In some cases, alcohol dehydrogenase (adhP) is derived from Escherichia coli. In some cases, alcohol dehydrogenase (PduQ) is derived from Propionibacterium freudenreichii. In some cases, alcohol dehydrogenase (ADH1) is derived from Saccharomyces cerevisiae. In some cases, alcohol dehydrogenase (ADH2) is derived from Saccharomyces cerevisiae. In some cases, alcohol dehydrogenase (ADH4) is derived from Saccharomyces cerevisiae. In some cases, alcohol dehydrogenase (ADH6) is derived from Saccharomyces cerevisiae. In some aspects, alcohol dehydrogenase (PduQ) is derived from Salmonella enterica. In some aspects, alcohol dehydrogenase (Adh) is derived from Sulfolobus tocodile. In some aspects, aldehyde dehydrogenase (acetylated) (PduP) is derived from Salmonella enterica, and alcohol dehydrogenase (ADH1) is derived from Saccharomyces cerevisiae.

[0115] (Table 12) Candidates for the conversion of propionyl-CoA to propionaldehyde and candidate conversions for propionaldehyde to 1-propanol TIFF2026094284000014.tif229143

[0116] Conveniently, the co-production pathway for butanone and 1-propanol is redox-neutral and ATP-positive, resulting in more efficient and higher yield production of the desired compound. Furthermore, the balanced pathway can be carried out under anaerobic conditions, which offers several advantages of the fermentation process compared to the aerobic process at the same yield: anaerobic fermenters are less expensive than aerobic fermentation, air compressors are expensive and mean higher costs, and anaerobic processes allow for larger fermenters, thus requiring fewer fermenters compared to the aerobic process for the same product production capacity.

[0117] In some aspects, at least a portion of the excess NAD(P)H produced by modified yeast in butanone production is utilized to supply NAD(P)H in 1-propanol production. While we do not wish to be bound by theory, it is thought that the simultaneous production of butanone and 1-propanol with a balanced redox ratio facilitates fermentation under anaerobic conditions without forming undesirable byproducts at significant levels, thereby avoiding a reduction in the yield of the desired product.

[0118] In some cases, the simultaneous production of butanone and 1-propanol is carried out using industrial ethanol-producing yeast strains. In some cases, industrial ethanol-producing yeast strains are operated to simultaneously produce butanone and 1-propanol under anaerobic fermentation conditions by diverting a portion of the carbon source to the production of butanone and 1-propanol while continuing to produce ethanol. In some cases, industrial ethanol-producing yeast strains retain substantially all of the performance and robustness of industrial ethanol yeast, thus enabling their use and successful introduction into existing industrial ethanol production activities.

[0119] Modified yeast The modified yeasts provided herein may include: - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to succinyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of fermentation carbon sources to 1,2-propanediols, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to lactic acid, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to β-alanine, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to threonine, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to citramaric acid, - One or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of fermentable carbon sources to malonic acid semialdehyde, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA. - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of threonine to 2-ketobutyrate (2-kB), - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of citramaric acid to 2-ketobutyric acid (2-kB), - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of β-alanine to malonic acid semialdehyde, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of malonate semialdehyde to 3-hydroxypropionic acid (3-HP), - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of lactate to acryl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of β-alanine to acryl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of 3-HP to acryl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of methylmalonyl-CoA to propionyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of 2-kB to propionyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acryl-CoA to propionyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde. - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of 1,2-propanediol to propionaldehyde, and / or - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0120] The modified microorganisms provided herein may include: - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to pyruvate, - One or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of fermentable carbon sources to malonic acid semialdehyde (MSA), - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of pyruvate to acetyl-CoA, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of MSA to acetyl-CoA; - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetyl-CoA to malonyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of malonyl-CoA to acetoacetyl-CoA, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetoacetyl-CoA to acetoacetic acid, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA (HMG-CoA), - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of HMG-CoA to acetoacetate, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetoacetic acid to acetone, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of acetone to 2-propanol, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of fermentable carbon sources to butyric acid, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of butyric acid to propane, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of propane to 2-propanol, - One or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of fermentation carbon sources to 2-propanol, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of 2-propanol to propene, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of fermentable carbon sources to butyryl-CoA, - One or more polynucleotides encoding enzymes in the pathway that catalyzes the conversion of butyrate to butanal, - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of butyryl-CoA to butanal, and / or - One or more polynucleotides encoding an enzyme in the pathway that catalyzes the conversion of butanal to 1-butanol.

[0121] In some embodiments, the yeast is Saccharomyces cerevisiae, Kluyveromyces lactis, or Pichia pastoris.

[0122] In some embodiments, the yeast is Saccharomyces cerevisiae, an industrial ethanol production yeast strain, i.e., a yeast strain already used in existing industrial ethanol fermentation processes and assets, such industrial yeast possessing appropriate and outstanding robustness and fermentation performance for ethanol production.

[0123] In some embodiments, the yeast is Saccharomyces cerevisiae, an industrial ethanol-producing yeast strain already used in existing industrial ethanol fermentation processes and assets, such processes and assets based on sugarcane, sugar beet, or maize as raw materials.

[0124] In some embodiments, the yeast is Saccharomyces cerevisiae, derived from existing maize-based ethanol fermentation processes and assets, or is an industrial ethanol-producing yeast strain used industrially in existing maize-based ethanol fermentation processes and assets.

[0125] In some embodiments, the yeast is further modified to include one or more tolerance mechanisms, for example, tolerance to the molecules produced (e.g., 1-propanol, acetone, 2-propanol, propene, 1-butanol, 2-butanol, methyl ethyl ketone, and / or methyl propionate), and / or organic solvents. Yeast modified to include such tolerance mechanisms can provide a means to increase the fermentation titer and / or control contamination in industrial-scale processes.

[0126] Host cells are transformed or transfected with the expression vectors or cloning vectors disclosed herein for the production of one or more enzymes, or with polynucleotides encoding one or more enzymes disclosed herein, and cultured in a conventional nutrient medium appropriately modified for promoter induction, selection of transformants, or amplification of genes encoding a desired sequence.

[0127] Host cells containing the desired nucleic acid sequence encoding the disclosed enzyme can be cultured in various media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing host cells. Furthermore, any of the culture media described in Ham et al., Meth. Enz. 58: 44, (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; or No. 5,122,469; WO 90 / 103430; WO 87 / 00195; or U.S. Reissue Patent No. 30,985 may be used as the culture medium for the host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphates), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamicin (GENTAMYCIN)), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or equivalent energy sources. Any other necessary supplements may be included in appropriate concentrations that would be considered known to those skilled in the art. Culture conditions, such as temperature and pH, are those previously used with host cells selected for expression and would be obvious to those skilled in the art.

[0128] Method for the simultaneous production of ethanol and its co-products Ethanol and one or more co-products may be produced by contacting any of the genetically modified yeasts provided herein with a fermentable carbon source. Such a method may preferably include the steps of: contacting the fermentable carbon source with yeast containing one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source into one or more intermediates in the production of the co-product and one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of one or more intermediates into the co-product in a fermentation medium; and expressing one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of the fermentable carbon source into one or more intermediates in the production of the co-product and one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of one or more intermediates into the co-product.

[0129] The fermentation products of this disclosure may be prepared by conventional processes for industrial sugarcane, sugar beets, or more preferably, maize ethanol production. In such processes, glucose and dextrose or another suitable carbon source may be derived from renewable cereal sources through the saccharification of starch-based raw materials, including cereals such as maize, wheat, rye, barley, oats, rice, and mixtures thereof. Suitable carbon sources also include, but are not limited to, glucose, fructose, and sucrose, or mixtures thereof with C5 sugars such as xylose and / or arabinose. The carbon source may be derived from renewable sugar sources such as sugarcane, sugar beets, cassava, sweet sorghum, and mixtures thereof.

[0130] The fermentation medium may further contain appropriate minerals, salts, cofactors, buffers, and other components suitable for the growth and maintenance of the culture.

[0131] Fermentation processes, such as those for maize ethanol production, typically consist of two stages: a yeast growth stage and a fermentation stage. During the yeast growth stage, the mass of yeast increases to a level suitable for the fermentation stage. Typically, the growth stage is carried out in a continuous seed tank. A suitable culture medium containing salt, nutrients, and a carbon source (e.g., hydrolyzed corn mash, sugarcane molasses, or any other low-cost carbon source) is brought into contact with active dried yeast (ADY), yeast slurry, or pressed yeast. Yeast growth is preferably carried out under aerobic conditions, but can also be carried out under anaerobic conditions. Once a suitable yeast concentration is reached, the material is transferred to a fermentation tank to begin the fermentation stage. During the fermentation stage, the carbon source is converted into a main product such as ethanol and other by-products derived from the yeast's natural metabolism. In the fermentation stage of maize ethanol production, mash prepared from maize ground by a dry or wet grinding process is used. The wet grinding process involves fractionating the maize into various components, with only the starch fraction being added to the fermentation process. The dry milling process involves grinding corn kernels into meal and then mixing the meal with water and enzymes. Generally, two different dry milling processes are used. The commonly used process ("conventional process") involves grinding the starch-containing material, then liquefying the gelatinized starch at high temperatures, usually using bacterial α-amylase, followed by simultaneous saccharification and fermentation (SSF). Another well-known process, often called the "raw starch hydrolysis" process (RSH process), involves grinding the starch-containing material, then simultaneously saccharifying and fermenting the granular starch at a temperature below the initial gelatinization temperature, usually in the presence of acid-bacterial α-amylase and glucoamylase (see, for example, U.S. Patent No. 8,962,286).

