Genetically modified yeast and fermentation process for production of ethanol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing ethanol. The genetically engineered yeast cell comprises an exogenous polynucleotide sequence encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO: 28, 32, 48, 52, 64, 68, 80, 92, and 96.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 371,692, filed on August 17, 2022, which is incorporated herein by reference in its entirety.

[0003] Reference to sequence listing submitted via Patent Center

[0004] The contents of the sequence listing XML file named "PT-1298-WO-PCT.xml", which was created on August 3, 2023 and electronically submitted via Patent Center with this application, and is 479,867 bytes in size, are incorporated herein by reference in its entirety. Background Art

[0005] Many fermentation raw materials are derived from plant sources (e.g., corn mash), in which carbohydrates are primarily in the form of starch polymers. The starch polymers in these raw materials must be processed into low molecular weight sugars that can be consumed by yeast and used to grow and produce bioproducts. Typical treatments include acid hydrolysis and / or enzymatic hydrolysis, in which the polymer chains are hydrolyzed to produce sugars that can be used by yeast. Starch degrading enzymes such as alpha amylase and glucoamylase can be added to convert the polymers into simple sugars. However, adding this enzyme can significantly increase the cost and complexity of the fermentation process.

[0006] It may be challenging to realize heterologous expression and functionality of enzymes in yeast to help starch hydrolysis, because it is difficult to know whether nucleic acids will be correctly expressed and whether functional enzymes will be formed, and whether the active form of enzymes will be secreted from cells. It is also challenging to engineer yeast to grow and produce bioproducts under non-optimal conditions such as high temperatures and high bioproduct titers. For example, although it is a well-known industrial process to produce ethanol by fermentation, it is difficult to minimize the formation of byproducts in order to reduce the dependence on supplementary enzymes, so that ethanol rates, titers and productivity are maintained under non-optimal conditions (e.g., temperature), and it may be difficult to minimize byproduct formation in technology. Increased ethanol concentration and unwanted byproduct accumulation may also be unfavorable to cell health. Summary of the invention

[0007] The present disclosure provides a genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 48, 52, 64, 68, 80, 92, and 96. The gapN enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 48, 52, and 64. The gapN enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 52, and 64. The gapN enzyme may be at least 85% identical to SEQ ID NO: 28; the gapN enzyme may be at least 85% identical to SEQ ID NO: 32; the gapN enzyme may be at least 85% identical to SEQ ID NO: 52; and / or the gapN enzyme may be at least 85% identical to SEQ ID NO: 64. The engineered yeast cell is capable of producing ethanol at a titer of at least 60 g / L, at least 80 g / L, at least 100 g / L, or at least 120 g / L ethanol after 48 hours, and wherein the glycerol production of the engineered yeast cell is reduced relative to the glycerol production in an equivalent yeast cell lacking the gapN enzyme.

[0008] The engineered yeast cell may additionally comprise an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 98 and 100. The engineered yeast cell may comprise a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene. The engineered yeast cell may comprise a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GDP) gene. The engineered yeast cell may additionally comprise an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme. The encoded GA enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1, 122, 123, and 124. The engineered yeast cell is capable of producing ethanol at a titer of at least 60 g / L, at least 80 g / L, at least 100 g / L, or at least 120 g / L ethanol after 48 hours, and wherein the glycerol production of the engineered yeast cell is reduced relative to the glycerol production in an equivalent yeast cell lacking the gapN enzyme.

[0009] Also provided is a genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme, the glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme being at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 28, 32, 48, 52, 64, 68, 80, 92 and 96; an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme, the alcohol dehydrogenase (ADH) enzyme being identical to SEQ ID NOs: NO: 98 and 100 at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical; exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme; and a deletion or disruption of at least one allele of a native GPP gene. The encoded GA enzyme may be at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NO: 1, 122, 123 and 124.

[0010] One or more of the exogenous polynucleotide sequences can be operably connected to a heterologous promoter or an artificial promoter. The promoter can be selected from the group consisting of: pyruvate decarboxylase (PDC) promoter, glyceraldehyde-3-phosphate dehydrogenase GAPDH (TDH3) promoter, translation elongation factor 1 (TEF1) promoter, URA3 promoter, S-adenosylmethionine transferase 2 (SAM2) promoter; alcohol dehydrogenase 1 (ADH1) promoter and 3-phosphoglycerate kinase (PGK1) promoter. One or more of the exogenous polynucleotide sequences can be operably connected to a heterologous terminator or an artificial terminator. The terminator can be selected from the group consisting of: different-1-cytochrome c (CYC1) terminator, URA3 terminator, PDC terminator, ADH1 terminator, TEF1 terminator or GAL10 terminator.

[0011] The yeast cell may be selected from the group consisting of: Saccharomyces species, Schizosaccharomyces species, Pichia species, Paffia species, Kluyveromyces species, Candida species, Talaromyces species, Brettanomyces species, Pachysolen species, Debaryomyces species, and Yarrowia species. The yeast cell may be a Saccharomyces cerevisiae cell.

[0012] The disclosure also provides a method for producing ethanol, the method comprises contacting substrate with engineered yeast as described herein, wherein the engineered yeast cell produces at least 60g / L, at least 80g / L, at least 100g / L or at least 120g / L ethanol after 48 hours, and wherein the glycerol production of the engineered yeast cell reduces with respect to the glycerol production in the equivalent yeast cell lacking the gapN enzyme. The substrate can include starch, glucose, sucrose, cellulose biomass or a combination thereof. The substrate can be obtained from wheat, corn or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The drawings illustrate generally by way of example and not by way of limitation various aspects described herein.

[0014] Figure 1 Screening of gapN biodiversity as outlined in Example 1 is shown.

[0015] Figure 2 Glycerol production in the deep well plate assay outlined in Example 3 is shown.

[0016] Figure 3 Ethanol production in the deep well plate assay outlined in Example 3 is shown.

[0017] Figure 4 Shown is the NADP redox balance using a combination of NADP-dependent gapN and NADP-dependent ADH enzymes in the production of ethanol from glucose.

[0018] Figure 5 Results of the ADH enzyme assay outlined in Example 4 are shown.

[0019] Figure 6 Glycerol and ethanol titers from the shake flask assay outlined in Example 6 are shown.

[0020] Figure 7 Glycerol and ethanol titers from the shake flask assay outlined in Example 6 are shown.

[0021] Figure 8 Glycerol and ethanol titers from the shake flask assay outlined in Example 6 are shown.

