Genetically modified yeast and fermentation processes for the production of ethanol

Abstract

Disclosed herein are genetically engineered yeast cells capable of producing ethanol. The genetically engineered yeast cells comprise an exogenous polynucleotide sequence encoding an alpha-amylase enzyme and an exogenous polynucleotide sequence encoding a pullulanase enzyme. Also disclosed herein are fermentation methods to produce ethanol using the genetically engineered cells described herein.

Description

PT-1891-WO-PCTGENETICALLY MODIFIED YEAST AND FERMENTATION PROCESSES FOR THE PRODUCTION OF ETHANOLCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 688.393, filed August 29, 2024, which is incorporated by reference herein in its entirety.REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA PATENT CENTER

[0002] The content of the Sequence Listing XML file of the sequence listing named “PT-1891-WO-PCT.xml” which is 256,138 bytes in size created on July 23, 2025 and electronically submitted via Patent Center herewith the application is incorporated by reference in its entirety.BACKGROUND

[0003] Many fermentation feedstocks are derived from plant sources (e.g., com mash) where the carbohydrates are predominantly in the form of starch polymers. The starch polymers in such feedstocks must be treated to low molecular weight sugars that can be consumed by the yeast and used for growth and bioproduct production. Typical treatments include acid and / or enzy matic hydrolysis where the polymer chain is hydrolyzed to generate the sugars that can be used by the yeast. Starch degrading enzymes such as alpha amylases and glucoamylases can be added to convert the polymer to simple sugars. However, such enzyme additions can add significant cost and complexity to the fermentation process.

[0004] Heterologous expression and functionality of enzymes in yeast to aid in starch hydrolysis can be challenging, as it is difficult to know if the nucleic acid will be expressed properly and a functional enzyme will form, and if an active form of the enzyme will be secreted from the cell. It is also challenging to engineer yeast for grow th and bioproduct production at non- optimal conditions, such as high temperatures, and in high bioproduct titers. For example, while ethanol production by fermentation is a well-known industrial process, maintaining ethanol rates, titers, and yields while at the same time engineering the yeast to reduce reliance on supplemental enzymes, growth under non-optimal conditions (e.g., temperature), and minimizing by-product formation can be technically difficult. Increased ethanol concentration and accumulation of undesirable byproducts can also be detrimental to cell health. Described herein are engineered cells that express heterologous alpha-amylase and / or pullulanase enzymes for use if fermentation methods to increase ethanol production.PT-1891-WO-PCTSUMMARY

[0005] The disclosure describes a genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding an alphaamylase enzyme 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:35-39, preferably at least one of SEQ ID NOs:35, 36, 38, and 39, most preferably SEQ ID NO:35; and an exogenous polynucleotide encoding a pullulanase enzyme 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:58- 60, preferably at least one of SEQ ID NOs:59 and 60, most preferably SEQ ID NO:58. The engineered cell may additionally comprise a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme 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: 13-17. The gapN enzyme may be 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: 13, 14, 16, and 17. The gapN enzyme may at least 85% identical to SEQ ID NO: 13, at least 85% identical to SEQ ID NO: 14, at least 85% identical to SEQ ID NO: 16, and / or at least 85% identical to SEQ ID NO: 17. The genetically engineered cell may additionally comprise an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme 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:22-23.

[0006] The genetically engineered yeast cell may include an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme. The GA enzyme may be 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:9-12.

[0007] The genetically engineered yeast cell may include one or more deletions or disruptions that improve ethanol production. The genetically engineered yeast cell may include a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene. The genetically engineered yeast cell may include a deletion or disruption of a native glycerol -3 -phosphate dehydrogenase (GDP) gene.

[0008] The genetically engineered yeast cell capable of producing ethanol may include an exogenous polynucleotide encoding an alpha-amylase enzyme 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:35-39, preferably at least one of SEQ ID NOs:35, 36, 38, and 39, most preferablyPT-1891-WO-PCTSEQ ID NO:35; an exogenous polynucleotide encoding a pullulanase enzyme 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:58-60, preferably at least one of SEQ ID NOs:59 and 60, most preferably SEQ ID NO:58; an exogenous polynucleotide encoding a glyceraldehyde-3 -phosphate dehydrogenase (gapN) enzyme 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: 13-21 ; an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme 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:22-23; an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme; and a deletion or disruption of at least 1 allele of a native GPP gene.

[0009] Any of the exogenous polynucleotide sequences of the genetically engineered yeast cells may be operably linked to a heterologous or artificial promoter 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-adenosyl methionine transferase 2 (SAM2) promoter; an alcohol dehydrogenase 1 (ADH1) promoter, and a 3 -phosphoglycerate kinase (PGK1) promoter; and / or operably linked to a heterologous or artificial terminator selected from the group consisting of an iso-l-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEFl terminator, or a GAL 10 terminator.

[0010] The engineered yeast cell may be a yeast cell selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffla spp., Khiyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp.. and Yarrowia spp.. The engineered yeast cell may be a Saccharomyces cerevisiae cell.

[0011] The engineered yeast cell may be capable of producing ethanol at a titer of at least 60, at least 80, at least 100, or at least 120 g / L ethanol after 48 hours. Ethanol production by the engineered yeast cell may be increased relative to ethanol production in an equivalent yeast cell lacking the alpha-amylase and pullulanase enzymes.

[0012] The disclosure also provides a method for producing ethanol, the method comprising contacting a substrate with an engineered yeast as described herein, where the engineered yeast cell produces at least 60, at least 80, at least 100, or at least 120 g / L ethanol after 48 hours. Ethanol production by the engineered yeast cell may be increased relative to ethanol production in an equivalent yeast cell lacking the alpha-amylase and pullulanase enzymes. The substrate mayPT-1891-WO-PCT comprise starch, glucose, sucrose, cellulosic biomass, or combinations thereof. The substrate may be obtained from wheat, com, or a combination thereof.BRIEF DESCRIPTION OF THE FIGURES

[0013] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.

[0014] FIG. 1 shows ethanol titer (g / L) and the titer and degree of polymerization (DP) of residual starch in the fermentation broth of the deep well assays outlined in Example 2.

[0015] FIG. 2 shows the titer and DP of residual starch in the fermentation broth assays outlined in Example 2.

[0016] FIG. 3 shows the titer and DP of residual starch in the deep well assays outlined in Example 3.

[0017] FIG. 4 shows the titer and DP of residual starch in the fermentation broth assays outlined in Example 3.DETAILED DESCRIPTION

[0018] Reference will now be made in detail to certain aspects of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0019] 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 nonexclusive “or” unless otherwise indicated. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference. In the event of inconsistent usages between this document and those documents so incorporated by reference, the usage in the incorporated reference should be considered supplementary to that of this document; for irreconcilable inconsistencies, the usage in this document controls.

[0020] V alues expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range were explicitly recited. For example, a range of “about 0.1% to about 5%” orPT-1891-WO-PCT“about 0.1% to 5%?’ should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement “about X to Y” has the same meaning as “about X to about Y,’" unless indicated otherwise. Likewise, the statement “about X, Y, or about Z has the same meaning as “about X, about Y. or about Z,’?unless indicated otherwise.