[0132] In various embodiments, fermentation takes place at temperatures ranging from approximately 15°C to approximately 60°C, preferably from 28°C to approximately 35°C. In various embodiments, the pH range of fermentation is from pH 2.0 to pH 9.0. In some cases, the initial pH conditions are from pH 6.0 to pH 8.0. Fermentation can be carried out under aerobic or anaerobic conditions. Maize ethanol fermentation is usually carried out under anaerobic or microaerobic conditions. In some embodiments, air can be supplied during fermentation.

[0133] The appropriate fermentation run time ranges from approximately 24 to 96 hours, such as approximately 36 to 72 hours. The fermentation run time will vary depending on the amount of yeast transferred from the growth stage and the amount of starch enzymes during mash preparation and the SSF or RSH process. Once the carbon source is depleted, the fermented mash is transferred to the downstream process (DPS) to purify the produced ethanol and other by-products of additional costs (e.g., soluble dry distillation grain residue (DDGS)).

[0134] The methods and compositions disclosed herein can be adapted to conventional fermentation bioreactors (e.g., batch, feed-in batch, cell recycling, and continuous fermentation).

[0135] In some embodiments, the yeasts provided herein (e.g., genetically modified yeasts) are cultured in a liquid fermentation medium (i.e., in-liquid culture) that results in the discharge of fermentation products into the fermentation medium. In one embodiment, the final fermentation product can be isolated from the fermentation medium using any suitable method known in the art.

[0136] In some embodiments, the formation of fermentation products occurs during the initial rapid growth period of the yeast. In one embodiment, the formation of fermentation products occurs during a second period in which the culture is maintained in a slow-growing or non-growing state. In one embodiment, the formation of fermentation products occurs during two or more growth periods of the yeast. In such embodiments, the amount of fermentation product formed per unit time is generally a function of the metabolic activity of the yeast, physiological culture conditions (e.g., pH, temperature, medium composition), and the amount of yeast present in the fermentation process.

[0137] Ethanol and the co-products of interest can be separated and purified by the approaches described in the following paragraphs, taking into consideration that many separation and purification methods are known in the art and are not intended to be limited by the following disclosures.

[0138] For general processing of fermentation broths containing ethanol and low-boiling molecules, various methods can be employed to remove biomass and / or separate ethanol and low-boiling molecules from the culture broth and its components. A sugar-based raw material stream is converted into ethanol and other co-products of interest in a fermenter, as disclosed herein. In one embodiment of this disclosure, ethanol and one or more low-boiling co-products are produced, and these products are obtained in both the gas phase (off-gas) and the liquid phase (broth). Products in the off-gas are recovered in an absorption column or other washing device to minimize the loss of ethanol and low-boiling volatile co-products. This stream, containing the products recovered from the off-gas and the broth, can be mixed for further processing. Alternatively, a solid removal step may be performed, including centrifugation, decanting, filtration, or a combination thereof, and an operating unit system may be implemented depending on the size of the solid particles present in the broth. Optionally, non-condensable gas removal may be adapted, including a flash unit, or a distillation unit, or an absorption unit, or a combination thereof. The mixture can then proceed directly to a distillation column system comprising one or more distillation columns, but depending on the properties of the low-boiling molecules, the system may further include one or more additional operating units, including extractive distillation, azeotropic distillation, flashing, adsorption and absorption, or a combination thereof. At the end of these steps, ethanol and volatile products are obtained in the specifications required for the particular application.

[0139] Regarding the general processing of fermentation broths containing ethanol and high-boiling molecules, various methods can be employed to remove biomass and / or separate ethanol and high-boiling molecules from the culture broth and its components. The process of isolating ethanol from one or more high-boiling co-products is carried out by distillation to remove volatile substances (especially ethanol), followed by crystallization, solvent extraction, chromatographic separation, adsorption, filtration, salt splitting, precipitation, acidification, ion exchange, evaporation, or a combination thereof to obtain purified high-boiling molecules.

[0140] The fermentation products are subjected to a centrifuge unit to settle the cells and insoluble contents. The liquid supernatant contains water, ethanol, and soluble co-products. Distillation is then applied to separate the volatile products (particularly ethanol) as vapor, while the high-boiling co-products and salts remain in the liquid aqueous phase. The stream containing the liquid phase is then subjected to separation of the salts in a process involving one or more of the possible processes, including but not limited to crystallization, chromatographic separation, solvent extraction, adsorption, salt decomposition, sedimentation, filtration (ultrafiltration, nanofiltration, and / or microfiltration), acidification, ion exchange, or other processes and combinations thereof. The stream containing the high-boiling products in solution may be concentrated in a single distillation column or by one-step evaporation or multi-step evaporation, depending on the relative volatility related to other co-products or water. For example, if the high-boiling products are dispersed in water, the products collect at the bottom of the column and the water is removed at the top. If high-boiling point co-products form azeotropic mixtures with water, a series of extraction units or molecular sieves may be required. The recovered products can be finished in a dryer to reduce humidity and improve stability for further storage.

[0141] In another embodiment, biomass from a carbon source (e.g., unfermented grain residue) is also part of the fermentation broth. The fermentation products are subjected to a distillation process to separate volatile products (particularly ethanol) as vapor, while high-boiling co-products, cellular debris, brewing grains from the carbon source, and salts remain in the liquid phase. The products in the liquid phase are subjected to a centrifugation unit to precipitate cellular debris, the insoluble portion of the brewing grains, and other insoluble components. The supernatant of the centrifugation process leads to the separation of salts and potential portions of the brewing grains from high-boiling molecules in a process involving one or more of the possible processes, including but not limited to crystallization, chromatographic separation, solvent extraction, adsorption, salt decomposition, precipitation, filtration (ultrafiltration, nanofiltration, and / or microfiltration), acidification, ion exchange, or other processes and combinations thereof. The streams containing both the soluble and insoluble portions of the brewing grains can be combined and subjected to an evaporator unit and / or dryer to reduce humidity and constitute dry brewing grains (DDGS) with soluble portions. Streams containing high-boiling point products in solution can be concentrated in a single distillation column, by one-step evaporation, or by multiple evaporation steps, depending on the relative volatility of other co-products or water. [Examples]

[0142] Example 1: Modification of ethanol-producing yeast for the production of 1-propanol Yeast can be genetically modified to produce 1-propanol from fermentable carbon sources, such as glucose.

[0143] In an exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to succinyl-CoA; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of succinyl-CoA to methylmalonyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of methylmalonyl-CoA to propionyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde; and (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0144] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to 1,2-propanediol; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of 1,2-propanediol to propionaldehyde; and (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0145] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to threonine or citramaric acid; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of threonine or citramaric acid to 2-ketobutyric acid (2-kB); (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of 2-kB to propionyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde; and (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0146] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to lactic acid or β-alanine; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of lactic acid or β-alanine to acrylyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acrylyl-CoA to propionyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde; and (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0147] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to β-alanine; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of β-alanine to malonic acid semialdehyde; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of malonic acid semialdehyde to 3-hydroxypropionic acid (3-HP); (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of 3-HP to acryl-CoA; (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acryl-CoA to propionyl-CoA; (vi) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde; and (vii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0148] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to oxaloacetate malonate semialdehyde; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of oxaloacetate to malonate semialdehyde; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of malonate semialdehyde to 3-hydroxypropionic acid (3-HP); (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of 3-HP to acryl-CoA; (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acryl-CoA to propionyl-CoA; (vi) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionyl-CoA to propionaldehyde; and (vii) One or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propionaldehyde to 1-propanol.

[0149] Alternatively, yeast lacking one or more enzymes (e.g., one or more catalytically active functional enzymes) for the conversion of a fermentable carbon source to 1-propanol is genetically modified to include one or more polynucleotides encoding an enzyme (e.g., a functional enzyme including any enzyme disclosed herein) in a pathway that lacks the ability to catalyze the conversion of a fermentable carbon source to 1-propanol.

[0150] Example 2: Modification of ethanol-producing yeast for the production of acetone, 2-propanol, propene, and / or 1-butanol. In an exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to pyruvate or malonic acid semialdehyde (MSA); (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of pyruvate or MSA to acetyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetyl-CoA to acetoacetic acid; and (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetic acid to acetone.

[0151] In an exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to pyruvate or malonic acid semialdehyde (MSA); (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of pyruvate or MSA to acetyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetyl-CoA to malonyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of malonyl-CoA to acetoacetyl-CoA; (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetyl-CoA to acetoacetic acid; and (vi) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetic acid to acetone.

[0152] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to pyruvate or malonic acid semialdehyde (MSA); (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of pyruvate or MSA to acetyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetyl-CoA to acetoacetyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA (HMG-CoA); (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of HMG-CoA to acetoacetic acid; and (vi) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetic acid to acetone.

[0153] In an exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to pyruvate or malonate semialdehyde (MSA); (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of pyruvate or MSA to acetyl-CoA; (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetyl-CoA to malonyl-CoA; (iv) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of malonyl-CoA to acetoacetyl-CoA; (v) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetyl-CoA to hydroxymethylglutaryl-CoA (HMG-CoA); (vi) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of HMG-CoA to acetoacetic acid; and (vii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetoacetic acid to acetone.