[0022] Fig. 9 Glycerol and ethanol titers from the Ambr15 assay outlined in Example 7 are shown. DETAILED DESCRIPTION

[0023] Reference will now be made in detail to certain aspects of the presently disclosed subject matter, examples of which are partially illustrated in the accompanying drawings.While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the illustrated subject matter is not intended to limit the claims to the disclosed subject matter.

[0024] In this document, the terms "a", "an" or "the" are used to include one or more than one, unless the context clearly dictates otherwise. The term "or" is used to refer to a non-exclusive "or" unless otherwise indicated. All publications, patents, and patent documents cited in this document are incorporated herein by reference in their entirety, as if individually incorporated by reference. In the event of an inconsistency between the usages of this document and those documents so incorporated by reference, the usage in the incorporated references should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0025] Values ​​expressed in range format should be interpreted in a flexible manner to include not only the values ​​explicitly listed as the limits of the range, but also all individual values ​​or subranges covered within the range, as if each value and subrange were explicitly listed. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not only about 0.1% to about 5%, but also individual values ​​(e.g., 1%, 2%, 3% and 4%) and subranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the specified range. Unless otherwise indicated, the statement "about X to Y" has the same meaning as "about X to about Y". Similarly, unless otherwise indicated, the statement "about X, Y or about Z" has the same meaning as "about X, about Y or about Z".

[0026] Unless expressly stated, ppm (parts per million), percentages and ratios are based on weight. Percentages based on weight are also referred to below as wt% or (wt)%.

[0027] The present disclosure relates to various recombinant cells engineered to produce ethanol via glucose, which recombinant cells also have reduced glycerol production. Typically, the recombinant cells described herein comprise heterologous nucleic acids encoding gapN enzymes, such as at least one of SEQ ID NOs: 28, 32, 48, 52, 64, 68, 80 and 92. The recombinant cells may additionally comprise heterologous nucleic acids encoding ADH enzymes, such as at least one of SEQ ID NOs: 99 and 100. The present disclosure further provides a fermentation method for producing ethanol using the genetically engineered cells described herein.

[0028] In general, recombinant cells as described herein are yeast cells. Non-limiting examples of yeast cells include yeast cells obtained from, for example, Saccharomyces species, Schizosaccharomyces species, Pichia species, Paffia species, Kluyveromyces species, Candida species, Talaromyces species, Brettanomyces species, Pachycystis species, Debaryomyces species, Yarrowia species, and industrial polyploid yeast strains. Suitable yeast cells may include, but are not limited to, Saccharomyces cerevisiae, Issatchensis orientalis, Pichia galeiformis, Pichia species YB-4149 (NRRL naming), Candida ethanolica, Pichia deserticola, Pichia membranifadens, or Pichia fermentans. Yeast cells may be ethanol-tolerant yeast strains, such as commercially available ethanol-tolerant yeast strains, such as RED STAR TM and ETHANOL RED TMYeast (Fermentis / Lesaffre, USA), FALI TM (Fleischmann's Yeast, USA), SUPERSTART and THERMOSACC TM Yeast (Ethanol Technology, Wisconsin, USA), BIOFERM TM AFT and XR (NABC-NorthAmerican Bioproducts Corporation, Georgia, USA), GERT STRAND (Gert Strand AB, Sweden), SUPERSTART TM (Alltech), ANGEL TM (China Angel Yeast Ltd) and FERMIOL TM (DSM Specialties). One of ordinary skill will appreciate that the requirements for selecting suitable yeast cells and recombinant yeast cells of the present disclosure are not limited to those explicitly listed herein. For example, examples of suitable host cells and recombinant cells capable of producing ethanol are described in U.S. Patent No. 10,724,023, U.S. Patent No. 10,334,288, U.S. Patent Publication No. 20200270644A1, U.S. Patent No. 11,111,482, U.S. Patent Publication No. 20190345471A1, U.S. Patent No. 11,041,218, U.S. Patent No. 11,306,330, and U.S. Patent Publication No. 20210062230A1, each of which is incorporated herein by reference in its entirety.

[0029] The recombinant cells described herein comprise one or more exogenous polynucleotide sequences encoding one or more exogenous polypeptides that, when expressed, improve the recombinant cells to ferment sucrose into lactate. The recombinant cells may alternatively or additionally comprise one or more genetic modifications that increase the expression of native polypeptides, wherein the increase in expression improves the recombinant cells to ferment sucrose into lactate.

[0030] As used herein, "exogenous" refers to genetic material or its expression products that originate from outside the host organism. For example, the exogenous genetic material or its expression products can be a modified form of genetic material native to the host organism, it can be derived from another organism, it can be a modified form of a component derived from another organism, or it can be a synthetically derived component. For example, the K. lactis invertase gene is exogenous when introduced into I. orientalis.

[0031] As used herein, "native" refers to genetic material or its expression product found in the genome of a wild-type cell of a host cell, excluding inter-individual mutations that do not affect function or expression.

[0032] As used herein, the terms "polypeptide" and "peptide" are used interchangeably and refer to the overall primary, secondary, tertiary and quaternary amino acid sequences and structures necessary to give the macromolecule its function and properties. As used herein, "enzyme" or "biosynthetic pathway enzyme" refers to a protein that catalyzes a chemical reaction. The description of any particular enzyme (either independently or as part of a biosynthetic pathway) is understood to include the cofactors, coenzymes and metals necessary for the enzyme to function properly. An overview of amino acids and their three letter and one letter symbols as understood in the art is provided in Table 1. The amino acid names, three letter symbols and one letter symbol are used interchangeably herein.

[0033] Table 1: Amino Acid Three-Letter and One-Letter Symbols

[0034]

[0035]

[0036] Variants or sequences having substantial identity or homology to the polypeptides described herein can be used in the disclosed engineered cells, compositions and methods. Such sequences can be referred to as variants or modified sequences. That is, the polypeptide sequence can be modified but still retains the ability to exhibit the desired activity. Typically, the variant or modified sequence can include or be greater than about 45%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity to the wild-type, naturally occurring polypeptide sequence or to a variant polypeptide as described herein.