[0021] Unless expressly stated, ppm (parts per million), percentage, and ratios are on a by weight basis. Percentage on a by weight basis is also referred to as wt% or % (wt) below.

[0022] This disclosure relates to various recombinant cells engineered to produce ethanol via glucose, said recombinant cells also expressing an alpha-amylase and a pullulanase. In general, the recombinant cells described herein include a heterologous nucleic acid sequence encoding an alpha-amylase enzyme, for example the alpha-amylase enzy me of at least one of SEQ ID NOs:35- 39, and include a heterologous nucleic acid sequence encoding a pullulanase enzyme, for example the pullulanase enzyme of at least one of SEQ ID NOs: 58-60. The recombinant cell may additionally include a heterologous nucleic acid encoding an ADH enzyme, for example, the ADH enzyme of at least one of SEQ ID NO:99 and 100, a heterologous nucleic acid encoding a gapN enzyme, for example the gapN enzyme of at least one of 13-21, and / or a heterologous nucleic acid encoding a GA enzyme, for example the GA enzyme of at least one of SEQ ID NOs:9-12. The disclosure further provides fermentation methods for the production of ethanol using the genetically engineered cells described herein.

[0023] In general, recombinant cells described herein are yeast cells. Non-limiting examples of yeast cells include yeast cells obtained from, e.g., Saccharomyces spp., Schizosaccharomyces spp., Pichia spp., Paffla spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., Yarrowia spp. and industrial polyploid yeast strains. Suitable yeast cells may include, but are not limited to, Saccharomyces cerevisiae, Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida ethanolica. Pichia deserticola. Pichia membranifadens, or Pichia fermentans. The yeast cell may be an ethanol tolerant yeast strain, for example, a commercially available ethanol tolerance yeast such as RED STAR™ and ETHANOL RED™ yeast (Fermentis / Lesaffre, USA), FALI™ (Fleischmann's Yeast, USA). SUPERSTART and THERMOSACC™ yeast (Ethanol Technology, Wis.. USA), BIOFERM™ AFT and XR (NABC-North American Bioproducts Corporation, GA, USA), GERT STRAND (Gert Strand AB, Sweden), SUPERSTART™ (Alltech), ANGEL™ (Angel Yeast Ltd, China) and FERMIOL™ (DSM Specialties). An ordinarily skilled artisanPT-1891-WO-PCT would understand the requirements for selection of a suitable yeast cell, and recombinant yeast cells of the present disclosure are not limited to those expressly recited herein. For example, suitable host cells and examples of recombinant cells capable of producing ethanol are described in US Patent No. 10,724,023, US Patent No. 10,334,288, US Patent Publication No. 20200270644A1, US Patent No. 11,111.482. US Patent Publication No. 20190345471A1, US Patent No. 1 1 ,041 ,218, US Patent No. 1 1 ,306,330, and US Patent Publication No. 20210062230A1, each of which is incorporated herein by reference in its entirety.

[0024] As used herein, ‘‘exogenous” refers to genetic material or an expression product thereof that originates from outside of the host organism. For example, the exogenous genetic material or expression product thereof 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, a K. lactis invertase gene is exogenous when introduced into S. cerevisiae.

[0025] As used herein, “native” refers to genetic material or an expression product thereof that is found, apart from individual-to-individual mutations which do not affect function or expression, within the genome of wild-type cells of the host cell.

[0026] As used herein, the terms “polypeptide” and “peptide” are used interchangeably and refer to the collective primary, secondary, tertiary, and quaternary amino acid sequence and structure necessary to give the recited macromolecule its function and properties. As used herein, “enzy me” or “biosynthetic pathway enzyme” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzy me, either independently or as part of a biosynthetic pathway is understood to include the co-factors, co-enzymes, and metals necessary for the enzyme to properly function. A summary of the amino acids and their three and one letter symbols as understood in the art is presented in Table 1. The amino acid name, three letter symbol, and one letter symbol are used interchangeably herein.Table 1. Amino Acid three and one letter symbolsPT-1891-WO-PCT

[0027] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the disclosed engineered cells, compositions, and methods. Such sequences can be referred to as variants or modified sequences. That is, a polypeptide sequence can be modified yet still retain the ability7to exhibit the desired activity7. Generally, the variant or modified sequence may include or be greater than about 45%. 45%, 50%, 55%, 60%, 65%. 70%, 75%, 80%, 85%. 90%, or 95% sequence identity with the wild type, naturally occurring polypeptide sequence, or with a variant polypeptide as described herein.

[0028] As used herein, the phrases “% sequence identity7,” “% identity7,” and “percent identity7,” are used interchangeably and refer to the percentage of residue matches betw een 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. Sequence alignment and generation of sequence identity7include global alignments and local alignments which are carried out using computational approaches. An alignment can be performed using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.31 software with default parameters. Amino acid % sequence identity between amino acid sequences can be determined using standard protein BLAST with the following defaultPT-1891-WO-PCT parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a uery range: 0; Matrix: BLOSUM62; Gap Costs: (Existence: 11, Extension: 1); Compositional adjustments: Conditional compositional score matrix adjustment; Filter: none selected; Mask: none selected. Nucleic acid % sequence identity between nucleic acid sequences can be determined using standard nucleotide BLAST with the following default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match / Mismatch Scores: 1, -2; Gap costs: Linear: Filter: Low complexity regions; Mask: Mask for lookup table only. A sequence having an identity score of XX% (for example, 80%) with regard to a reference sequence using the NCBI BLAST version 2.2.31 algorithm with default parameters is considered to be at least XX% identical or, equivalently, have XX% sequence identity to the reference sequence.

[0029] Polypeptide or polynucleotide sequence identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, 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 contiguous residues. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.

[0030] The polypeptides disclosed herein may include “variant’" polypeptides, “mutants,” and “derivatives thereof.” As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of a polypeptide as it occurs in nature as distinguished from variant or mutant forms. As used herein, a “variant,” “mutant,” or “derivative” refers to a polypeptide molecule having an amino acid sequence that differs from a reference protein or polypeptide molecule. A variant or mutant may have one or more insertions, deletions, or substitutions of an amino acid residue relative to a reference molecule.

[0031] The amino acid sequences of the polypeptide variants, mutants, derivatives, or fragments as contemplated 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 that are a substitution of an amino acid for a different amino acid where the substitution is predicted to interfere least with thePT-1891-WO-PCT properties of the reference polypeptide. In other words, conservative amino acid substitutions substantially conserve the structure and the function of the reference polypeptide. Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, 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 bulk of the side chain.

[0032] As used herein, terms “polynucleotide,” “polynucleotide sequence,” and “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides 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 or the antisense strand. The DNA polynucleotides may be a cDNA or a genomic DNA sequence.

[0033] A polynucleotide is said to encode a polypeptide if, in its native state or when manipulated by methods known to those skilled in the art, it can be transcribed and / or translated to produce the polypeptide or a fragment thereof. The anti-sense strand of such a polynucleotide is also said to encode the sequence.