[0154] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to acetone; and (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of acetone to isopropanol (2-propanol).

[0155] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to butyrate; (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of butyrate to propane; and (iii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of propane to 2-propanol.

[0156] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of a fermentable carbon source to 2-propanol; and (ii) one or more polynucleotides encoding an enzyme in a pathway that catalyzes the conversion of 2-propanol to propene.

[0157] In another exemplary method, yeast is genetically engineered by any method known in the art to include: (i) one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of a fermentable carbon source to butyric acid or butyryl-CoA; (ii) one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of butyric acid or butyryl-CoA to butanal; and (iii) one or more polynucleotides encoding enzymes in a pathway that catalyzes the conversion of butanal to 1-butanol.

[0158] Alternatively, yeast lacking one or more enzymes (e.g., one or more catalytically active functional enzymes) for the conversion of a fermentable carbon source to acetone, 2-propanol, propene, and / or 1-butanol is genetically modified to include one or more polynucleotides encoding an enzyme (e.g., a functional enzyme including any enzyme disclosed herein) in a pathway that lacks the ability to catalyze the conversion of a fermentable carbon source to acetone, 2-propanol, propene, and / or 1-butanol.

[0159] Example 3: Fermentation of glucose by genetically modified ethanol-producing yeast to produce 1-propanol, acetone, 2-propanol, propene, and / or 1-butanol. Using the genetically modified yeast produced in Example 1 or Example 2 above, a carbon source is fermented to produce 1-propanol, acetone, 2-propanol, propene, and / or 1-butanol.

[0160] In an exemplary method, a pre-sterilized culture medium containing a fermentable carbon source (e.g., 9 g / L glucose, 1 g / L KH2PO4, 2 g / L (NH4)2HPO4, 5 mg / L FeSO4·7H2O, 10 mg / L MgSO4·7H2O, 2.5 mg / L MnSO4·H2O, 10 mg / L CaCl2·6H2O, 10 mg / L CoCl2·6H2O, and 10 g / L yeast extract) is filled into a bioreactor.

[0161] During fermentation, anaerobic conditions are maintained, for example, by sparging nitrogen through the culture medium. A suitable temperature for fermentation (e.g., about 30°C) is maintained using any method known in the art. A nearly physiological pH (e.g., about 6.5) is maintained, for example, by the automatic addition of sodium hydroxide. The bioreactor is stirred, for example, at about 50 rpm. Fermentation is allowed to complete.

[0162] Example 4: Fermentation of glucose by genetically modified ethanol-producing yeast for the production of ethanol and low-boiling point co-products. Using the genetically modified yeast produced in Example 1 or Example 2 above, a carbon source is fermented to produce ethanol and one or more low-boiling point co-products such as 1-propanol, 2-propanol, acetone, methyl ethyl ketone, ethyl acetate, isopropyl acetate, ethane, and propene.

[0163] In an exemplary method, a pre-sterilized culture medium containing a fermentable carbon source (e.g., 9 g / L glucose, 1 g / L KH2PO4, 2 g / L (NH4)2HPO4, 5 mg / L FeSO4·7H2O, 10 mg / L MgSO4·7H2O, 2.5 mg / L MnSO4·H2O, 10 mg / L CaCl2·6H2O, 10 mg / L CoCl2·6H2O, and 10 g / L yeast extract) is filled into a bioreactor.

[0164] During fermentation, if used, anaerobic conditions are maintained, for example, by sparging nitrogen through the culture medium. A suitable temperature for fermentation (e.g., about 30°C) is maintained using any method known in the art. A nearly physiological pH (e.g., about 6.5) is maintained, for example, by the automatic addition of sodium hydroxide. The bioreactor is stirred, for example, at about 50 rpm. Fermentation is allowed to complete.

[0165] Example 5: Fermentation of glucose by genetically modified ethanol-producing yeast for the production of ethanol and high-boiling point co-products. Using the genetically modified yeast produced in Example 1 or Example 2 above, a carbon source is fermented to produce ethanol and one or more high-boiling point co-products such as monoethylene glycol, n-butanol, 3-hydroxypropionic acid, adipic acid, diethanolamine, and 1,3-propanediol.

[0166] In an exemplary method, a pre-sterilized culture medium containing a fermentable carbon source (e.g., 9 g / L glucose, 1 g / L KH2PO4, 2 g / L (NH4)2HPO4, 5 mg / L FeSO4·7H2O, 10 mg / L MgSO4·7H2O, 2.5 mg / L MnSO4·H2O, 10 mg / L CaCl2·6H2O, 10 mg / L CoCl2·6H2O, and 10 g / L yeast extract) is filled into a bioreactor.

[0167] During fermentation, if used, anaerobic conditions are maintained, for example, by sparging nitrogen through the culture medium. A suitable temperature for fermentation (e.g., about 30°C) is maintained using any method known in the art. A nearly physiological pH (e.g., about 6.5) is maintained, for example, by the automatic addition of sodium hydroxide. The bioreactor is stirred, for example, at about 50 rpm. Fermentation is allowed to complete.

[0168] Example 6: Effects of high concentrations of C3 and C4 alcohols on yeast Alcohol tolerance experiments were conducted in Saccharomyces cerevisiae to understand the negative effects of n-propanol (i.e., 1-propanol), 2-propanol, and 2-butanol compared to ethanol. Ethanol, a natural product (or native product) produced in sugar-ethanol fermentation, is generally well-tolerated by yeasts such as S. cerevisiae. However, existing approaches to producing non-natural chemicals, such as C3, C4, or C5 alcohols, ketones, organic acids, or other non-natural products (e.g., alcohols other than ethanol), by using genetically modified yeast are usually negatively affected by the high toxicity of such non-natural chemicals or alcohols (e.g., n-propanol and 2-propanol) to yeast cell growth and / or performance compared to ethanol.

[0169] Several concentrations of n-propanol, 2-propanol, 2-butanol, and ethanol were tested in yeast cultures. The experiments were conducted using the industrial ethanol-producing yeast strain PE-2 in a 250 mL shaking flask containing 50 mL of YNB medium with 40 g / L glucose at 32°C. The culture was then subjected to initial OD (Oxygen Demand) testing. 600nm The experiment was conducted at OD = 12 (optical density) for 8-9 hours. Samples were taken at appropriate intervals and analyzed by HPLC to measure glucose, ethanol, n-propanol, 2-propanol, and 2-butanol. The experiment was performed in pairs according to the parameters in Table 13. Conditions in which no sugar consumption was observed were considered lethal concentrations and were excluded from the analysis.

[0170] (Table 13) Experimental design - Yeast tolerance to C2, C3, and C4 alcohols TIFF2026094284000015.tif189143

[0171] The degree of glucose consumption inhibition was evaluated for various alcohol concentrations. Samples were tested 2 hours after inoculation. At this point, glucose had not been completely consumed. Linear regression curves are shown in Figure 6, and the results are provided in Table 14.

[0172] (Table 14) Dependence of sugar consumption inhibition on alcohol concentration TIFF2026094284000016.tif71160

[0173] As shown in Figure 6, unnatural n-propanol, 2-propanol, and 2-butanol negatively affect S. cerevisiae, with this effect being even greater at higher concentrations. As shown in Table 14, 2-butanol showed 2.94 times greater inhibition than ethanol. On the other hand, 2-propanol showed 1.45 times greater inhibition than ethanol. N-propanol showed an effect intermediate with 2-propanol and 2-butanol, exhibiting 2.16 times greater inhibition than ethanol. These results demonstrate how unnatural products such as n-propanol, 2-propanol, and 2-butanol can exacerbate the negative effects on yeasts such as Saccharomyces cerevisiae, impairing aspects of ethanol fermentation performance, such as sugar consumption profiles and therefore productivity.

[0174] Example 7: Simulation of industrial ethanol yeast fermentation performance, with ethanol as the main component and 1-propanol and 2-propanol produced simultaneously at non-toxic concentrations. Laboratory simulations were conducted to study the effects of co-products, or non-natural products produced in yeast along with ethanol, on yeast sugar-ethanol fermentation. Two conditions were tested: i) Condition 1, where industrial ethanol-producing yeast produced ethanol from added sugar in the culture medium, with ethanol exogenously added to reach the expected final ethanol titer; and ii) Condition 2, where the same industrial ethanol-producing yeast produced ethanol from added sugar in the culture medium, with concentrated solutions of n-propanol and 2-propanol (50 / 50 wt%) exogenously added to the culture medium to reach the same final titer concentrations as in Condition 1. Experiments were carried out using a 1 L bioreactor with a final volume of 0.7 L. pH was controlled to 4.5 by adding 25% w / w NaOH, at a temperature of 32°C, and stirring at 300 rpm. The industrial ethanol-producing yeast strain used was PE-2. The initial pitch was 0.7 g / L DWC, and the culture medium was YNB, which does not contain amino acids. The final sugar concentration was 224 g / L glucose. The experiment was conducted under sterile conditions.

[0175] The bioreactor was first filled with 650 ml of YNB medium and sugar, and after stabilizing the pH and temperature, a 50 mL suspension of yeast inoculated into the bioreactor was added. Next, 130 mL of 160 g / L concentrated ethanol solution was added for condition 1, and 130 mL of 177 g / L concentrated n-propanol and 2-propanol solution was added for condition 2. Each solution followed the following profile: 0.2 mL / min for the first 10 hours after inoculation; 0.4 mL / min from 11 to 15 hours; 0.6 mL / min from 16 to 40 hours; and 0.2 mL / min from 41 to 46 hours. This profile was added to simulate the ethanol production profile of PE-2 yeast. Fermentation was terminated after a 70-hour fermentation run. Samples were taken at appropriate intervals to measure ethanol, 1-propanol, 2-propanol, and glucose. The results are presented in Table 15 and Figure 7.