[0037] As used herein, the phrases "% sequence identity", "% identity" and "percent identity" are used interchangeably and refer to the percentage of residue matches between at least two amino acid sequences or at least two nucleic acid sequences aligned using a standardized algorithm. Methods of amino acid and nucleic acid sequence alignment are well known. The generation of sequence alignments and sequence identities includes global alignments and local alignments performed using computational methods. Alignments can be performed using BLAST (National Center for Biological Information (NCBI) Local Alignment-Based Search Tool) version 2.2.31 with default parameters. The amino acid sequence identity % between amino acid sequences can be determined using standard protein BLAST with the following default parameters: maximum target sequence: 100; short query: automatically adjust parameters for short input sequences; expected threshold: 10; word length: 6; maximum number of matches within the query range: 0; matrix: BLOSUM62; gap penalty: (existence: 11, extension: 1); component adjustment: conditional component score matrix adjustment; filter: not selected; mask: not selected. The % nucleic acid sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Maximum target sequence: 100; Short query: Automatically adjust parameters for short input sequences; Expected threshold: 10; Word length: 28; Maximum number of matches within query range: 0; Match / mismatch score: 1, -2; Gap penalty: linear; Filter: Low complexity region; Mask: Mask for lookup table only. Sequences with an identity score of XX% (e.g., 80%) relative to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters are considered to be at least XX% identical, or equivalently, to have XX% sequence identity with the reference sequence.

[0038] Polypeptide or polynucleotide sequence identity can be measured over the length of an entire defined polypeptide sequence (e.g., as defined by a particular SEQ ID number), or can be measured over a shorter length, for example, over the length of a fragment (e.g., a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70, or at least 150 consecutive residues) taken from a larger defined polypeptide sequence. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown in this article, table, figure, or sequence listing can be used to describe the length over which a percentage identity can be measured.

[0039] The polypeptides disclosed herein may include "variant" polypeptides, "mutants" and "derivatives thereof". As used herein, the term "wild type" is a term understood by those skilled in the art and means the typical form of the polypeptide in nature, as distinguished from a variant or mutant form. As used herein, a "variant", "mutant" or "derivative" refers to a polypeptide molecule having an amino acid sequence that is different from a reference protein or polypeptide molecule. A variant or mutant may have an insertion, deletion or substitution of one or more amino acid residues relative to a reference molecule.

[0040] The amino acid sequence of the polypeptide variant, mutant, derivative or fragment considered herein may include conservative amino acid substitutions relative to a reference amino acid sequence. For example, a variant, mutant, derivative or fragment polypeptide may include conservative amino acid substitutions relative to a reference molecule. "Conservative amino acid substitutions" are those substitutions in which the amino acid is substituted with a different amino acid, wherein the substitution is predicted to interfere with the properties of the reference polypeptide to the minimum. In other words, conservative amino acid substitutions substantially retain the structure and function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the region of the substitution, such as a beta sheet or alpha helical conformation, (b) the charge and / or hydrophobicity of the molecule at the site of the substitution, and / or (c) the volume of the side chain.

[0041] As used herein, the terms "polynucleotide," "polynucleotide sequence," and "nucleic acid sequence" and "nucleic acid" are used interchangeably and refer to a nucleotide sequence or any fragment thereof. These phrases also refer to DNA or RNA of natural or synthetic origin, which may be single-stranded or double-stranded and may represent the sense strand or the antisense strand. The DNA polynucleotide may be a cDNA or a genomic DNA sequence.

[0042] If in its natural state or when operated by methods known to those skilled in the art, a polynucleotide can be transcribed and / or translated to produce a polypeptide or fragment thereof, then the polynucleotide is said to encode a polypeptide. The antisense strand of such a polynucleotide is also considered to encode the sequence.

[0043] Those skilled in the art understand the degeneracy of the genetic code and that a variety of polynucleotides can encode the same polypeptide. In some aspects, the polynucleotide (i.e., a polynucleotide encoding a non-heme iron-binding protein polypeptide) can be codon-optimized for expression in a specific cell, which includes but is not limited to a plant cell, a bacterial cell, a fungal cell, or an animal cell. Although polypeptides encoded by polynucleotide sequences found in corals are disclosed herein, any polynucleotide sequence encoding the desired form of the polypeptides described herein can be used. Therefore, non-naturally occurring sequences can be used. These sequences may be desirable, for example, to enhance expression in a heterologous expression system of a polypeptide or protein. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencils, paper, genetic codes, and human hands can also be used to generate degenerate coding sequences.

[0044] Recombinant cells as described herein may comprise deletions or destructions in one or more natural genes. The phrase "deletion or destruction" refers to the state of natural genes in recombinant cells, which have a completely eliminated coding region (deletion) or gene, its promoter or its terminator modification (such as deletion, insertion or mutation) so that the gene no longer produces an active expression product, produces an expression product of a severely reduced amount (e.g., reduced by at least 75% or reduced by at least 90%) or produces an expression product of severely reduced activity (e.g., reduced by at least 75% or reduced by at least 90%). Deletion or destruction can be achieved by genetic engineering methods, forced evolution, mutagenesis and / or selection and screening. The natural gene to be deleted or destroyed can be replaced with an exogenous nucleic acid of interest for expressing an exogenous gene product (e.g., polypeptide, enzyme, etc.).

[0045] The recombinant cells described herein may have a deletion or disruption of one or more native genes encoding enzymes involved in glycerol synthesis. The deletion or disruption of one or more of these glycerol biosynthetic pathway enzymes reduces the ability of the cell to produce glycerol, thereby increasing the fermentation yield of ethanol.

[0046] The recombinant cells described herein may include a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene. The native GPP gene encodes an enzyme that catalyzes the hydrolysis of glycerol-3-phosphate to glycerol. When the host cell contains multiple GPP genes, it is preferred to delete or disrupt at least one of them, and more preferably to disrupt all of them to more completely eliminate the ability of the host cell to produce glycerol. In Saccharomyces cerevisiae, there are two GPP paralogs, referred to as Gpp1p (SEQ ID NO: 120, encoded by the GPP1 gene) and Gpp2p (SEQ ID NO: 121, by the GPP2 gene). When the recombinant cell is a Saccharomyces cerevisiae cell, the cell may include a deletion or disruption of a GPP gene, which encodes an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identical to at least one of SEQ ID NOs: 120 and 121. Methods for deletion or disruption of the GPP gene of Saccharomyces cerevisiae are known and described in the art and are exemplified herein.

[0047] The recombinant cells described herein may comprise a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GPD) gene. The deletion or disruption of a native GPD gene improves acetic acid consumption by providing a larger pool of reducing equivalents to the cell to help oxidize and reduce acetic acid to ethanol. When the host cell contains multiple GPD genes, it is preferred to delete or disrupt at least one of them, more preferably to disrupt all of them. In Saccharomyces cerevisiae, there are two glycerol-3-phosphate dehydrogenases, called Gpd1p (encoded by GPD1) and Gpd2p (encoded by GPD2).