[0034] Those of skill 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 polynucleotides (i.e., polynucleotides encoding a non-heme iron-binding protein polypeptide) may be codon-optimized for expression in a particular cell including, without limitation, a plant cell, bacterial cell, fungal cell, or animal cell. While polypeptides encoded by polynucleotide sequences found in various host organisms are disclosed herein any polynucleotide sequences may be used which encodes a desired form of the polypeptides described herein. Thus, non-naturally occurring sequences may be used. These may be desirable, for example, to enhance expression in heterologous expression systems of polypeptides or proteins. Computer programs for generating degenerate coding sequences are available and can be used for this purpose. Pencil, paper, the genetic code, and a human hand can also be used to generate degenerate coding sequences.

[0035] The recombinant cells described herein may include deletions or disruptions in one or more native genes. The phase “deletion or disruption” refers to the status of a native gene in the recombinant cell that has either a completely eliminated coding region (deletion) or a modification of the gene, its promoter, or its terminator (such as be a deletion, insertion, or mutation) so that the gene no longer produces an active expression product, produces severely reduced quantities of the expression product (e.g., at least a 75% reduction or at least a 90% reduction) or produces an expression product with severely reduced activity (e.g., at least 75% reduced or at least 90%PT-1891-WO-PCT reduced). The deletion or disruption can be achieved by genetic engineering methods, forced evolution, mutagenesis, and / or selection and screening. The native gene to be deleted or disrupted may be replaced with an exogenous nucleic acid of interest for the expression of an exogenous gene product (e.g., polypeptide, enzy me, and the like).

[0036] The recombinant cell described herein may have a deletion or disruption of one or more native genes encoding an enzyme involved in the synthesis of glycerol. Deletion or disruption of one or more of these glycerol biosynthetic pathway enzy mes decreases the ability of the cell to produce glycerol, thereby increasing fermentation production of ethanol.

[0037] The recombinant cells described herein may include a deletion or disruption of a native glycerol-3- phosphate phosphatase (GPP) gene. The native GPP gene(s) encode an enzyme that catalyzes the hydrolysis of glycerol-3-phosphate into glycerol. When the host cell contains multiple GPP genes, it is preferred to delete or disrupt at least one of them and more preferred to disrupt all of them to more completely eliminate the host cell’s ability to produce glycerol. In S. cerevisiae. there are two GPP paralogs, referred to as Gpplp (SEQ ID NO: 1). encoded by the GPP 1 gene, and Gpp2p (SEQ ID NO: 2), encoded by the GPP 2 gene. When the recombinant cell is a S. cerevisiae cell, the cell may include a deletion or disruption of a GPP gene encoding an amino acid sequence 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: 1 and 2. Methods for the deletion or disruption of the GPP genes of S. cerevisiae are known and described in the art and are exemplified herein.

[0038] The recombinant cells described herein may include a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GPD) gene. Deletion or disruption of a native GPD gene improves acetate consumption by providing the cell with a greater pool of reducing equivalents to assist in the oxido-reduction of acetate to ethanol. When the host cell contains multiple GPD genes, it is preferred to delete or disrupt at least one of them and more preferred to disrupt all of them. In S. cerevisiae, there are two glycerol-3-phosphate dehydrogenases, referred to as Gpdlp, encoded by GPD1. and Gpd2p, encoded by GPD2.

[0039] The recombinant cells described herein may include one or more genetic modifications in which an exogenous nucleic acid is integrated into the genome of the host cell. One of skill in the art know how to select suitable loci in a yest genome for integration of the exogenous nucleic acid. For example, in an S. cerevisiae host cells, suitable interaction loci may include, but are not limited to, the GPP1 loci (defined as the loci flanked by SEQ ID NO:3 and SEQ ID NO:4), the DLD1 loci (defined as the loci flanked by SEQ ID NO:5 and SEQ ID NO:6), and the GPD1 lociPT-1891-WO-PCT(defined as the loci flanked by SEQ ID NO:7 and SEQ ID NO:8). Other suitable integration loci may be determined one of skill in the art. Furthermore, one of skill in the art would recognize how to use sequences to design primers to verify correct gene integration at the chosen locus.

[0040] The recombinant cells described herein are capable of producing ethanol and contain an exogenous polynucleotide sequence encoding an alpha-amylase enzyme. The alpha-amylase enzyme may be any suitable enzyme with alpha-amylase activity. The exogenous polynucleotide sequence may be an exogenous alpha-amylase gene.

[0041] As used herein "alpha-amylase gene’' refers to any gene or polynucleotide that encodes a polypeptide with alpha-amylase activity. As used herein "alpha-amylase enzy me” refers to a polypeptide with alpha-amylase activity. As used herein, "al ph a- amylase activity” refers to the ability to catalyze the hydrolysis of internal alpha- 1,4-glycosidic linkages in polysaccharides. The alpha symbol “a” is commonly used in the art in place of the word alpha and they have the same meaning. The alpha-amylase may be from any suitable source organism or may be synthetic. Suitable alpha-amylase enzymes may include, but are not limited to, enzymes of EC 3.2. 1.1. Suitable alpha-amylase enzymes may be the alpha-amylase enzy mes from Aspergillus niger. Schwanniomyces occidentalism Bacillus subtilis, Thermoactinomyces vulgaris. Butyrivibrio fibrisolvens and the like. The alpha-amylase gene may encode an amino acid sequence 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:35-39. The alpha-amylase gene may encode an amino acid sequence 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:35, 36, and 38.

[0042] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from an Aspergillus niger gene encoding the amino acid sequences of SEQ ID NO:35. The exogenous polynucleotide may7encode an amino acid sequence with 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:35.

[0043] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Schwanniomyces occidentalis gene encoding the amino acid sequences of SEQ ID NO:36. The exogenous polynucleotide may encode an amino acid sequence with 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:36.

[0044] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Bacillus subtilis gene encoding the amino acid sequences of SEQ ID NO:37. ThePT-1891-WO-PCT exogenous polynucleotide may encode an amino acid sequence with 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:37.

[0045] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Thermoactinomyces vulgaris gene encoding the amino acid sequences of SEQ ID NO:38. The exogenous polynucleotide may encode an amino acid sequence with 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 38.

[0046] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Butyrivibrio fibrisolvens gene encoding the amino acid sequences of SEQ ID NO:39. The exogenous polynucleotide may encode an amino acid sequence with 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:39.

[0047] The recombinant cells descnbed herein are capable of producing ethanol and include an exogenous polynucleotide sequence encoding a pullulanase enzy me. The recombinant cells described herein are capable of producing ethanol and may include both an exogenous polynucleotide sequence encoding a pullulanase enzyme and an exogenous polynucleotide sequence encoding an alpha-amylase enzyme. The pullulanase enzyme may be any suitable enzyme with pullulanase activity'. The exogenous polynucleotide sequence may be an exogenous pullulanase gene.

[0048] As used here, “pullulanase gene’' refers to any gene or polynucleotide that encodes a polypeptide with pullulanase activity. As used herein “pullulanase enzyme” refers to a polypeptide with pullulanase activity.” As used herein “pullulanase activity” refers to the ability' to catalyze the hydrolysis of alpha- 1,6 glucosidic linkages in polysaccharides. The pullulanase enzy me may be from any suitable source organism or may be synthetic. Suitable pullulanase enzymes may include, but are not limited to, enzymes of EC 3.2.1.41. Suitable pullulanase enzy mes may be the pullulanase enzymes from Paenibacillus sp. FSL H8-0259, Fonsecaea nubica. Pseudobacteroides celhilosolvens , and the like. The pullulanase gene may encode an amino acid sequence 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:58-60.