[0176] (Table 15) Ethanol-yeast fermentation with added alcohol TIFF2026094284000017.tif44170

[0177] As shown in Table 15, the yeast fermentation parameters for ethanol yield and volume productivity were similar under both conditions tested. In other words, the yeast ethanol fermentation profile of yeast cultures exposed to ethanol alone (Condition 1) was similar to that of those exposed to a mixture of n-propanol and 2-propanol (Condition 2). Compared to Condition 1, under Condition 2 (where 16.5% of the final total alcohol during fermentation was C3 alcohol, i.e., a combination of n-propanol and 2-propanol), the effect on ethanol yield and volume productivity was observed to be minimal or nonexistent. Furthermore, as shown in Figure 7, sugar consumption and ethanol production were similar under both test conditions. These results demonstrate that the test concentrations of n-propanol and 2-propanol avoid impairing yeast fermentation performance during ethanol fermentation, as assessed by ethanol fermentation yield and volume productivity.

[0178] Example 8: Recombinant ethanol-producing yeast that co-produces 3-hydroxypropionic acid along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain was genetically modified to co-produce 3-hydroxypropionic acid (3-hydroxypropionic acid) as the main component along with ethanol by switching the carbon flow from glucose as the carbon source. Saccharomyces cerevisiae is not naturally able to produce 3-hydroxypropionic acid from glucose. Therefore, the metabolic pathway and target enzyme for 3-hydroxypropionic acid production were heterologously expressed in Saccharomyces cerevisiae yeast (strain W303). Furthermore, the yeast strain was modified to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branch point by reducing the half-life of the PYK1 enzyme through the deletion of wild-type pyruvate kinase (PYK1) and the expression of the PYK1 enzyme downregulated using weaker promoters (pNUP57 and pMET25ΔF), thereby reducing the carbon flow from PEP to pyruvate and better controlling the amount of naturally produced ethanol.

[0179] As shown in Table 16, recombinant yeast strains YS_001 and YS_002 had 3-hydroxypropionic acid pathway-producing genes integrated into their genomes, including AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castaneum, PYD4 (PYD4.Lk) from L. kluyveri, and YDFG (YDFG.Ec) from Escherichia coli. Furthermore, these strains possess E. coli-derived PEP.CK (PEPCK.Ec), which is overexpressed to switch the carbon flow from PEP to oxaloacetate (OAA). All 3-hydroxypropionic acid biosynthesis pathway genes were codon-optimized to be optimally expressed in yeast under varying promoter strengths and varying gene copy numbers.

[0180] Ethanol fermentation was performed in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Stirring was performed at 135 rpm in an incubator with a shaking diameter of 50 mm. 3-hydroxypropionic acid, ethanol, glycerol, and glucose were measured after 48 hours of fermentation using standard analytical methods and instruments, and the results are shown in Table 16.

[0181] (Table 16) Simultaneous production of 3-hydroxypropionic acid with ethanol as the main component during anaerobic ethanol fermentation from glucose TIFF2026094284000018.tif101156

[0182] The recombinant yeast strain YS_001, using a moderately strong promoter (pMET25ΔF) for PYK1 expression, allowed for appropriate control of the sugar-to-glucan ratio to either ethanol as the main component or 3-hydroxypropionic acid as a byproduct at non-toxic levels, resulting in the desired sugar-ethanol fermentation profile. Despite genetic modification involving switching the carbon flow from glucose to either ethanol or 3-hydroxypropionic acid and introducing heterologous genes for the production of ethanol and non-natural 3-hydroxypropionic acid, YS_001 was able to consume all supplied glucose and showed very good cell proliferation, reaching a final OD600 of 61. The YS_001 recombinant yeast strain was capable of co-producing ethanol at a high concentration of 29 g / L and 3-hydroxypropionic acid at 4.7 g / L. In summary, the results in Table 16 indicate that 3-hydroxypropionic acid was co-produced with ethanol as the main component during sugar-ethanol fermentation, where the product ratio was controlled to maintain ethanol properties while producing 3-hydroxypropionic acid at low and non-toxic concentrations.

[0183] The results presented herein were demonstrated using recombinant yeast strain W303, but other Saccharomyces cerevisiae yeast strains, including industrial yeasts such as PE-2, CAT-1, BG-1, and Ethanol Red yeast strains widely used in industrial sugarcane-ethanol fermentation processes and maize-ethanol fermentation processes, can also be used.

[0184] Example 9: Recombinant ethanol-producing yeast that co-produces 1-propanol along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain was genetically modified to simultaneously produce 1-propanol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. Saccharomyces cerevisiae is normally only able to produce residual amounts of 1-propanol via the Ehrlich pathway, which is involved in branched-chain amino acid metabolism. The biosynthetic metabolic pathway and target enzymes for 1-propanol production were heterologously expressed in the W303 yeast strain. Furthermore, the yeast strain was modified to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branch point by reducing the half-life of the PYK1 enzyme through the deletion of wild-type pyruvate kinase (PYK1) and the expression of a downregulated PYK1 enzyme using a weak promoter such as pNUP57, thereby reducing the carbon flow from PEP to pyruvate and better controlling the amount of naturally produced ethanol.

[0185] As shown in Table 17, recombinant yeast strains YS_003 and YS_004 incorporated 1-propanol pathway-producing genes into their genomes in various copies, including AAT2 from S. cerevisiae (AAT2.Sc), PAND from T. castanum (PAND.Tca), PYD4 from L. kruiberi (PYD4.Lk), YDFG from Escherichia coli (YDFG.Ec), HPD1 from C. albicans (HPD1.Ca), PCT from C. propionicum (PCT.Cp), HPCD and ACR from R. pomeroi (HPCD.Rp and ACR.Rp), and PDUP from S. enterica (PDUP.Sen). All 1-propanol biosynthesis pathway genes were codon-optimized for optimal expression in yeast, and the constructed recombinant yeast strains overexpressed E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA). YS_003 and YS_004 had different 3-hydroxypropionic acid dehydrogenase candidates (3HPDH) involved in the conversion of MSA to 3-hydroxypropionic acid. YS_003 overexpressed the NADPH-dependent 3HPDH enzyme (YDFG.Ec), while YS_004 overexpressed the NADPH-dependent 3HPDH enzyme (HPD1.Cal).

[0186] The ethanol fermentation test was performed in a 125 mL fermentation flask sealed with a silicone cap pierced with two 1 mL pipette tips with filters, in the presence of 25 mL of rich medium containing 40 g / L glucose. An additional 40 g / L of glucose was added 24 hours after growth. Stirring was performed at 180 rpm in an incubator with a shaking diameter of 50 mm. 1-propanol, ethanol, glycerol, and glucose were measured after 48 hours of fermentation using standard analytical methods and instruments (GC / MS-MS), and the results are shown in Table 17.

[0187] (Table 17) Simultaneous production of 1-propanol with ethanol as the main component during ethanol fermentation from glucose TIFF2026094284000019.tif107159

[0188] The YS_003 and YS_004 recombinant yeast strains produced 1-propanol and, despite genetic modifications that switched the carbon flow from glucose, were able to consume all supplied glucose, showing relatively good cell proliferation reaching final OD600s of 43 and 59, respectively. During ethanol fermentation, the YS_003 and YS_004 recombinant yeast strains were able to produce 0.71 g / L and 1.13 g / L of 1-propanol, respectively, while producing ethanol as the main component at a high titer of 28–30 g / L depending on a glucose supply of 80 g / L. While we do not wish to be constrained by theory, the increased 1-propanol production in YS_004 is thought to be due to a higher copy number of aspartate decarboxylase and overexpression of NADH-dependent 3-hydroxypropionate dehydrogenase enzyme.

[0189] The results presented herein were demonstrated using recombinant yeast strain W303, but other Saccharomyces cerevisiae yeast strains, including industrial yeasts such as PE-2, CAT-1, BG-1, and Ethanol Red yeast strains widely used in industrial sugarcane-ethanol fermentation processes and maize-ethanol fermentation processes, can also be used.

[0190] Example 10: Recombinant ethanol-producing yeast that co-produces acetone along with ethanol as the main component during ethanol fermentation from glucose. Ethanol-producing S. cerevisiae yeast strains were genetically modified to simultaneously produce acetone as the main component along with ethanol by switching the carbon flow from glucose as the carbon source. The metabolic pathway and target enzymes for acetone production were heterologously expressed in the W303 yeast strain. As shown in Table 18, recombinant yeast strains YS_006 and YS_007 were derived from YS_005 and had acetone pathway-producing genes integrated into their genomes, including AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castanum, PYD4 (PYD4.Lk) from L. kruiberi, MSD (MSD.PA or MSD.Cal) from Pseudomonas aeruginosa and C. albicans, ERG10 (ERG10.Sc) from S. cerevisiae, ATOAD (ATOA.EC and ATOD.Ec) from Escherichia coli, ADC (ADC.Ca) from C. acetobutyricum, PTA (PTA.Cg) from C. glutamicum, and ACK (ACK.Ec) from Escherichia coli. All acetone biosynthesis pathway genes were codon-optimized for optimal expression in yeast, and the constructed recombinant yeast strain overexpressed E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA).