[0048] Recombinant cells as described herein may comprise one or more genetic modifications, wherein exogenous nucleic acids are integrated into the genome of the host cell. Those skilled in the art will appreciate how to select suitable loci in the yeast genome for integration of exogenous nucleic acids. For example, in a saccharomyces cerevisiae host cell, suitable interaction loci may include but are not limited to GPP1 loci (defined as loci flanked by SEQ ID NO:136 and SEQ ID NO:137), DLD1 loci (defined as loci flanked by SEQ ID NO:138 and SEQ ID NO:139) and GPD1 loci (defined as loci flanked by SEQ ID NO:140 and SEQ ID NO:141). Those skilled in the art may determine other suitable integration loci. In addition, those skilled in the art will appreciate how to use sequences to design primers to verify the correct gene integration at the selected locus.

[0049] The recombinant cells described herein are capable of producing ethanol and comprise an exogenous polynucleotide sequence encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme. The gapN enzyme can be any suitable enzyme having glyceraldehyde-3-phosphate dehydrogenase activity. The exogenous polynucleotide sequence can be an exogenous glyceraldehyde-3-phosphate dehydrogenase (gapN) gene.

[0050] "Glyceraldehyde-3-phosphate dehydrogenase gene" and "gapN gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having glyceraldehyde-3-phosphate dehydrogenase activity. As used herein, "glyceraldehyde-3-phosphate dehydrogenase activity" refers to a gene that catalyzes the conversion of D-glyceraldehyde-3-phosphate and NADP +The ability to convert into 3-phospho-D-glyceric acid and NADPH. The gapN enzyme can be from any suitable organism source, or can be synthetic. Suitable gapN enzymes can include but are not limited to enzymes classified under Enzyme Commission (EC) No. 1.2.1.9, also referred to in the art as "NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase". Suitable gapN enzymes can be gapN enzymes from Streptococcus pyogenes, Pseudomonas fluorescens, Brevibacillus laterosporus, Arabidopsis thaliana, Chryseobacterium gleum, Streptococcus mutans, Streptococcus henryi, Lactobacillus delbrueckii, Bacillus cereus, etc. The gapN gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 48, 52, 64, 68, 80, 92, and 96. The gapN gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 48, 52, and 64. The gapN gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 28, 32, 52, and 64.

[0051] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus pyogenes gene encoding the amino acid sequence of SEQ ID NO: 28. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 28.

[0052] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Pseudomonas fluorescens gene encoding the amino acid sequence of SEQ ID NO: 32. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 32.

[0053] The recombinant cell may comprise an exogenous polynucleotide that is or is derivable from a Brevibacillus laterosporus gene encoding the amino acid sequence of SEQ ID NO: 48. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 48.

[0054] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from an Arabidopsis thaliana gene encoding the amino acid sequence of SEQ ID NO: 52. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 52.

[0055] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Chryseobacterium mycotoxins gene encoding the amino acid sequence of SEQ ID NO: 64. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 64.

[0056] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a mutans Streptococcus gene encoding the amino acid sequence of SEQ ID NO: 68. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 68.

[0057] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus henylica gene encoding the amino acid sequence of SEQ ID NO: 80. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to SEQ ID NO: 80.

[0058] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Lactobacillus delbrueckii gene encoding the amino acid of SEQ ID NO: 92. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 92.

[0059] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Bacillus cereus gene encoding the amino acid of SEQ ID NO: 96. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 96.

[0060] The recombinant cells described herein are capable of producing ethanol, and the recombinant cells include an exogenous polynucleotide sequence encoding a gapN enzyme, and may additionally include an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme. The ADH enzyme may be any suitable enzyme having NADP-dependent alcohol dehydrogenase activity. The exogenous polynucleotide sequence may be an exogenous alcohol dehydrogenase (ADH) gene.

[0061] "Alcohol dehydrogenase gene" and "ADH gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having alcohol dehydrogenase activity. As used herein, "alcohol dehydrogenase activity" refers to the catalysis of the conversion of acetaldehyde and NADH or NADPH into ethanol and NADPH. + or NADP + As used herein, "NADP-dependent alcohol dehydrogenase activity" refers to the ability to catalyze the conversion of acetaldehyde and NADPH into ethanol and NADP + The ADH enzyme may be derived from any suitable source, or may be synthetic. Suitable ADH enzymes may be ADH enzymes from Rhodotorula toruloides, Candida maltosa, and the like. The ADH gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 98 and 100.

[0062] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Candida maltosa gene encoding the amino acid sequence of SEQ ID NO: 98. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:98.

[0063] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhodosporidium toruloides gene encoding the amino acid sequence of SEQ ID NO: 100. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 100.

[0064] Recombinant cells as described herein are capable of producing ethanol, and the recombinant cells comprise an exogenous polynucleotide sequence encoding a gapN enzyme, may optionally comprise an exogenous polynucleotide sequence encoding an ADH enzyme, and may additionally comprise an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme. The GA enzyme may be any suitable enzyme with glucoamylase activity. The exogenous polynucleotide sequence may be an exogenous glucoamylase (GA) gene.

[0065] "Glucoamylase gene" and "GA gene" are used interchangeably herein and refer to any gene or polynucleotide encoding a polypeptide having glucoamylase activity. As used herein, "glucoamylase activity" refers to the ability to catalyze the hydrolysis of terminal 1,4-linked α-D-glucose residues from the non-reducing end of the amylose chain to release free glucose. The GA enzyme may be from any suitable organism source, or may be synthetic. Suitable glucoamylase enzymes may include, but are not limited to, enzymes of EC 3.2.1.3. Suitable GA enzymes may be GA enzymes from Saccharmycopsis fibuligera, Rhizopus delemar, Rhizopus microspoons, Rhizopus oryzae, and the like. The GA gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1, 122, 123, and 124. The GA gene may encode an amino acid that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 1, 123, and 124. Additional GA genes and GA enzyme sequences are known and described in the art, such as a GA gene encoding a GA enzyme with a modified leader / signal sequence. See, e.g., U.S. Patent No. 10,364,421, U.S. Patent No. 10,724,023, U.S. Patent Publication No. 20190345471A1, and U.S. Patent No. 11,306,330, each of which is incorporated herein by reference in its entirety.

[0066] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus microsporus gene encoding the amino acid sequence of SEQ ID NO: 1. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 124.

[0067] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Saccharomyces cerevisiae gene encoding the amino acid sequence of SEQ ID NO: 122. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 122.

[0068] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus delbrueckii gene encoding the amino acid sequence of SEQ ID NO: 123. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 123.

[0069] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus oryzae gene encoding the amino acid sequence of SEQ ID NO: 124. The exogenous polynucleotide may encode an amino acid sequence that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 124.