[0049] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Paenibacillus sp. FSL H8-0259 gene encoding the amino acid sequences of SEQ ID NO:58. The exogenous polynucleotide may encode an amino acid sequence with at least 70%,PT-1891-WO-PCT 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:58.

[0050] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Fonsecaea nubica gene encoding the amino acid sequences of SEQ ID NO:59. The exogenous polynucleotide may encode an amino acid sequence with 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:59.

[0051] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from aPseudobacteroid.es cellulosolvens gene encoding the amino acid sequences of SEQ ID NO:60. The exogenous polynucleotide may encode an amino acid sequence with 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:60.

[0052] The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzyme and may include an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme. The GA enz me may be any suitable enzyme with glucoamylase activity. The exogenous polynucleotide sequence may be an exogenous glucoamylase (GA) gene.

[0053] A “glucoamylase gene" and ‘'GA gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with glucoamylase activity. As used herein, “glucoamylase activity” refers to the ability to catalyze the hydrolysis of the terminal 1,4-linked alpha-D-glucose residue from the non-reducing end of an amylose chain to release free glucose. As used herein “glucoamylase enzyme” and “GA enzyme” are used interchangeably and refer to a polypeptide with glucoamylase activity. The GA enzyme can be from any suitable source organism or may be synthetic. Suitable glucoamylase enzy mes may include, but are not limited to, enzymes of EC 3.2. 1.3. Suitable GA enzymes may be the GA enzymes from Saccharomycopsis fibuligera. Rhizopus delemar. Rhizopus microsporus, Rhizopus or zae. and the like. The GA gene may encode an amino acid 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:9-12. The GA gene may encode an amino acid 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:9 and 10. Additional GA genes and GA enzyme sequences are known and described in the art, such as GA genes encoding GA enzymes with modified leader / signal sequences. See, for example, US PatentNo. 10,364,421,PT-1891-WO-PCTUS Patent No. 10,724,023, US Patent Publication No. 20190345471 Al, and US Patent No. 1 1,306,330, each of which is incorporated by reference herein in its entirety.

[0054] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus microsporus gene encoding the amino acid sequences of SEQ ID NO: 9. The exogenous polynucleotide may encode an amino acid sequence with 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:9.

[0055] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Saccharomycopsis flbuligera gene encoding the amino acid sequences of SEQ ID NOTO. The exogenous polynucleotide may encode an amino acid sequence with 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 NOTO.

[0056] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus delemar gene encoding the amino acid sequences of SEQ ID NOT 1. The exogenous polynucleotide may encode an amino acid sequence with 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 NOTE

[0057] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhizopus oryzae gene encoding the amino acid sequences of SEQ ID NOT 2. The exogenous polynucleotide may encode an amino acid sequence with 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: 12.

[0058] The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzyme and may include an exogenous polynucleotide sequence encoding aglyceraldehyde-3-phosphate dehydrogenase (gapN) enzy me. The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzyme and may include an exogenous polynucleotide sequence encoding a gapN enzyme and an exogenous polynucleotide sequence encoding a GA enzyme. The gapN enzyme may be any suitable enzyme with glyceraldehyde-3-phosphate dehydrogenase activity. The exogenous polynucleotide sequence may be an exogenous glyceraldehyde-3 -phosphate dehydrogenase (gapN) gene.PT-1891-WO-PCT

[0059] A “glyceraldehyde-3-phosphate dehydrogenase gene” and "gapN gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with glyceraldehyde-3 -phosphate dehydrogenase activity. As used herein “glyceraldehyde-3- phosphate dehydrogenase activity” refers to the ability to catalyze the conversion of D- glyceraldehyde 3-phosphate and NADP+to 3-phospho-D-gly cerate and NADPH. As used herein, “glyceraldehyde-3-phosphate dehydrogenase enzyme” and "gapN enzyme” are used interchangeably and refer to a polypeptide with glyceraldehyde-3-phosphate dehydrogenase activity. The gapN enzyme can be from any suitable source organism or may be synthetic. Suitable gapN enzymes may include, but are not limited to, enzymes categorized under Enzyme Commission (EC) number 1.2. 1.9, also known in the art as '‘NADP-dependent nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase.” Suitable gapN enzymes may be the gapN enzymes from Streptococcus pyogenes, Pseudomonas fluorescens , Brevibacillus laterosporus . Arabidopsis thaliana. Chryseobacterium gleum, Streptococcus mutans, Streptococcus henryi, Lactobacillus delbrueckii. Bacillus cereus, and the like. The gapN gene may encode an amino acid 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: 13-21. The gapN gene may encode an amino acid 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: 13-17. The gapN gene may encode an amino acid 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: 13, 14, 16, andl7.

[0060] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus pyogenes gene encoding the amino acid sequences of SEQ ID NO: 13. The exogenous polynucleotide may encode an amino acid sequence with 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: 13.

[0061] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Pseudomonas fluorescens gene encoding the amino acid sequences of SEQ ID NO: 14. The exogenous polynucleotide may encode an amino acid sequence with 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: 14.

[0062] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Brevibacillus laterosporus gene encoding the amino acid sequences of SEQ ID NO: 15. The exogenous polynucleotide may encode an amino acid sequence with at least 70%, atPT-1891-WO-PCT least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO: 15.

[0063] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from aArabidopsis thaliana gene encoding the amino acid sequences of SEQ ID NO: 16. The exogenous polynucleotide may encode an amino acid sequence with 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: 16.

[0064] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Chryseobacterium gleum gene encoding the amino acid sequences of SEQ ID NO: 17. The exogenous polynucleotide may encode an amino acid sequence with 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: 17.

[0065] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus mutans gene encoding the amino acid sequences of SEQ ID NO: 18. The exogenous polynucleotide may encode an amino acid sequence with 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: 18.

[0066] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Streptococcus henryi gene encoding the amino acid sequences of SEQ ID NO: 19. The exogenous polynucleotide may encode an amino acid sequence with 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: 19.

[0067] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Lactobacillus delbrueckii gene encoding the amino acid sequences of SEQ ID NO:20. The exogenous polynucleotide may encode an amino acid sequence with 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:20.

[0068] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Bacillus cereus gene encoding the amino acid sequences of SEQ ID NO:21. The exogenous polynucleotide may encode an amino acid sequence with 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:21.PT-1891-WO-PCT

[0069] The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzyme and may include an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme. The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzy me and may include an exogenous polynucleotide sequence encoding an ADH enzyme and an exogenous polynucleotide sequence encoding a gapN enzyme. The recombinant cells described herein are capable of producing ethanol, include an exogenous polynucleotide sequence encoding a pullulanase enzyme and / or an exogenous polynucleotide sequence encoding an alpha-amylase enzy me and may' include an exogenous polynucleotide sequence encoding an ADH enzyme, an exogenous polynucleotide sequence encoding a gapN enzyme, and an exogenous polynucleotide sequence encoding a GA enzyme. The ADH enzyme may be any suitable enzyme with NADP-dependent alcohol dehydrogenase activity’. The exogenous polynucleotide sequence may be an exogenous alcohol dehydrogenase (ADH) gene.