[0191] Ethanol fermentation was performed in a 125 mL fermentation flask with a silicone cap fitted with two filter pipette tips in the presence of 25 mL of rich medium containing 80 g / L glucose. Agitation was maintained at 135 rpm in an incubator with a shaking diameter of 50 mm. Acetone, ethanol, and glucose were measured after 48 hours of fermentation using standard analytical methods and instruments (GC / MS-MS headspace). Since the parent strain YS_005 lacks heterologous genes and related enzymes involved in acetone biosynthesis, strain YS_005 was used as a negative control in the fermentation assay.

[0192] (Table 18) Simultaneous production of acetone with ethanol as the main component during ethanol fermentation from glucose TIFF2026094284000020.tif73157

[0193] The YS_006 and YS_007 recombinant yeast strains were able to consume all supplied glucose despite genetic modification that switched the carbon flow from glucose to ethanol and introduced heterologous genes for ethanol and acetone production, showing good cell proliferation reaching a final OD600 of over 65. While maintaining ethanol performance by reaching a high titer of approximately 35 g / L of ethanol, very close to the amount produced by the acetone-incapable YS_005 strain, the YS_006 and YS_007 recombinant yeast strains were able to produce 0.7 g / L and 1.0 g / L of acetone, respectively. These results also demonstrate the expected increase in acetone production in the YS_007 strain, which includes additional copies of PAND.Tca and MSD.Pa, thought to promote the conversion of β-alanine to MSA and the conversion of MSA to acetyl-CoA, a major acetone precursor, although we do not wish to be constrained by theory.

[0194] The results presented herein were demonstrated using recombinant yeast strain W303, but other Saccharomyces cerevisiae yeast strains, including industrial yeasts such as PE-2, CAT-1, BG-1, and Ethanol Red yeast strains widely used in industrial sugarcane-ethanol fermentation processes and maize-ethanol fermentation processes, can also be used.

[0195] Example 11: Recombinant ethanol-producing yeast that co-produces 2-propanol along with ethanol as the main component during ethanol fermentation from glucose. Ethanol-producing S. cerevisiae yeast strains were genetically modified to simultaneously produce 2-propanol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. The metabolic pathway and target enzymes for 2-propanol production were heterologously expressed in the W303 yeast strain. As shown in Table 19, recombinant yeast strain YS_008 had 2-propanol pathway-producing genes integrated into its genome, including AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castanum, PYD4 (PYD4.Lk) from L. kruiberi, MSD (MSD.PA or MSD.Cal) from Pseudomonas aeruginosa and C. albicans, ERG10 (ERG10.Sc) from S. cerevisiae, ATOAD (ATOA.EC and ATOD.Ec) from Escherichia coli, ADC (ADC.Pp) from P. polymyxa, PTA (PTA.Cg) from C. glutamicum, ACK (ACK.Ec) from Escherichia coli, and IPDH1 (IPDH1.Cbe) from C. beijelinkyi. All 2-propanol biosynthesis pathway genes were codon-optimized for optimal expression in yeast, and the constructed recombinant yeast strains overexpressed E. coli-derived PEP.CK (PEPCK.Ec) to similarly switch the carbon flow from PEP to oxaloacetate (OAA).

[0196] Ethanol fermentation was performed in a 125 mL fermentation flask with a silicone cap and two filter pipette tips inserted, in the presence of 25 mL of rich medium containing 80 g / L glucose. Agitation was maintained at 135 rpm in an incubator with a shaking diameter of 50 mm. 2-propanol, ethanol, and glucose were measured after 48 hours of fermentation using standard analytical methods and instruments (GC / MS-MS).

[0197] (Table 19) Simultaneous production of 2-propanol with ethanol as the main component during ethanol fermentation from glucose TIFF2026094284000021.tif42158

[0198] The YS_008 recombinant yeast was able to reach a final OD600 of 100 despite genetic modification involving switching the carbon flow from glucose to ethanol and introducing heterologous genes for the production of ethanol and 2-propanol. The YS_008 recombinant yeast produced 1.42 g / L of 2-propanol and 39 g / L of ethanol, and was able to maintain good ethanol performance based on the supplied glucose g / L.

[0199] The results presented herein were demonstrated using recombinant yeast strain W303, but other Saccharomyces cerevisiae yeast strains, including industrial yeasts such as PE-2, CAT-1, BG-1, and Ethanol Red yeast strains widely used in industrial sugarcane-ethanol fermentation processes and maize-ethanol fermentation processes, can also be used.

[0200] Example 12: Recombinant ethanol-producing yeast that co-produces both 1-propanol and 2-propanol along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain was genetically modified to co-produce 1-propanol and 2-propanol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. The metabolic pathways and target enzymes for 1-propanol and 2-propanol production were heterologously expressed in the W303 yeast strain. Furthermore, the yeast strain was modified to downregulate the innate ethanol-producing metabolic pathway at the pyruvate branch point by reducing the half-life of the PYK1 enzyme through the deletion of wild-type pyruvate kinase (PYK1) and the expression of the PYK1 enzyme downregulated using a weak promoter such as pNUP57, thereby reducing the carbon flow from PEP to pyruvate and better controlling the amount of naturally produced ethanol.

[0201] As shown in Table 20, recombinant yeast strain YS_009 is derived from AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castanum, PYD4 (PYD4.Lk) from L. kruiberi, YDFG (YDFG.Ec) from Escherichia coli, YDF1 (YDF1.Sc) from S. cerevisiae, PCT (PCT.Cp) from C. propionicum, HPCD and ACR (HPCD.Rp and ACR.Rp) from R. pomeroi, PDUP (PDUP.Sen) from S. enterica, MSD (MSD.Pa or MSS.Ca) from Pseudomonas aeruginosa and C. albicans, ERG10 (ERG10.Sc) from S. cerevisiae, and ATOAD from Escherichia coli. Genes producing 1-propanol and 2-propanol pathways were integrated into the genome, including (ATOA.Ec and ATOD.Ec), ADC from P. polymixa (ADC.Pp), PTA from C. glutamicum (PTA.Cg), ACK from E. coli (ACK.Ec), and IPDH1 from C. beijelinky (IPDH1.Cbe). Furthermore, YS_009 was overexpressed with PEP.CK from E. coli (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA). All 1-propanol and 2-propanol biosynthesis pathway genes were codon-optimized to be optimally expressed in yeast under varying promoter strengths and varying gene copy numbers.

[0202] (Table 20) Simultaneous production of 1-propanol and 2-propanol with ethanol as the main component during ethanol fermentation from glucose. TIFF2026094284000022.tif48128

[0203] The YS_009 recombinant yeast strain was assayed in a 0.7 L bioreactor in the presence of 0.2 L YPD medium supplied with approximately 250 g / L of glucose. At the very beginning of fermentation, stirring was maintained at 500 rpm with aeration at 0.125 vvm. Ethanol, 1-propanol, acetone, 2-propanol, and glucose were measured using GC-MS / FID, and the results are shown in Table 21.

[0204] (Table 21) Simultaneous production of 1-propanol and 2-propanol with ethanol as the main component during ethanol fermentation from glucose. TIFF2026094284000023.tif45163

[0205] The YS_009 recombinant yeast was able to consume most of the supplied glucose and showed a high cell density, reaching an OD600 of 150 at 40 hours of fermentation. The YS_009 recombinant yeast was able to produce 1.5 g / L of 1-propanol and 2-propanol, along with a high-titer ethanol of 101 g / L, after 56 hours of fermentation. Furthermore, most of the carbon source from glucose was converted to ethanol, and a small portion of the carbon source was converted to 1-propanol and 2-propanol at non-toxic final concentrations. Glycerol was measured at a final titer of 1.4 g / L after 56 hours of fermentation.

[0206] The results presented herein were demonstrated using recombinant yeast strain W303, but other Saccharomyces cerevisiae yeast strains, including industrial yeasts such as PE-2, CAT-1, BG-1, and Ethanol Red yeast strains widely used in industrial sugarcane-ethanol fermentation processes and maize-ethanol fermentation processes, can also be used.

[0207] Example 13: Recombinant ethanol-producing yeast that co-produces acrylic acid along with ethanol as the main component during ethanol fermentation from glucose. We will genetically modify ethanol-producing S. cerevisiae yeast strains to simultaneously produce acrylic acid along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. We will heterologously express the acrylic acid biosynthesis metabolic pathway and target enzymes via 3-hydroxypropionic acid in experimental yeast strain W303, and also in industrial ethanol-producing yeast strains PE-2 and Red. Furthermore, we will modify the yeast strains to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branching point.

[0208] Recombinant yeast strains incorporate genes from the acrylic acid production pathway into their genomes, including AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castanum, PYD4 (PYD4.Lk) from L. kruiberi, YDFG (YDFG.Ec) from E. coli, HPD1 (HPD1.Ca) from C. albicans, PCT (PCT.Cp) from C. propionicum, HPCD (HPCD.Rp) from R. pomeroi, and acyl-CoA hydrolase YciA (YciA.Ec) from E. coli. All acrylic acid biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under the control of various promoter strengths and various gene copy numbers.