[0070] The exogenous nucleic acid in the recombinant cell described herein can be under the control of a promoter. For example, the exogenous nucleic acid can be operably connected to a heterologous promoter or an artificial promoter. Suitable promoters are known and described in the art. Promoters can include, but are not limited to, pyruvate decarboxylase (PDC1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (TDH3 herein; annotated with EC 1.2.1.12; SEQ ID NO: 125), translation elongation factor 1 (TEF1; SEQ ID NO: 128), URA3 (SEQ ID NO: 126), S-adenosylmethionine transferase 2 (SAM2; SEQ ID NO: 129), alcohol dehydrogenase 1 (ADH1; SEQ ID NO: 130) and 3-phosphoglycerate kinase (PGK1; SEQ ID NO: 127).

[0071] The exogenous nucleic acid in the recombinant cells described herein can be under the control of a terminator. For example, the exogenous nucleic acid can be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. Terminators can include, but are not limited to, iso-1-cytochrome c (CYC1; SEQ ID NO: 131), URA3 (SEQ ID NO: 132), PDC, ADH1 (SEQ ID NO: 134), TEF1 (SEQ ID NO: 135), and ScGAL10 (SEQ ID NO: 133).

[0072] A promoter or terminator is "operably linked" to a given polynucleotide (eg, a gene) if its position relative to the polynucleotide in the genome or expression cassette is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0073] The polypeptides described herein may be provided as part of a construct. As used herein, the term "construct" refers to a recombinant polynucleotide, including but not limited to DNA and RNA, which may be single-stranded or double-stranded and may represent a sense strand or an antisense strand. A recombinant polynucleotide is a polynucleotide formed by a laboratory method, which includes a polynucleotide sequence derived from at least two different natural sources, or they may be synthetic. Therefore, a construct may include a new modification to an endogenous gene introduced by, for example, a genome editing technique. A construct may also include a recombinant polynucleotide produced using, for example, a recombinant DNA method. A construct may be a vector including a promoter operably connected to a polynucleotide encoding a heat-labile non-heme iron-binding polypeptide. As used herein, the term "vector" refers to a polynucleotide capable of transporting another polynucleotide connected thereto. A vector may be a plasmid, which refers to a circular double-stranded DNA loop, into which an additional DNA fragment may be integrated.

[0074] The present disclosure also provides a fermentation method for producing ethanol using the recombinant cells described herein. The fermentation method includes the step of fermenting a substrate using the genetically engineered yeast described herein to produce ethanol. As will be appreciated by those skilled in the art, the fermentation method may include additional steps. Non-limiting examples of additional process steps include maintaining the temperature of the fermentation broth within a predetermined range, adjusting the pH during fermentation, and separating ethanol from the fermentation broth.

[0075] The fermentation substrate may include starch. Starch may be obtained from natural sources such as plant sources. Starch may also be obtained from raw materials with high starch or sugar content, including but not limited to corn, sweet sorghum, fruit, sweet potato, rice, barley, sugar cane, beet, wheat, cassava, potato, tapioca, arrowroot, pea or sago. The fermentation substrate may come from lignocellulosic biomass such as wood, wheat straw, grass, or algae biomass such as microalgae and macroalgae. The fermentation substrate may include cellulose or lignocellulosic biomass. The fermentation substrate may come from grass, tree or agricultural and forestry residues such as corn cobs and corn stalks, straw, sawdust and wood chips. The fermentation substrate may also include sugar, such as glucose (dextrose) or sucrose. The fermentation substrate may include dry milled ethanol raw materials, such as corn mash. The fermentation substrate may include liquefied corn mash (LCM). The fermentation substrate may include corn wet milled raw materials, such as lightly soaked water / liquefied matter (LSW / LQ).

[0076] The culture medium used to ferment the engineered yeast described herein can be supplemented with various components. For example, the culture medium used to ferment the engineered yeast described herein can be supplemented with a glucoamylase, such as a glucoamylase Spirizyme TM (Novozymes, Bagsværde, Denmark).

[0077] Fermentation process can be operated under various conditions. Fermentation temperature, i.e. the temperature of the fermented liquid during processing, can be ambient temperature. Alternatively or additionally, fermentation temperature can be maintained within a predetermined range. For example, fermentation temperature can be maintained within the range of 25°C to 40°C, 27°C to 38°C or 30°C to 35°C. Fermentation temperature can be maintained at a temperature of, for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween. However, it will be appreciated by those skilled in the art that fermentation temperature is not limited to any specific range described herein and can be appropriately modified.

[0078] The pH of the culture medium described herein can be controlled for optimal ethanol production. The pH of the culture or fermentation mixture of the engineered cells described herein can be in the range between 4.0 and 6.0. The pH of at least a portion of the incubation can be maintained at 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0. The pH can be maintained in the range between 5.0 and 5.5.

[0079] The engineered yeast can be cultured for approximately 24 hours to 72 hours. For example, the engineered yeast can be cultured for about 12 hours, 18 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 hours, 31 hours, 32 hours, 33 hours, 34 hours, 35 hours, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 hours, 46 hours, 47 hours, 48 ​​hours, 49 hours, 50 hours, 51 hours, 52 hours, 53 hours, 54 hours, 55 hours, 56 hours, 57 hours, 58 hours, 59 hours, 60 hours, 61 hours, 62 hours, 63 hours, 64 hours, 65 hours, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 78 hours, 80 hours, 90 hours, 96 hours, or more than 96 hours. The engineered yeast described herein can be cultured for about 48 hours to 72 hours. The culture (fermentation) time of about 48 hours is a representative time for commercial-scale ethanol fermentation processes. Therefore, the 48-hour time point can be used to compare the fermentation performance of different yeast strains.

[0080] Reaction parameters can be measured or adjusted during ethanol production. Non-limiting examples of reaction parameters include biological parameters (e.g., growth rate, cell size, cell number, cell density, cell type or cell state, etc.), chemical parameters (e.g., pH, redox potential, concentration of reaction substrates and / or products, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentration, metabolite concentration, ethanol concentration, fermentation substrate concentration, concentration of oligopeptides, concentration of amino acids, concentration of vitamins, concentration of hormones, concentration of additives, serum concentration, ionic strength, concentration of ions, relative humidity, molar concentration, molar osmotic pressure concentration, other chemicals such as buffers, adjuvants or reaction byproducts), physical / mechanical parameters (e.g., density, conductivity, degree of agitation, pressure and flow rate, shear stress, shear rate, viscosity, color, turbidity, light absorption, mixing rate, conversion rate, and thermodynamic parameters such as temperature, light intensity / quality, etc.). Sensors for measuring parameters described herein are well known to those of ordinary skill in the art.