[0070] An “alcohol dehydrogenase gene” and “ADH gene” are used interchangeably herein and refer to any gene or polynucleotide that encodes a polypeptide with alcohol dehydrogenase activity. As used herein, “alcohol dehydrogenase activity” refers to the ability to catalyze the conversion of acetaldehyde and NADH or NADPH to ethanol and NAD+or NADP+. As used herein, NADP-dependent alcohol dehydrogenase activity” refers to the ability7to catalyze the conversion of acetaldehyde and NADPH to ethanol and NADP+. The ADH enzyme may be derived from any suitable source or may be synthetic. Suitable ADH enzymes may be the ADH enzymes omRhodotorula toruloides, Candida maltosa, and the like. The ADH gene may encode an amino acid 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:22 and 23.

[0071] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Candida maltosa gene encoding the amino acid sequences of SEQ ID NO:22. The exogenous polynucleotide may encode an amino acid sequence with 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:22.

[0072] The recombinant cell may comprise an exogenous polynucleotide that is or may be derived from a Rhodotorula toruloides gene encoding the amino acid sequences of SEQ ID NO:23. The exogenous polynucleotide may encode an amino acid sequence with at least 70%, atPT-1891-WO-PCT least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to SEQ ID NO:23.

[0073] The exogenous nucleic acids in the recombinant cells described herein may be under the control of a promoter. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Promoters may include, but are not limited to, pyruvate decarboxylase (PDC1 ), glyceraldehyde- 3-phosphate dehydrogenase (GAPDH) (TDH3 herein; annotated in EC 1.2.1.12; SEQ ID NO:24), translational elongation factor 1 (TEF1; SEQ ID NO:25), URA3 (SEQ ID NO:26), S-adenosyl methionine transferase 2 (SAM2; SEQ ID NO:27), alcohol dehydrogenase 1 (ADH1; SEQ ID NO:28) and 3-phosphogly cerate kinase (PGK1; SEQ ID NO:29).

[0074] The exogenous nucleic acids in the recombinant cells described herein may be under the control of a terminator. For example, the exogenous nucleic acid may be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. Terminators may include, but are not limited to. iso- 1 -cytochrome c (CYC1; SEQ ID NO:30), URA3 (SEQ ID NO:31), PDC, ADH1 (SEQ ID NO:32), TEF1 (SEQ ID NO:33), ScGALlO (SEQ ID NO:34), and ribosomal 60S subunit protein L3 (RPL3; SEQ ID NO:75).

[0075] A promoter or terminator is “operably linked” to a given polynucleotide (e.g., a gene) if its position in the genome or expression cassette relative to said polynucleotide is such that the promoter or terminator, as the case may be, performs its transcriptional control function.

[0076] The polypeptides described herein may be provided as part of a construct. As used herein, the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double-stranded and may represent the sense or the antisense strand. Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies. The construct may be a vector including a promoter operably linked to the polynucleotide encoding the thermolabile non-heme iron-binding polypeptide. As used herein, the term “vector” refers to a polynucleotide capable of transporting another polynucleotide to which it has been linked. The vector may be a plasmid, which refers to a circular double-stranded DNA loop into which additional DNA segments may be integrated.PT-1891-WO-PCT

[0077] The disclosure also provides fermentation methods for the production of ethanol using the recombinant cells described herein. The fermentation methods include the step of fermenting a substrate using the genetically engineered yeasts described herein to produce ethanol. The fermentation method can include additional steps, as would be understood by a person skilled in the art. 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 isolating the ethanol from the fermentation broth.

[0078] The fermentation substrate can comprise a starch. Starch can be obtained from a natural source, such as a plant source. Starch can also be obtained from a feedstock with high starch or sugar content, including, but not limited to com, sweet sorghum, fruits, sweet potato, rice, barley, sugar cane, sugarbeets, wheat, cassava, potato, tapioca, arrowroot, peas, or sago. The fermentation substrate may be from lignocellulosic biomass such as wood, straw, grasses, or algal biomass, such as microalgae and macroalgae. The fermentation substrate may include cellulosic or lignocellulosic biomass. The fermentation substrate may be from grasses, trees, or agricultural and forestry residues, such as com cobs and stalks, rice straw, sawdust, and wood chips. The fermentation substrate can also comprise a sugar, such as glucose (dextrose) or sucrose. The fermentation substrate may comprise a dry grind ethanol feedstock, such as com mash. The fermentation substrate can comprise a liquefied com mash (LCM). The fermentation substrate may comprise a com wet mill feedstock, such as Light Steep Water / Liquifact (LSW / LQ).

[0079] Media for fermentation of the engineered yeast described herein can be supplemented with various components. For example, media for fermentation of the engineered yeast described herein can be supplemented with a glucoamylase, e.g., the glucoamylase SPIRIZYME™ (Novozymes, Bagsvaerd, Denmark) or the glucoamylase DISTILLASE® DXT (Dupont Industrial Biosciences).

[0080] The fermentation process can be run under various conditions. The fermentation temperature, i.e.. the temperature of the fermentation broth during processing, may be ambient temperature. Alternatively, or additionally, the fermentation temperature may be maintained within a predetermined range. For example, the fermentation temperature can be maintained in the range of 25 °C to 40 °C, 27 °C to 38 °C, or 30 °C to 35 °C. The fermentation temperature may be maintained at a temperature of, e.g., 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C, or any value in between. However, a skilled artisan will recognize that the fermentation temperature is not limited to any specific range recited herein and may be modified as appropriate.PT-1891-WO-PCT

[0081] The pH of a culture medium described herein may be controlled for optimal ethanol production. The pH of the culture or a fermentation mixture of an engineered cell described herein may be in the range of between 4.0 and 6.0. The pH may be maintained for at least part of the incubation 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 may be maintained at a range between 5.0 and 5.5.

[0082] The engineered yeast may be cultured for approximately 24-72 hours. For example, the engineered yeast may be cultured for approximately 12, 18, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72. 73. 74. 75, 78, 80, 90, 96 hours, or more than 96 hours. The engineered yeast described herein may be cultured for approximately 48 to 72 hours. A culture (fermentation) time of about 48 hours is a representative time for commercial-scale ethanol fermentation processes. Accordingly, a 48 hour time point can be used to compare the fermentation performance of different yeast strains.

[0083] Reaction parameters can be measured or adjusted during the production of ethanol. 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 substrate and / or product, concentration of dissolved gases, such as oxygen concentration and CO2 concentration, nutrient concentrations, metabolite concentrations, ethanol concentration, fermentation substrate concentration, concentration of an oligopeptide, concentration of an amino acid, concentration of a vitamin, concentration of a hormone, concentration of an additive, serum concentration, ionic strength, concentration of an ion, relative humidity, molarity, osmolarity, concentration of other chemicals, for example buffering agents, adjuvants, or reaction by-products), 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, as well as thermodynamic parameters, such as temperature, light intensity / quality, etc.). Sensors to measure the parameters described herein are well known to one of ordinary skill in the art.