[0209] These recombinant yeast strains may also be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, the PYK1 enzyme may be downregulated using promoters of varying strengths, preferably weak promoters, to reduce the PYK1 enzyme half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0210] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. Acrylic acid, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. Acrylic acid is co-produced with ethanol as the main component in the g / L range.

[0211] Example 14: Recombinant ethanol-producing yeast that co-produces propionic acid along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain will be genetically modified to simultaneously produce propionic acid along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. The propionic acid biosynthesis metabolic pathway and target enzymes via 3-hydroxypropionic acid will be heterologously expressed in the W303 yeast strain, as well as in the industrial ethanol-producing yeast strains PE-2 and Ethanol Red. Furthermore, the yeast strain will be modified to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branching point.

[0212] Recombinant yeast strains incorporate propionic acid production pathway genes into their genomes, including AAT2 (AAT2.Sc) from S. cerevisiae, PAND (PAND.Tca) from T. castanum, PYD4 (PYD4.Lk) from L. kruybergii, YDFG (YDFG.Ec) from E. coli, HPD1 (HPD1.Ca) from C. albicans, PCT (PCT.Cp) from C. propionicum, HPCD (HPCD.Rp) from R. pomeroy, and ACR (ACR.Rp) from R. pomeroy, where PCT.Cp is involved in the CoA activation of 3-hydroxypropionic acid and CoA transfer from propionyl-CoA to other molecules that release propionic acid. All propionic acid biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under varying promoter strengths and varying gene copy numbers.

[0213] These recombinant yeast strains may be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, using a weak promoter, the PYK1 enzyme may be downregulated to reduce the PYK1 enzyme half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0214] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. Propionic acid, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. Propionic acid is produced with ethanol as the main component at concentrations of 5 g / L, 10 g / L, 15 g / L or higher.

[0215] Example 15: Recombinant ethanol-producing yeast that co-produces butanone along with ethanol as the main component during ethanol fermentation from glucose. We will genetically modify an ethanol-producing S. cerevisiae yeast strain to simultaneously produce butanone along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. Butanone can be produced via propionyl-CoA and acetyl-CoA condensation, where both intermediates are derived from malonic acid semialdehyde. This biosynthetic metabolic pathway and target enzyme will be heterologously expressed in the W303 strain, and also in the widely used industrial ethanol-producing yeast strains PE-2 and Red. Furthermore, we will modify the yeast strain to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branching point.

[0216] These recombinant yeast strains include AAT2 (AAT2.Sc) derived from S. cerevisiae, PAND (PAND.Tca) derived from T. castanum, PYD4 (PYD4.Lk) derived from L. kruiberi, YDFG (YDFG.Ec) derived from E. coli, HPD1 (HPD1.Ca) derived from C. albicans, PCT (PCT.Cp) derived from C. propionicum, HPCD and ACR (HPCD.Rp and ACR.Rp) derived from R. pomeroi, MSD (MSD.Pa or MSD.Ca) derived from C. albicans or Pseudomonas aeruginosa, β-ketothiolase BktB (BtkB.Cn) derived from C. nekator, ATOAD (ATOA.Ec and ATOD.Ec) derived from E. coli, and ADC derived from C. acetobutyricum or P. polymixa. The butanone production pathway genes, including (ADC.Ca or ADC.Pp), are incorporated into the genome. All butanone biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under varying promoter strengths and varying gene copy number controls.

[0217] These recombinant yeast strains may be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, using a weak promoter, the PYK1 enzyme may be downregulated to reduce its half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0218] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. Butanone, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. Butanone with ethanol as the main component is co-produced at concentrations of 5 g / L, 10 g / L, 15 g / L, or higher.

[0219] Example 16: Recombinant ethanol-producing yeast that co-produces 2-butanol along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain is genetically modified to simultaneously produce 2-butanol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. 2-butanol can be produced from MSA-derived butanone, as described in the previous example. The 2-butanol biosynthesis metabolic pathway and target enzymes are heterologously expressed in the W303 yeast strain, and also in the widely used industrial ethanol-producing yeast strains, PE-2 and Ethanol Red. Furthermore, the yeast strain is modified to downregulate the innate ethanol-producing metabolic pathway at the pyruvate branching point.

[0220] These recombinant yeast strains include AAT2 (AAT2.Sc) derived from S. cerevisiae, PAND (PAND.Tca) derived from T. castanum, PYD4 (PYD4.Lk) derived from L. kruiberi, YDFG (YDFG.Ec) derived from Escherichia coli, HPD1 (HPD1.Ca) derived from C. albicans, PCT (PCT.Cp) derived from C. propionicum, HPCD and ACR (HPCD.Rp and ACR.Rp) derived from R. pomeroi, MSD (MSD.Pa or MSD.Ca) derived from C. albicans or Pseudomonas aeruginosa, β-ketothiolase BktB (BtkB.Cn) derived from C. nekator, ATOAD (ATOA.Ec and ATOD.Ec) derived from Escherichia coli, and ADC derived from C. acetobutylcum or P. polymixa. The 2-butanol production pathway genes, including (ADC.Ca or ADC.Pp) and the secondary alcohol dehydrogenase ADH (ADH.Lb) from L. brevis, are incorporated into the genome. All 2-butanol biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under the control of various promoter strengths and various gene copy numbers.

[0221] These recombinant yeast strains may also be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, using a weak promoter, the PYK1 enzyme may be downregulated to reduce its half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0222] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. 2-butanol, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. 2-butanol is co-produced with ethanol as the main component at concentrations of 5 g / L, 10 g / L, 15 g / L, or higher.

[0223] Example 17: Recombinant ethanol-producing yeast that co-produces propyl acetate along with ethanol as the main component during ethanol fermentation from glucose. Ethanol-producing S. cerevisiae yeast strains are genetically modified to simultaneously produce propyl acetate along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. Propyl acetate can be produced by esterification of 1-propanol and acetyl-CoA. The propyl acetate biosynthesis metabolic pathway and target enzymes are heterologously expressed in the W303 strain, as well as in the industrial ethanol-producing yeast strains PE-2 and Ethanol Red. Furthermore, yeast strains are modified to downregulate the innate ethanol-producing metabolic pathway at the pyruvate branching point.

[0224] These recombinant yeast strains include AAT2 (AAT2.Sc) derived from S. cerevisiae, PAND (PAND.Tca) derived from T. castanum, PYD4 (PYD4.Lk) derived from L. kruiberi, YDFG (YDFG.Ec) derived from Escherichia coli, HPD1 (HPD1.Ca) derived from C. albicans, PCT (PCT.Cp) derived from C. propionicum, HPCD and ACR (HPCD.Rp and ACR.Rp) derived from R. pomeroyi, MSD (MSD.Pa or MSD.Ca) derived from C. albicans or Pseudomonas aeruginosa, PDUP (PDUP.Sen) derived from S. enterica, ADH1 (ADH1.Sc) derived from S. cerevisiae, MSD (MSD.Ca or MSD.Pa) derived from C. albicans or Pseudomonas aeruginosa, and alcohol O-acetyltransferase 1 ATF1 derived from S. cerevisiae. The genes for the propyl acetate production pathway, including (ATF1.Sc), are incorporated into the genome. All genes for the propyl acetate biosynthesis pathway are codon-optimized to be optimally expressed in yeast under the control of various promoter strengths and various gene copy numbers.

[0225] These recombinant yeast strains may be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, by using a weak promoter such as pMET25DF or pNUP57, the PYK1 enzyme may be downregulated to reduce its half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0226] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. Propyl acetate, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. Propyl acetate is co-produced in the g / L range, with ethanol as the main component.

[0227] Example 18: Recombinant ethanol-producing yeast that co-produces 2,3-butanediol along with ethanol as the main component during ethanol fermentation from glucose. The ethanol-producing S. cerevisiae yeast strain will be genetically modified to simultaneously produce 2,3-butanediol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. The 2,3-butanediol biosynthesis metabolic pathway and target enzymes will be heterologously expressed in the W303 strain, and also in the industrial ethanol-producing yeast strains PE-2 and Red.

[0228] These recombinant yeast strains incorporate 2,3-butanediol production pathway genes into their genomes, including acetolactate synthase ALS (ALS.Pp) from P. polymixa, acetolactate decarboxylase (ALD.Bs) from Bacillus subtilis, and 2,3-butanediol dehydrogenase (BDH.Ca) from C. autoethanogenum. All 2,3-butanediol biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under varying promoter strengths and varying gene copy numbers. Beyond the expression of enzymes competing for the same substrate, pyruvate, further carbon diversion from ethanol to 2,3-butanediol can be achieved by genetic manipulation that reduces pyruvate decarboxylase (PDC) activity, such as by using weak promoters and / or deleting one or more isozymes.

[0229] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. The mixture is stirred at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. 2,3-butanediol, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. 2,3-butanediol is co-produced with ethanol as the main component at concentrations of 5 g / L, 10 g / L, 15 g / L, or higher.

[0230] Example 19: Recombinant ethanol-producing yeast that co-produces succinic acid along with ethanol as the main component during ethanol fermentation from glucose. We will genetically modify an ethanol-producing S. cerevisiae yeast strain to simultaneously produce succinic acid along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. We will heterologously express the succinic acid biosynthesis metabolic pathway and target enzymes in experimental yeast strain W303, and also in industrial ethanol-producing yeast strains PE-2 and Red. Furthermore, we will modify the yeast strain to downregulate the natural ethanol-producing metabolic pathway at the pyruvate branching point.