[0081] Fermentation processes can be associated with various characteristics, such as, but not limited to, fermentation production rate, approach fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of yeast and / or the genetic modification of yeast used in the fermentation process. These characteristics can be affected by regulating the fermentation process conditions. These characteristics can be regulated via a combination of yeast selection or modification and the selection of fermentation process conditions.

[0082] The ethanol production rate of the process can be at least 1.0 g L -1 h -1 , at least 1.5 g L -1 h -1 , or at least 2.0 g L - 1 h -1 , at least 2.5 g L -1 h -1 , at least 3.0 g L -1 h -1 or at least 3.5 g L -1 h -1 The final ethanol titer of the process may be at least 60 g / L, at least 80 g / L, at least 100 g / L, or at least 120 g / L. The final glycerol titer of the process may be less than 10 g / L, less than 8 g / L, less than 6 g / L, less than 4 g / L, or less than 3 g / L.

[0083] Example

[0084] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise stated, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should never be interpreted as being limited to the following examples, but should be interpreted as covering any and all changes that become apparent due to the teachings provided herein.

[0085] Example 1: gapN biodiversity

[0086] Previous work has demonstrated the effectiveness of Bacillus cereus gapN for reducing glycerol byproduct formation and increasing ethanol titer in Saccharomyces cerevisiae (see, e.g., U.S. Patent Application Publication No. US 2021 / 006230, which is incorporated herein by reference in its entirety). However, there is still room for further improvement in reducing glycerol and increasing ethanol production. Surveying the biodiversity of gapN revealed less than 1,000 annotated gapN genes, primarily in the genera Streptococcus (230 gapN genes), Pseudomonas (94), Bacillus (95), and Clostridium (96). Figure 1 ). Based on this analysis, 24 gapN genes were selected for further analysis.

[0087] Example 2: Genetically modified yeast strain - gapN

[0088] strain 1-1

[0089] Strain 1-17 described by Poynter et al. (U.S. Patent Application Publication No. US 2021 / 006230, published on March 4, 2021, which is incorporated herein by reference in its entirety) is a ura3-positive (ura3+) and amdS-positive (amdS+) Saccharomyces cerevisiae host strain, and in which both alleles of the cytosine deaminase (FCY1) gene are knocked out and replaced by an expression cassette of Rhizopus microsporus glucoamylase of SEQ ID NO: 1 with a TDH3 promoter and a CYC1 terminator. In this article, strain 1-1 refers to strain 1-17 of U.S. Application Publication No. US2021 / 006230 by Poynter et al.

[0090] Strain 1-2

[0091] Strain 1-18 described by Poynter et al. (U.S. Patent Application Publication No. US 2021 / 006230, published on March 4, 2021, which is incorporated herein by reference in its entirety) is a ura3-negative (ura3-) and amdS-negative (amdS-) Saccharomyces cerevisiae host strain, and in which both alleles of the cytosine deaminase (FCY1) gene are knocked out and replaced by an expression cassette of Rhizopus microsporus glucoamylase of SEQ ID NO: 1 with a TDH3 promoter and a CYC1 terminator. In this article, strain 1-2 refers to strain 1-18 of U.S. Application Publication No. US2021 / 006230 by Poynter et al.

[0092] Strains 1-3

[0093] Strain 1-25 described by Poynter et al. (U.S. Patent Application Publication No. US 2021 / 006230, published on March 4, 2021, which is incorporated herein by reference in its entirety) is a ura3+ and amdS+ Saccharomyces cerevisiae host strain, in which both alleles of the FCY1 gene are knocked out and replaced by an expression cassette of Rhizopus microsporus glucoamylase of SEQ ID NO: 1 with a TDH3 promoter and a CYC1 terminator, and in which both alleles of the GPP1 gene are replaced by an expression cassette of Bacillus cereus glyceraldehyde-3-phosphate dehydrogenase (gapN) of SEQ ID NO: 96. In this article, strain 1-3 refers to strain 1-25 of Poynter et al. International Application Publication No. US2021 / 006230.

[0094] Strains 1-4 to 1-27

[0095] Strain 1-2 was transformed with the "upstream fragments" and "downstream fragments" shown in Table 2 to produce strains 1-4 to 1-27. Each "upstream fragment" contains i) 5'GPP1 flanking sequence; ii) ScTDH3 promoter; iii) gene encoding gapN shown; iv) ScCYC1 terminator; v) loxP site; vi) ScURA3 promoter; and vii) 5' portion of the ScURA open reading frame. Each "downstream fragment" contains i) 3'GPP1 flanking sequence; ii) ScTDH3 promoter; iii) gene encoding gapN shown; iv) ScPGK1 terminator, v) loxP site; vi) ScURA3 terminator; and vii) 3' portion of the ScURA open reading frame. Transformants were selected on synthetic nutrient-deficient medium (ScD-Ura) plates lacking uracil. The resulting transformants were streaked on ScD-Ura plates for single colony isolation. Single colonies were selected. Correct integration of the indicated gene encoding gapN was verified by PCR, and the PCR-verified isolates were designated with the strain identifiers summarized in Table 2. The resulting strains 1-4 to 1-20, 1-23, 1-24, 1-26, and 1-27 included two copies of the indicated gapN expression cassette in tandem on a single allele of the GPP1 gene. The resulting strains 1-21, 1-22, and 1-25 included one copy of the indicated gapN expression cassette in tandem on a single allele of the GPP1 gene and one copy of the Bacillus cereus gapN expression cassette.

[0096] Table 2 .

[0097]

[0098]

[0099] *These strains include an expression cassette for one copy of the gapN shown and one copy of the B. cereus gapN of SEQ ID NO:96 in tandem on a single allele of the GPP1 gene.

[0100] Example 3: Deep hole measurement

[0101] Strains 1-1 (negative control), 1-3 (positive control), and 1-4 to 1-27 were run in 96-deep-well plates to determine ethanol and glycerol production.

[0102] The strain was scraped onto the ScD-ura plate and incubated at 30°C until a single colony was visible (2-3 days). The cells from the ScD-ura plate were scraped into 20g / L YPD medium and grown overnight at 30°C and 800rpm in a 96 deep well plate. The 2mL shake flask medium containing sterilized rapeseed oil was added to each well of the 96 deep well plate and then inoculated immediately. The shake flask medium consisted of 725g partially hydrolyzed corn starch, 150g filtered sterilized (0.2μm) light immersion water, 10g water, 25g glucose and 1g urea. The strain was incubated at 35°C with shaking at 250rpm in an orbital shaker for about 70 hours. Samples were collected and analyzed for ethanol and glycerol concentrations by HPLC. The average values ​​of 6 wells per strain are reported in Table 3 and Figure 2 and Figure 3 middle.