[0084] The fermentation process can be associated with various characteristics, such as, but not limited to, fermentation production rate, pathway fermentation yield, final titer, and peak fermentation rate. These characteristics can be affected by the selection of the yeast and / or genetic modification of the yeast used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via aPT-1891-WO-PCT combination of yeast selection or modification and the selection of fermentation process conditions.

[0085] The ethanol production rate of the process may be at least 1.0, at least 1.5, or at least 2.0, at least 2.5, at least 3.0, or at least 3.5 g L1h'1. The final ethanol titer of the process may be at least 80 g / L. at least 100, at least 120. or at least 140 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.EXAMPLES

[0086] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1 : Saccharomyces cerevisiae Strain EngineeringStrain 1-1

[0087] Strain 23 described by Jauert et al. (PCT Patent Application Publication No. WO2018 / 204798, published November 8. 2018, incorporated herein by reference in its entirety) is a Saccharomyces cerevisiae host strain that is ura3 positive (lira 3+) and amdS positive (amclS+) and in which both alleles of the cytosine deaminase (FCY1) gene are knocked out and replaced with an expression cassette for i) the Rhizopus microsporus glucoamylase of SEQ ID NO:9 with a TDH3 promoter and a CYC1 terminator and ii) the Magnaporthe grisea trehalase under the control of a SAM2 promoter and a GAL 10 terminator. Herein, Strain 1-1 refers to Strain 23 of Jauert et al. PCT Patent Application Publication No. WO2018 / 204798.Strain 1-2

[0088] Strain 1-1 was transformed with SEQ ID NO:76. Transformants were selected on synthetic complete media plates containing 3.5 g / L of p-fluorophenylalanine and 1 g / L L-tyrosine (ScD-PFP). Resulting transformants were struck out for single colony isolation and a single colony was selected. The ura3- and amdS- PCR verified isolate was designated strain 1-2.PT-1891-WO-PCTStrain 1-3

[0089] Strain 1-2 was transformed with SEQ ID NO:77. SEQ ID NO:77 contained the ScURA3 promoter (SEQ ID NO:26), the ScURA3 gene, and the ScURA3 terminator (SEQ ID NO:31). Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. A single PCR verified isolate was designated strain 1-3.Strain 1-4

[0090] Strain 1-2 was transformed with SEQ ID NO:78 and SEQ ID NO:79. SEQ ID NO:78 contained an expression cassette for the Rhizopus microspores glucoamylase of SEQ ID NO: 9 under the control of the TDH3 promoter (SEQ ID NO:24) and the CYC1 terminator (SEQ ID NO:30) and a 5’ portion of the Saccharomyces cerevisiae URA3 gene under the control of the URA3 promoter (SEQ ID NO:26). SEQ ID NO:79 contained a 3’ portion of the Saccharomyces cerevisiae URA3 gene with the URA3 terminator (SEQ ID NO:31). an expression cassette for the Rhizopus microsporus glucoamylase of SEQ ID NO:9 under the control of the PGK promoter (SEQ ID NO:29) and the GAL10 terminator (SEQ ID NO:34) and the 5’ portion of the Saccharomyces cerevisiae SAM2 promoter. Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. A single PCR verified isolate was designated strain 1-4.Strain 1-5

[0091] Strain 1-4 was transformed with SEQ ID NO:80 and SEQ ID NO:81. SEQ ID NO:80 contained a 3' portion of the Aspergillus nidulans amdS gene with the ADH terminator (SEQ ID NO:32), an expression cassette for the Rhizopus microsporus glucoamylase of SEQ ID NO:9 under the control of the PGK promoter (SEQ ID NO:290 and the GAL10 terminator (SEQ ID NO:34), and the 5’ portion of the Saccharomyces cerevisiae SAM2 promoter. SEQ ID NO:81 contained an expression cassette for the Rhizopus microsporus glucoamylase of SEQ ID NO:9 under the control of the TDH3 promoter (SEQ ID NO:240 and the CYC1 terminator (SEQ ID NO:30) and the 5’ portion of the Aspergillus nidulans amdS gene with a TEF1 promoter (SEQ ID NO:25). Transformants were selected on YNB + acetamide plates then struck out for single colony isolation. A single PCR verified isolate was designated strain 1-5.PT-1891-WO-PCTStrain 1-6

[0092] Strain 1-5 was transformed with SEQ ID NO: 82. SEQ ID NO: 82 was an episomal plasmid including a G418 resistance gene (SEQ ID NO: 86) under the control of the TEF1 promoter (SEQ ID NO:25) and the GAL10 terminator (SEQ ID NO:34), a Cre recombinase gene (SEQ ID NO: 87) under the control of the PGK promoter (SEQ ID NO:29) and the CYC1 terminator (SEQ ID NO:30). Transformants were selected on YPD + 200 mg / L geneticin (G418) and grown for 2 days at 30°C. Transformants were selected, diluted 1 / 10, and plated on YPD+200 mg / L G418 to obtain single colony isolates. Isolates were screened for loss of the URA3 (ScD- ura plates) and amdS marker (on YNB / acetamide plates). Plates were grown overnight or for 2 days (YNB acetamide) at 30 °C. Strains that had lost the amdS marker were grown overnight in a YPD medium in deep-well plates with no selection to cure the G418 plasmid, and plated for singles on YPD agar plates and grown overnight at 30 °C. The ura3- and amdS- PCR verified isolate was designated strain 1-6.Strain 1-7

[0093] Strain 1-6 was transformed with SEQ ID NO:77. SEQ ID NO:77 contained the ScURA3 promoter (SEQ ID NO:26), the ScURA3 gene, and the ScURA3 terminator (SEQ ID NO:31). Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. A single PCR verified isolate was designated strain 1-7.Example 2: Alpha- Amylase Assays

[0094] Alpha-amylase candidates, 24 in total, were chosen based on previous analysis (not show) and assayed to determining the degree of polymerization (DP) of the remining starch in a fermentation broth. The alpha-amylase candidates are outlined in Table 2.

[0095] Gene sequences encoding each of the alpha-amylase candidates outlined in Table 2 were cloned into the plasmid of SEQ ID NO:58. Each cloned plasmid was then transformed into the Saccharomyces cerevisiae strain Ethanol Red® (Strain 2-1). Table 2 also outlines the strains number produced by the transformation of Ethanol Red® (Strain 2-1).PT-1891-WO-PCTTable 2.Deep Well AssayL0096] Seed cultures of strains 1-7, 2-4, 2-2, 2-8, 2-9, 2-10, 2-3, 2-11, 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, and 2-19 were produced by inoculating 600 pl of synthetic complete medium with 50 g / L dextrose (ScD50; 6.7 g / L yeast nitrogen base without amino acids, 50 g / L dextrose,PT-1891-WO-PCT and 2 g / L amino acid mixture) and incubating at 30 °C and 800 rpm for 24 hours. Deep well plates were filled with 700 pl of assay media (850g partially hydrolyzed com starch, 1 0g light steep water, 25g dextrose, and 1g urea) in each of the 96 wells. Each well was inoculated to an Optical Density at 600nm (OD600) of 0. 1 using the 24-hour seed culture. Deep well plates were incubated at 30 °C and 800 rpm for 24 hours. Table 3 and FIG. 1 show results for the 24-hour deep well plate cultures, including the titer of ethanol produced as well as the degree of polymerization (DP) of the remaining starch in the fermentation broth.Table 3.