[0231] These recombinant yeast strains incorporate succinate production pathway genes into their genomes, including malate dehydrogenase Mdh (MDH.Rd) derived from R. delemar, and fumarate reductases FumC and FumABCD (FUMC.Ec and FUMABCD.Ec) derived from E. coli. All heterologous genes are codon-optimized to be optimally expressed in yeast under varying promoter strengths and gene copy number control.

[0232] These recombinant yeast strains may also be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, by using a weak promoter such as pMET25DF, the PYK1 enzyme may be downregulated to reduce its half-life, thereby reducing the carbon flow from PEP to pyruvate and better controlling the amount of naturally produced ethanol.

[0233] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. Maintain stirring at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. Succinic acid, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. Succinic acid is co-produced in the g / L range, with ethanol as the main component.

[0234] Example 20: Recombinant ethanol-producing yeast that co-produces 1,4-butanediol along with ethanol as the main component during ethanol fermentation from glucose. Ethanol-producing S. cerevisiae yeast strains are genetically modified to co-produce 1,4-butanediol along with ethanol as the main component by switching the carbon flow from glucose as the carbon source. The 1,4-butanediol biosynthesis metabolic pathway and target enzymes are heterologously expressed in strain W303, and also in industrial ethanol-producing yeast strains such as PE-2, BG-1, CAT-1, and Red, continuing to downregulate the innate ethanol-producing metabolic pathway at the pyruvate branch point, as demonstrated.

[0235] These recombinant yeast strains incorporate genes for the 1,4-butanediol production pathway into their genomes, including malate dehydrogenase Mdh (MDH.Rd) from R. delemar, fumarate FumC, fumarate reductase FumABCD, and succinyl-CoA synthetase SucCD (FUMC.Ec, FUMABCD.Ec, and SUCCD.Ec) from Escherichia coli, CoA-dependent succinate semialdehyde dehydrogenase SucD, 4-hydroxybutyrate dehydrogenase 4bdh, and CoA-acyltransferase Cat2 (SUCD.Pg, 4HBDH.Pg, and CAT2.Pg) from P. gingivalis, and CoA-dependent aldehyde dehydrogenase ALD and alcohol dehydrogenase ADH (ALD.Ca and ADH.Ca) from C. acetobutyricum. All 1,4-butanediol biosynthesis pathway genes are codon-optimized to be optimally expressed in yeast under the control of promoters of varying strengths and gene copy numbers.

[0236] These recombinant yeast strains may be overexpressed with E. coli-derived PEP.CK (PEPCK.Ec) to switch the carbon flow from PEP to oxaloacetate (OAA), and optionally, by using a weak promoter such as pMET25DF, the PYK1 enzyme may be downregulated to reduce its half-life, thereby reducing the carbon flow from PEP to pyruvate and allowing for better control of the amount of naturally produced ethanol.

[0237] The fermentation test is carried out in a 125 mL fermentation flask in the presence of 25 mL of YPD medium containing 80 g / L glucose. The mixture is stirred at 135 rpm in an incubator with a shaking diameter of 50 mm at 30–35°C. 1,4-butanediol, ethanol, glycerol, and glucose are measured after 48 hours of fermentation using standard instruments and analytical methods. 1,4-butanediol is co-produced with ethanol in the g / L range.

[0238] Example 21: Recombinant ethanol-producing yeast that co-produces one or more co-products under industrial ethanol fermentation conditions based on industrial sugarcane raw materials. Industrial ethanol-producing S. cerevisiae yeast strains are genetically modified to produce ethanol along with one or more co-products during an industrial ethanol fermentation process from sugarcane as a carbon source. These are preferably industrial ethanol-producing S. cerevisiae strains already used industrially in sugarcane-ethanol fermentation processes, including strains PE-2, BG-1, and CAT-1.

[0239] This genetically modified S. cerevisiae yeast strain can be obtained as described in the previous examples, so as to be able to produce ethanol with one or more co-products at non-toxic concentrations. This genetically modified S. cerevisiae yeast strain can produce ethanol with 1-propanol, acetone, 2-propanol, or a combination thereof. This genetically modified S. cerevisiae yeast strain can produce ethanol with 1-propanol, acetone, 2-propanol, or a combination thereof at non-toxic concentrations for industrial ethanol-producing yeast strains PE-2, BG-1, and CAT-1.

[0240] This genetically modified S. cerevisiae yeast strain can co-produce ethanol, 1-propanol, acetone, 2-propanol, or a combination thereof, from industrial sugarcane material through small-scale fermentation tests mimicking industrial sugarcane-ethanol fermentation conditions. This genetically modified S. cerevisiae yeast strain is tested in a 500 mL flask using 200 mL of sugarcane molasses with a TRS (total reducing sugars) content of 170 g / L. 70 mL of yeast suspension (inoculation solution) containing approximately 100 g / L (DWC) is mixed with 140 mL of molasses. The flask is stoppered with an airlock type S (to promote anaerobic conditions). The culture is then carried out at 32°C and 150 rpm for 8 hours. At the end of the culture, the fermentation liquid (beer) is centrifuged, and the yeast pellet is separated from the clarified fermentation liquid. The yeast pellet is resuspended in 74 mL of the clarified fermentation liquid. Samples are taken from the clarified fermentation broth and from the resuspended yeast. A new cycle is then started by mixing 140 mL of molasses (170 g / L TRS) with 70 mL of resuspended yeast (4 mL used as a sample). This procedure is repeated for 20 cycles. Samples at the end of each fermentation are taken for HPLC, GC-MS / FID, and standard analytical methods known to those skilled in the art. Glucose, sucrose, ethanol, glycerol, 1-propanol, acetone, and 2-propanol are measured. This genetically modified S. cerevisiae yeast strain exhibits very similar robustness and performance (ethanol yield and potency, etc.) for industrial ethanol fermentation to that expected from its parent industrial ethanol-producing yeast strains PE-2, BG-1, and CAT-1. Alcohol yield is approximately 0.43–0.46 grams of total alcohol per gram of sugar, while the total ethanol potency is approximately 60–80 g / L. Ethanol is present in an amount of approximately 80–85% by weight based on the total weight of the alcohol produced. Meanwhile, the total concentration of the resulting alcohols (n-propanol, 2-propanol, and acetone) is approximately 15–20% by weight.These results demonstrate a process for producing industrial ethanol-producing yeast that has been genetically modified to enable its use in the production of ethanol as a main component at non-toxic concentrations, along with 1-propanol, acetone, and / or 2-propanol, without compromising the robustness and fermentation performance of the parent yeast suitable for industrial production.

[0241] Example 22: Recombinant ethanol-producing yeast that co-produces one or more co-products under industrial ethanol fermentation conditions based on industrial corn raw materials. An industrial ethanol-producing S. cerevisiae yeast strain is genetically modified to produce ethanol along with one or more co-products during an industrial ethanol fermentation process from maize raw material as a carbon source. This is preferably an industrial ethanol-producing S. cerevisiae strain already used industrially in maize-ethanol fermentation processes, such as the Ethanol Red® (Leaf-Lesaffre) strain.

[0242] This genetically modified S. cerevisiae yeast strain is obtained as described in the previous examples, so as to be able to produce ethanol with one or more co-products at non-toxic concentrations. This genetically modified S. cerevisiae yeast strain can produce ethanol with 1-propanol, acetone, 2-propanol, or a combination thereof. This genetically modified S. cerevisiae yeast strain can produce ethanol with 1-propanol, acetone, 2-propanol, or a combination thereof at concentrations that are non-toxic to industrial ethanol-producing yeast strains such as Ethanol Red® (Leaf-Lesaffre) strain.

[0243] This genetically modified S. cerevisiae yeast strain can co-produce ethanol, 1-propanol, acetone, 2-propanol, or a combination thereof, from industrial maize material through small-scale fermentation tests mimicking industrial maize-ethanol fermentation conditions. This genetically modified S. cerevisiae yeast strain is tested in a 3.5 L bioreactor using 1 L of partially hydrolyzed corn mash. An appropriate amount of glucoamylase enzyme is added, and 0.5 g / L of fresh yeast is inoculated. The initial pH is adjusted to 4.5, but there is no control during fermentation. The temperature is set to 35°C with stirring at 300 rpm. The culture is run for 72 hours, and samples are taken at appropriate intervals. The experiment is performed in sets of three.

[0244] Sugars, glucose, ethanol, glycerol, 1-propanol, acetone, and 2-propanol were measured using HPLC, GC-MS / FID, and other standard analytical methods. This genetically modified S. cerevisiae yeast strain exhibits robustness and performance (such as ethanol yield and potency) for industrial ethanol fermentation that is remarkably similar to industrial ethanol-producing yeast strains. The alcohol yield is approximately 0.43–0.46 grams of total alcohol per gram of sugar, while the total ethanol potency is approximately 120–150 g / L. Ethanol is present in an amount of approximately 80–85% by weight based on the total weight of alcohol produced. Meanwhile, the total concentration of alcohols (n-propanol, 2-propanol, and acetone) is obtained at approximately 15–20% by weight. These results demonstrate a process for producing industrial ethanol-producing yeast that has been genetically modified to enable its use in the production of ethanol as a main component at non-toxic concentrations, along with 1-propanol, acetone, and / or 2-propanol, without compromising the robustness and fermentation performance of the parent yeast suitable for industrial production.