[0103] Table 3 .

[0104]

[0105] Some strains show glycerol levels similar to control strain 1-1, indicating that the gapN homologue introduced does not reduce the glycerol yield in the ethanol fermentation reaction. However, relative to control strain 1-1, some strains do show glycerol yields of reduced levels, and the reduced levels are equal to or better than the glycerol levels of the comparison strain 1-3 expressing bacillus cereus. Especially, compared with strains 1-1 and 1-3, strains 1-15, 1-16 and 1-19 show improved glycerol reduction. Strain 1-10, 1-11, 1-20, 1-23 and 1-26 show improved glycerol reduction relative to strain 1-1, but are at the same level as strain 1-3. Comparing strains 1-27 and 1-3, both have two copies of bacillus cereus gapN expression set, but one has a copy (1-3) at each allele of two GPP1 alleles, and the other has two copies (1-27) in front and behind on a single allele. The reduced glycerol production in both 1-3 and 1-27 relative to 1-1 demonstrates that the position of the gapN expression cassette can be variable and that a beneficial reduction in glycerol production can be seen even when one GPP1 allele is present.

[0106] Example 4: ADH Biodiversity

[0107] The biodiversity of alcohol dehydrogenase (ADH) enzymes was investigated and 41 were selected for testing. The gene encoding the selected ADH enzyme was cloned into Saccharomyces cerevisiae, the biomass was grown, the resulting cells were lysed, and the cell-free extract was assayed using ethanol and NAD / NADP as substrates. Although this reaction is opposite to the expected ADH in vivo activity, it is a suitable characterization of the NAD and NADP preference of the enzyme. Without wishing to be bound by any particular theory, embodiment or mode of action, it is believed that the use of ADH enzymes engineered yeast cells as cofactors matched to a given gapN will improve both glycerol reduction and ethanol production in the cell due to redox balance (see Figure 4 ).

[0108] like Figure 5 As shown, seven wells assayed (shown in boxes) showed higher levels of activity when NADP was used as a cofactor. Additional information about these top hits is provided in Table 4. Based on these results, ADH enzymes from Candida maltosa (SEQ ID NOs: 98 and 99) and Rhodosporidium toruloides (SEQ ID NOs: 100 and 101) were selected for further in vivo testing.

[0109] Table 4 .

[0110]

[0111] Example 5: Genetically modified strains - gapN and ADH

[0112] Strains 2-1 to 2-16

[0113] Strain 1-2 was transformed with the "5'gapN and ADH expression cassette" and "3'selectable marker cassette" shown in Table 5 to produce strains 2-1 to 2-8. Each "5'gapN and ADH expression cassette" contains i) 5'GPP1 flanking sequence; ii) PGK1 promoter; iii) gene encoding the gapN enzyme shown; iv) ADH1 terminator; v) TDH3 promoter; vi) gene encoding the ADH enzyme shown; vii) CYC terminator; and viiii) 5' portion of the ScURA open reading frame. The "3'selectable marker cassette" contains i) 3'GPP1 flanking sequence; ii) loxP site; iii) ScURA terminator; and vi) 3' portion of the ScURA open reading frame. Transformants were selected on ScD-Ura plates. The resulting transformants were streaked on ScD-Ura plates for single colony isolation. Single colonies were selected. Correct integration of the gapN and ADH expression cassettes shown was verified by PCR, and the PCR-verified isolates were designated with the strain identifiers outlined in Table 2. More than one PCR-verified isolate (e.g., "sister" isolates) are indicated by a letter following the strain number. For example, strain 2-1 has three sister isolates: strain 2-1a, strain 2-1b, and strain 2-1c.

[0114] The resulting strains 2-1 to 2-8 included a single copy of the gapN and ADH expression cassettes in tandem on a single allele of the GPP1 gene.

[0115] Strains 2-9 to 2-16

[0116] The parent strains shown in Table 5 were transformed with the "5'gapN and ADH expression cassette" and "3' selectable marker cassette" shown in Table 5 to produce strains 2-9 to 2-16. Each "5'gapN and ADH expression cassette" contains i) 5'GPP1 flanking sequence; ii) PGK1 promoter; iii) a gene encoding the indicated gapN enzyme; iv) ADH1 terminator; v) TDH3 promoter; vi) a gene encoding the indicated ADH enzyme; vii) a CYC terminator; and viiii) a 5' portion of the amdS open reading frame. The "3' selectable marker cassette" contains i) 3'GPP1 flanking sequence; ii) a loxP site; iii) an amdS terminator; and vi) a 3' portion of the amdS open reading frame. Transformants were selected on YNB+acetamide plates. The resulting transformants were streaked on YNB+acetamide plates for single colony isolation. Single colonies were selected. Correct integration of the gapN and ADH expression cassettes shown was verified by PCR, and the PCR-verified isolates were designated with the strain identifiers outlined in Table 2. More than one PCR-verified isolate (e.g., "sister" isolates) are indicated by a letter following the strain number. For example, strain 2-9 has two sister isolates: strain 2-9a and strain 2-9b.

[0117] The resulting strains 2-9 to 2-16 included two copies of the gapN and ADH expression cassettes, one on each of the two alleles of the GPP1 gene.

[0118]

[0119] Example 6: Shake flask assay

[0120] Strains 1-1, 1-3, and 2-1 to 2-8 were run in shake flasks to determine ethanol and glycerol production.

[0121] The strain was scraped onto the ScD-ura plate and incubated at 30°C until a single colony was visible (2-3 days). The cells from the ScD-ura plate were scraped into a sterile shake flask medium and the optical density (OD600) was measured. The optical density was measured using a Genesys 20 spectrophotometer (Thermo Scientific) at a wavelength of 600nm and a pathlength of 1cm. The shake flask was inoculated with a cell slurry to achieve an initial OD600 of 0.1. 50mL of shake flask medium was added to a 250mL baffled shake flask sealed with an air stopper containing 4mL of sterilized rapeseed oil and then inoculated immediately. The shake flask medium consisted of 725g partially hydrolyzed corn starch, 150g of filtered sterilized (0.2 μm) lightly immersed water, 10g of water, 25g of glucose and 1g of urea. The strain was incubated at 30°C in an orbital shaker with 100rpm for 72 hours under shaking. At the end of the fermentation, samples were collected and the metabolite concentrations in the broth were analyzed by HPLC.