[0097] Based on the 24-hour deep well plate assay, expression of the alpha-amylase enzymes of SEQ ID NOs:35, 36, and 37, in strains 2-2. 2-3, and 2-4, respectively, resulted in a decrease inPT-1891-WO-PCTDP4+ starches to less than 100 g / L in the fermentation broth compared to the No Cells control while maintaining an ethanol titer above 25 g / L.Fermentation Broth Assay

[0098] Strain 1-7 was grown in shake flasks with 30 ml media (850g partially hydrolyzed com starch, 150g light steep water, 25g dextrose, and 1g urea) at 30 °C, 250 rpm, and 8% relative humidity. Glucoamylase enzyme was added after 5 hours of grow th and agitation was decreased to 100 rpm. After 72 hours, the fermentation broth was spun and filter sterilized to remove any cells. Each well of a 96-deep well plate was filled with 300 pL of this fermentation broth and to this, 300 pL of fermentation broth from the deep well cultures (as described above under '‘Deep Well Assay”) of strains 2-5, 2-20, 2-6, 2-21, 2-16, 2-22, 2-23, 2-24, 2-8, and 2-2 were added in separate wells. The 96-well plates were incubated at 30 °C and 800 rpm for 24 hours. Results shown in Table 4 and FIG. 2 report the titer of dextrose and the DP2, DP3, and DP4 starches in the broth after incubation.Table 4.

[0099] In the fermentation broth assay, the alpha amylase enzymes secreted from the indicated strain were introduced to a new fermentation broth with a different starch profile. The resulting DP starch profiles from the various enzymes tested are also therefore different. For example, thePT-1891-WO-PCT alpha-amylase enzymes of SEQ ID NOs:35, 38, 39, 40, and 56, secreted from strains 2-2, 2-5, 2- 6, 2-7, and 2-23, respectively, were selected for further analysis based on their unique starch profiles.Example 3: Pullulanase Assays

[0100] Pullulanase candidates (17 in total) were chosen based on previous analysis (not show) and assayed to determining the degree of polymerization (DP) of the remining starch in a fermentation broth. The pullulanase candidates are outlined in Table 5.Gene sequences encoding each of the pullulanase candidates outlined in Table 5 were cloned into the plasmid of SEQ ID NO:58. Each cloned plasmid was then transformed into the Saccharomyces cerevisiae strain Ethanol Red® (Strain 2-1). Table 5 also outlines the strain number produced by the transformation of Ethanol Red® (Strain 2-1). The pullulanase sequences include a leader sequence to promote secretion of the enzymes into the fermentation broth. The recited SEQ ID NO: of the pullulanase enzymes include either the Pho5 leader sequence of SEQ ID NO: 101 or the Mfa-2 leader sequence of SEQ ID NO: 102.Table 5.PT-1891-WO-PCTDeep Well Assay

[0101] Seed cultures of strains 1-7, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 3-11, 3-1, 3-12, 3-13, and 3-14 were produced by inoculating 600 pL of ScD50 medium and incubating at 30 °C and 800 rpm for 24 hours. Deep well plates were filled with 700 pL of assay media (850g partially hydrolyzed com starch, 150g light steep water, 25g dextrose, and 1g urea) in each of the 96 wells. Each well was inoculated to an OD600 of 0. 1 using the 24-hour seed culture. Deep well plates were incubated at 30 °C and 800 rpm for 24 hours. Table 6 and FIG. 3 show results for the 24- hour deep well plate cultures, including the titer of residual glucose as well as the DP of the remaining starch in the fermentation broth.Table 6.PT-1891-WO-PCT

[0102] Based on the 24-hour deep well plate assay, expression of the pullulanase enzymes of SEQ ID NOs:70, 68, 66, and 64, in strains 3-13, 3-11, 3-9, and 3-7, respectively, were selected for additional testing.Fermentation Broth Assay

[0103] Strain 1 -7 was grown in shake flasks with 50 ml media (590g partially hydrolyzed com starch, 180g light steep water, 72g dextrose, and 158g water) at 30 °C, 100 rpm, and 80% relative humidity. After 72 hours, the fermentation broth was spun and filter sterilized to remove any cells. Each well of a 96-deep well plate was filled with 300 pL of this fermentation broth and to this, 300 pL of fermentation broth from the deep well cultures (as described above under '‘Deep Well Assay”) of strains 3-3, 3-2, 3-15, 3-16, 3-1, 3-10, and 3-17 were added in separate wells. The 96- well plates were incubated at 30 °C and 800 rpm for 24 hours. Results shown in Table 7 and FIG. 4 report the titer of dextrose and the DP2, DP3, and DP4+ starches in the broth after incubation.Table 7.

[0104] In the fermentation broth assay, the pullulanase enzymes secreted from the indicated strain were introduced to a new fermentation broth with a different starch profile. The resulting DP starch profiles from the various enzymes tested are also therefore different. The pullulanase enzymes of SEQ ID NOs:60, 59, 72, 58, and 74 secreted from strains 3-3, 3-2, 3-15, 3-1, and 3- 17, respectively, were selected for further analysis based on their unique starch profiles relative to what was seen in the deep well assay and additional data not shown.PT-1891-WO-PCTExample 4: Saccharomyces cerevisiae Strain Engineering

[0105] The seven alpha-amylase and nine pullulanase candidates outlined in Table 8 were chosen for further shake flask fermentation analysis based on the experiments outlined above (Examples 2 & 3). Gene sequences encoding the alpha-amylase and pullulanase candidates were cloned into the plasmid of SEQ ID NO:58. which included the TDH3 promoter of SEQ ID NO:24. Each cloned plasmid was then transformed in strain 1 -2, as outlined in Table 8.Table 8.Example 5: Shake Flask Fermentation Assays

[0106] Strains 4-1 through 4-16 and 1-3 were assayed in shake flasks to assess ethanol production and glucose consumption.

[0107] Cells from each of the strains tested were added to 50 rnL of medium (590g partially hydrolyzed com starch, 180g light steep water, 72g dextrose, and 158g water) in a 250 mL baffled flask such that each initial culture contained cells at a concentration of 0. 1 as measured by OD600.PT-1891-WO-PCTFlasks were incubated at 30 °C, 100 rpm, and 80% relative humidity for 72 hours. Samples of fermentation broth at 72 hours were analyzed by HPLC for the titer of ethanol and total glucose equivalents. Results are reported in Table 9.