[0245] Unless otherwise stated, all numerical values ​​indicating the quantities and properties of components used herein and in the claims, such as molecular weight and reaction conditions, should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise stated, the numerical parameters expressed herein and in the appended claims are approximations that may vary depending on the desired properties sought by this disclosure. At a minimum, and not as an attempt to limit the application of the doctrine of equivalents to the claims, each numerical parameter should be interpreted in light of the reported significant figures and by applying common rounding techniques.

[0246] Although it has been stated that the numerical ranges and parameters representing the broad scope of this disclosure are approximations, the numerical values ​​shown in the specific examples are reported as precisely as possible. However, each numerical value inherently contains a certain degree of error that inevitably arises from the standard deviation observed in each test measurement.

[0247] In the descriptions of this disclosure (particularly in the claims below), the terms “a,” “an,” “the,” and similar reference subjects are to be interpreted as encompassing both singular and plural, unless otherwise specifically stated herein or unless explicitly contradicted by the context. The descriptions of value ranges herein are intended merely as a convenient way of indicating each individual value included within that range. Unless otherwise specifically stated herein, each individual value is incorporated herein as if it were individually described herein. All methods herein may be performed in any appropriate order, unless otherwise specifically stated herein or unless explicitly contradicted by the context. The use of any examples or illustrative language provided herein (e.g., “such as”) is intended merely to better illustrate this disclosure and does not impose any limitation on the scope of this disclosure beyond what is stated in the claims. The absence of language herein is to be interpreted as indicating any element not described in the claims but essential for the implementation of this disclosure.

[0248] Groupings of other elements or aspects of the present disclosure disclosed herein should not be construed as limiting. Each member of a group may be referred to individually or in any combination with other members of the group or other elements found herein and may be recited in the claims. One or more members of a group may be expected to be included in or deleted from the group for convenience and / or patentability reasons. If such inclusion or deletion occurs, this specification includes the modified group and is thus considered to meet the requirements of all Markush group recitations used in the appended claims.

[0249] This specification describes certain aspects of the present disclosure, including what is presently known to the inventors as the best mode for carrying out the present disclosure. Of course, variations to these described aspects will be apparent to those skilled in the art upon reading the foregoing description. The inventors anticipate that those skilled in the art will use such variations as appropriate, and the inventors intend for the present disclosure to be implemented in a manner different from that specifically described herein. Accordingly, the present disclosure includes any modifications and equivalents permitted by the applicable law of the subject matter recited in the claims appended hereto. Further, unless otherwise specifically described herein or clearly contradicted by context, any combination of the above elements in all possible variations is included in the present disclosure.

[0250] The specific aspects disclosed herein may be further limited in the claims by use of the phrases consisting of and / or consisting essentially of. The aspects of the present disclosure so recited in the claims are inherent to or clearly described in this specification and are practicable.

[0251] It will be understood that the aspects of the disclosure disclosed herein are illustrative of the principles of the disclosure. Other modifications that may be used are included within the scope of the disclosure. Accordingly, other configurations of the disclosure may be used in accordance with the teachings herein, although these are examples and not limited to those shown herein. Accordingly, the disclosure is not strictly limited to those shown and described herein.

[0252] While this disclosure has been described and explained herein with reference to various specific materials, procedures, and examples, it will be understood that this disclosure is not limited to specific combinations of materials and procedures chosen for its purposes. Numerous variations of such details may be implied and will be recognized by those skilled in the art. This specification and the examples are intended to be illustrative only, and the true scope and spirit of this disclosure are shown in the appended claims. All references, patents, and patent applications referenced herein are incorporated herein by reference in their entirety.

Claims

1. A method for producing ethanol and one or more co-products, comprising the following steps: (a) The step of bringing a fermentable carbon source into contact with ethanol-producing yeast in a fermentation medium; (b) a step of fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more co-products from the fermentable carbon source, and the ethanol produced is present at a higher mg / mL concentration than the co-products produced; and (c) The step of isolating the ethanol and the one or more co-products. Includes, A method wherein the yeast is a recombinant yeast genetically modified to produce one or more coproducts.

2. The method according to claim 1, wherein the carbon source is glucose or dextrose.

3. The method according to claim 1 or 2, wherein the carbon source is derived from a renewable cereal source obtained by saccharification of starch-based raw materials, such as corn, wheat, rye, barley, oats, rice, or a mixture thereof.

4. The method according to any one of the claims, wherein the carbon source is derived from renewable sugars, such as sugarcane, sugar beet, cassava, sweet sorghum, or a mixture thereof.

5. The method according to any one of the claims, wherein the ethanol-producing yeast is Saccharomyces cerevisiae.

6. The method according to claim 5, wherein Saccharomyces cerevisiae is an industrial strain, any common strain used in the ethanol industry, a typical laboratory strain, or any strain resulting from a typical method of crossing strains.

7. The method according to any one of the claims, wherein the co-product is produced at a concentration that is non-toxic to ethanol-producing yeast.

8. The method according to any one of the claims, wherein the produced ethanol is present in an amount of at least 70% by weight, for example, at least 75% by weight, at least 80% by weight, at least 85% by weight, at least 90% by weight, or at least 95% by weight, based on the total weight of the produced ethanol and co-products.

9. The method according to any one of the claims, wherein the fermentation is carried out as a batch process, a feed batch process, or a continuous process.

10. The method according to any one of the claims, wherein the fermentation is carried out under anaerobic conditions at a temperature of approximately 15°C to approximately 60°C for approximately 24 hours to approximately 96 hours.

11. The method according to any one of the claims, wherein the fermentation is carried out in an industrial ethanol plant, preferably in an existing industrial ethanol plant.

12. The method according to any one of the claims, wherein one or more co-products are selected from the group consisting of alcohols other than ethanol; ketones; glycols; ethers; esters; diamines; carboxylic acids; amino acids; dienes; and alkenes.

13. The method according to any one of the claims, wherein one or more co-products are selected from the group consisting of 1-butanol, 2-butanol, isobutanol, methanol, n-propanol, isopropanol, isoamyl alcohol, acetone, methyl ethyl ketone, methyl propionate, 1,3-propanediol, monoethylene glycol, propylene glycol, citric acid, lactic acid, succinic acid, adipic acid, acetic acid, glutamic acid, propionic acid, franzicarboxylic acid, 2,4-franzicarboxylic acid, 2,5-franzicarboxylic acid, 3-hydroxypropionic acid, acrylic acid, itaconic acid, glutamic acid, ethyl acetate, isopropyl acetate, propyl acetate, isoprenol, 1,3-butanediol, 1,4-butanediol, 2,3-butanediol, diethanolamine, tryptophan, threonine, methionine, lysine, serine, tyrosine, butadiene, isoprene, ethane, and propene.

14. The method according to any one of the claims, wherein the step of isolating ethanol and one or more co-products comprises a process selected from distillation, adsorption, crystallization, absorption, electrodialysis, solvent extraction, ion exchange resin chromatography, evaporation, or a combination thereof.

15. A method for producing ethanol and one or more co-products, comprising the following steps: (a) The step of bringing a fermentable carbon source into contact with ethanol-producing yeast in a fermentation medium; (b) a step of fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more low-boiling point coproducts from the fermentable carbon source, and the ethanol produced is present at a higher mg / mL concentration than the coproducts produced; and (c) The step of isolating the ethanol and the one or more low-boiling point co-products. Includes, A method wherein the yeast is a recombinant yeast genetically modified to produce one or more coproducts.

16. The method according to claim 15, wherein the low boiling point co-product has a boiling point of 100°C or less at a standard pressure of 100 kPa (1 bar), for example, 99°C or less, 98°C or less, 97°C or less, 95°C or less, 90°C or less, 85°C or less, 80°C or less, 75°C or less, 70°C or less, 65°C or less, or 60°C or less.

17. The method according to any one of claims 15 or 16, wherein one or more low-boiling point coproducts are selected from acetone, 1-propanol, 2-propanol, or a combination thereof.

18. The method according to any one of claims 15, 16, or 17, wherein the step of isolating ethanol and one or more low-boiling point co-products is performed by a continuous distillation unit.

19. A method for producing ethanol and one or more co-products, comprising the following steps: (a) The step of bringing a fermentable carbon source into contact with ethanol-producing yeast in a fermentation medium; (b) a step of fermenting the yeast in the fermentation medium, wherein the yeast produces ethanol and one or more high-boiling point coproducts from the fermentable carbon source, and the ethanol produced is present at a higher mg / mL concentration than the coproducts produced; and (c) The step of isolating the ethanol and the one or more high-boiling point co-products. Includes, A method wherein the yeast is a recombinant yeast genetically modified to produce one or more high-boiling point coproducts.

20. The method according to claim 19, wherein the high-boiling point co-product has a boiling point of over 100°C at a standard pressure of 100 kPa (1 bar), for example, over 105°C, over 110°C, over 120°C, over 130°C, over 140°C, over 150°C, over 160°C, over 170°C, over 180°C, over 190°C, over 200°C, over 210°C, over 220°C, over 230°C, over 240°C, or over 250°C.

21. The method according to any one of claims 19 or 20, wherein the step of isolating ethanol and one or more high-boiling point co-products is carried out by distillation and a process selected therefrom from crystallization, solvent extraction, chromatographic separation, salt decomposition, precipitation, acidification, ion exchange, evaporation, or a combination thereof.

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