[0122] The fermentation results are reported in Tables 6 to 8 and Figures 6 to 8 Many of the tested strains showed improved ethanol titers, even though glycerol titers were not reduced. This could be used to increase the utilization of acetaldehyde by NADPH-dependent ADH enzymes. Strains that included a combination of S. pyogenes gapN and Candida maltosa ADH showed the lowest residual glucose at the end of fermentation. Likewise, strains that included a combination of A. glutinosus gapN and Candida maltosa or Rhodosporidium toruloides ADH also showed lower glycerol and higher ethanol titers, despite higher residual glucose levels.

[0123] Table 6 .

[0124]

[0125] Table 7 .

[0126]

[0127] Table 8.

[0128]

[0129] Example 7: Ambr15 assay

[0130] Strains 1-1, 1-3, and 2-9 to 2-15 were run in Ambr15 to determine ethanol and glycerol production.

[0131] The strain was scraped onto ScD-ura or YPD plates and incubated at 30°C until visible single colonies (2-3 days). The cells from ScD-ura or YPD plates were scraped into sterile culture medium and optical density (OD600) was measured. Model Genesys 20 spectrophotometers (Thermo Scientific) were used to measure optical density at 600nm wavelength and 1cm pathlength. Ambr15 reaction vessels were inoculated with cell slurry to make initial OD600 reach 0.2. The fermentation medium consisted of 295g partially hydrolyzed corn starch, 90g filtered sterilized (0.2 μm) lightly soaked water, 79g sterile water and 36g 500g / L sterile glucose. Under continuous stirring, 12mL fermentation medium was added to each bioreactor. The strain was incubated at 30°C under 450rpm stirring. Air was supplied with 2.4smlpm from the time of inoculation to 14 hours, and then reduced to 0smlpm for the remaining fermentation. Nitrogen was supplied at 0.01 smlpm from 14 hours to the end of the fermentation. In experiments requiring exogenous glucoamylase, 45 μL of DuPont TM A 1: 100 dilution of DXT was prepared. Samples were collected and analyzed by HPLC for ethanol and glycerol concentrations.

[0132] The results of the Ambr15 assay are reported in Tables 9 and Fig. 9 The only combination of enzymes that produced lower glycerol titers compared to the positive control strain 1-3 was the combination of Chryseobacterium glutinosus gapN and Candida maltosa ADH. The 25% glycerol reduction between strains 1-3 and strains 2-11 is consistent with the results of Example 6. Glycerol reduction was improved by 45% relative to the parent strain 1-1. It is unclear why some strains produce higher glycerol levels than the parent strain 1-1. In general, ethanol titers were equal to or greater than both the parent strain (1-1) and the positive control strain (1-3).

[0133] Table 9 .

[0134]

Claims

1. A genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising: An exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 64, 28, 32, 48, 52, 68, 80, 92, and 96.

2. The engineered yeast cell of claim 1, wherein the gapN enzyme is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 64, 28, 32, 48 and 52.

3. The engineered yeast cell of claim 1 or claim 2, wherein the gapN enzyme is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 64, 28, 32 and 52.

4. The engineered yeast cell according to any one of claims 1 to 3, wherein The gapN enzyme is at least 85% identical to SEQ ID NO:64; The gapN enzyme is at least 85% identical to SEQ ID NO:28; The gapN enzyme is at least 85% identical to SEQ ID NO:32; and / or The gapN enzyme is at least 85% identical to SEQ ID NO:

52.

5. The engineered yeast cell of any one of claims 1 to 4, wherein the engineered yeast cell further comprises an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 98 and 100.

6. The engineered yeast cell of any one of claims 1 to 5, wherein the engineered yeast cell comprises a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene.

7. The engineered yeast cell of any one of claims 1 to 6, wherein the engineered yeast cell comprises a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GDP) gene.

8. The engineered yeast cell of any one of claims 1 to 7, wherein the engineered yeast cell additionally comprises an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme.

9. A genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising: an exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 64, 28, 32, 48, 52, 68, 80, 92, and 96; an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme that is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NOs: 98 and 100; an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme; and Deletion or disruption of at least one allele of the native GPP gene.

10. The engineered yeast cell of claim 8 or 9, wherein the encoded GA enzyme is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99% or 100% identical to at least one of SEQ ID NOs: 122, 1, 123 and 124.

11. The engineered yeast cell of any one of claims 1 to 10, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous promoter or an artificial promoter.

12. The engineered yeast cell of claim 11, wherein the promoter is selected from the group consisting of: a pyruvate decarboxylase (PDC) promoter, a glyceraldehyde-3-phosphate dehydrogenase GAPDH (TDH3) promoter, a translation elongation factor 1 (TEF1) promoter, a URA3 promoter, an S-adenosylmethionine transferase 2 (SAM2) promoter; an alcohol dehydrogenase 1 (ADH1) promoter, and a 3-phosphoglycerate kinase (PGK1) promoter.

13. The engineered yeast cell of any one of claims 1 to 12, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous terminator or an artificial terminator.

14. The engineered yeast cell of claim 13, wherein the terminator is selected from the group consisting of: iso-1-cytochrome c (CYC1) terminator, URA3 terminator, PDC terminator, ADH1 terminator, TEF1 terminator or GAL10 terminator.

15. The engineered yeast cell of any one of claims 1 to 14, wherein the yeast cell is selected from the group consisting of: Saccharomyces species, Schizosaccharomyces species, Pichia species, Paffia species, Kluyveromyces species, Candida species, Talaromyces species, Brettanomyces species, Pachysolensis species, Debaryomyces species, and Yarrowia species.

16. The engineered yeast cell of any one of claims 1 to 15, wherein the yeast cell is a Saccharomyces cerevisiae cell.

17. according to the engineered yeast cell of any one of claims 1 to 16, wherein said engineered yeast cell is capable of producing ethanol with a titer of at least 60g / L, at least 80g / L, at least 100g / L, or at least 120g / L ethanol after 48 hours, and the glycerol production of said engineered yeast cell is reduced relative to the glycerol production in an equivalent yeast cell lacking said gapN enzyme.

18. A method for producing ethanol, the method comprising: The substrate is contacted with an engineered yeast cell according to any one of claims 1 to 17, wherein the engineered yeast cell produces at least 60 g / L, at least 80 g / L, at least 100 g / L or at least 120 g / L ethanol after 48 hours, and the glycerol production of the engineered yeast cell is reduced relative to the glycerol production in an equivalent yeast cell lacking the gapN enzyme.

19. The method of claim 18, wherein the substrate comprises starch, glucose, sucrose, cellulosic biomass, or a combination thereof.

20. The method of claim 18 or 19, wherein the substrate is obtained from wheat, corn or a combination thereof.

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