[0108] Since glucose can be present as free glucose or as linked monomers in polysaccharides, glucose measurements need to account for both. The total glucose equivalents measurement determines the total glucose present at the end of fermentation, including both free glucose and glucose bound in polysaccharides, for example maltose and isomaltose. Total glucose equivalence is measured using the following method. First, free glucose is measured with high performance liquid chromatography (HPLC) using refractive index (RI) detection. Glucose is measured in triplicate for each sample. Separation of polysaccharides from the sample is completed with a Bio Rad 87H column using a 10 mM H2SO4 mobile phase. Acid hydrolysis of the polysaccharide fraction is performed in triplicate in 6% (v / v) trifluoroacetic acid at 121°C for 15 minutes. The resulting glucose after hydrolysis is measured by the same HPLC method. The total glucose equivalents present in each sample is the amount of glucose measured after acid hydrolysis plus the amount of free glucose initially measured.Table 9.PT-1891-WO-PCT

[0109] The results demonstrate that expression of several alpha-amylase and pullulanase candidates resulted in a decrease in total glucose equivalents remaining after 72 hours, including those of SEQ ID NOs:38, 60, 70. 58. and 68. Expression of the candidates of SEQ ID NOs:39 and 60 also resulted in statistically significant increases in the titer of ethanol production by the strain.Example 6: Saccharomyces cerevisiae Strain Engineering

[0110] Saccharomyces cerevisiae strains were engineered to contain a gene encoding one of the alpha-amylase candidates and a gene encoding one of the pullulanase candidates under the control of a strong and / or weak promoter, as outlined in Table 10. These strains also expressed the gapN enzy me from Chryseobacterium gleum (SEQ ID NO: 17), the alcohol dehydrogenase enzyme from Candida mallosa (SEQ ID NO:22), and the glucoamylase enzyme from Rhizopus microsporus (SEQ ID NO:9).

[0111] The parent strain 5-1 was a ura3' Ethanol Red strain containing two copies of a gene encoding the R. microsporus glucoamylase of SEQ ID NO:9 under the control of the TDH3 promoter and CYC1 terminator and integrated at the FYC1 locus. Control strain 5-0 was a ura3 Ethanol Red strain containing two copies of a gene encoding the R. microsporus glucoamylase of SEQ ID NO:9 under the control of the TDH3 promoter and a CYC 1 terminator and integrated at the FYC1 locus.

[0112] SEQ ID NO:83 contained a 5’ GPP1 flanking sequence (SEQ ID NO:3); a PGK1 promoter (SEQ ID NO:29); a gene encoding the C. gleum gapN enzyme of SEQ ID NO: 17; an ADH1 terminator (SEQ ID NO:32); a TDH3 promoter (SEQ ID NO:24): a gene encoding the C. maltosa ADH enzyme of SEQ ID NO:22; a CYC1 terminator (SEQ ID NO:30); and a 5’ portion of a ScURA3 expression cassette.

[0113] SEQ ID NO:84 contained a 5’ GPP1 flanking sequence (SEQ ID NO:3), a PGK1 promoter (SEQ ID NO:29); a gene encoding the A. thaliana gapN enzyme of SEQ ID NO: 16; an ADH1 terminator (SEQ ID NO:32); a TDH3 promoter (SEQ ID NO:24); a gene encoding the C. maltosa ADH enzyme of SEQ ID NO:22; a CYC1 terminator (SEQ ID NO:30); and a 5’ portion of a ScURA3 expression cassette.PT-1891-WO-PCT

[0114] SEQ ID NO:85 contained a 3’ portion of an ScURA3 expression cassette and a 3’ GPP1 flanking sequence (SEQ ID NO:4).

[0115] SEQ ID NO:88 contained a 3’ portion of the ScURA3 gene and the URA3 terminator (SEQ ID NO:31) and a sequence encoding the Aspergillus niger alpha-amylase of SEQ ID NO:35 under the control of the TEF1 promoter (SEQ ID NO:25) and the TEF1 terminator (SEQ ID NO:33).

[0116] SEQ ID NO:89 contained a 5’ homology sequence to the TEF1 terminator, a sequence encoding the pullulanase of SEQ ID NO:58 under the control of the TDH3 promoter (SEQ ID NO:24) and the CYC1 terminator (SEQ ID NO30) and a 3’ GPP1 flanking sequence (SEQ ID NO:4).

[0117] SEQ ID NO:92 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Aspergillus niger alpha-amylase of SEQ ID NO:35 under the control of the SAM2 promoter (SEQ ID NO:27) and the TEF1 terminator (SEQ ID NO:33).

[0118] SEQ ID NO:93 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Schwanniomyces occidentalis alphaamylase of SEQ ID NO:36 under the control of the SAM2 promoter (SEQ ID NO:27) and the TEF1 terminator (SEQ ID NO:33).

[0119] SEQ ID NO:94 contained a 5’ homology sequence to the TEF1 terminator, a sequence encoding the Psuedobacterodies cellulosolvens pullulanase of SEQ ID NO: 60 under the control of the SAM2 promoter (SEQ ID NO:27) and the CYC1 terminator (SEQ ID NO30) and a 3' GPP1 flanking sequence (SEQ ID NO:4).

[0120] SEQ ID NO:95 contained a 5’ homology sequence to the TEF1 terminator, a sequence encoding the Fonsecaea nubica pullulanase of SEQ ID NO:59 under the control of the SAM2 promoter (SEQ ID NO:27) and the CYC1 terminator (SEQ ID NO30) and a 3’ GPP1 flanking sequence (SEQ ID NO:4).

[0121] SEQ ID NO:96 contained a 5’ homology sequence to the TEF1 terminator, a sequence encoding the Paenibacillus sp. FSL H8-0259 pullulanase of SEQ ID NO: 58 under the control of the SAM2 promoter (SEQ ID NO:27) and the CYC1 terminator (SEQ ID NO30) and a 3’ GPP1 flanking sequence (SEQ ID NO:4).

[0122] SEQ ID NO:97 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Butyrivibrio flbrisolvens alpha-PT-1891-WO-PCT amylase of SEQ ID NO:39 under the control ofthe TEFl promoter (SEQ ID NO:25) and the TEF1 terminator (SEQ ID NO:33).

[0123] SEQ ID NO:98 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Bacillus subtilis alpha-amylase of SEQ ID NO: 37 under the control of the TEF 1 promoter (SEQ ID NO:25) and the TEF 1 terminator (SEQ ID NO:33).

[0124] SEQ ID NO:99 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Schwanniomyces occidentalis alphaamylase of SEQ ID NO:36 under the control of the TEF 1 promoter (SEQ ID NO:25) and the TEF 1 terminator (SEQ ID NO:33).

[0125] SEQ ID NO: 100 contained a 5’ homology sequence to the TEF1 terminator, a sequence encoding the Fonsecaea nubica pullulanase of SEQ ID NO:59 under the control of the TDH3 promoter (SEQ ID NO:24) and the CYC1 terminator (SEQ ID NO30) and a 3' GPP1 flanking sequence (SEQ ID NO:4).

[0126] SEQ ID NO: 103 contained a 3’portion of the ScURA3 gene under the control of the URA3 terminator (SEQ ID NO:31) and a gene encoding the Thermoaciinomyces vulgaris alphaamylase of SEQ ID NO:38 under the control of the TEF1 promoter (SEQ ID NO:25) and the TEF 1 terminator (SEQ ID NO:33).

[0127] The indicated parent strain was transformed with the sequences outline in Table 10. Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. PCR verified isolates were designated as the indicated strain number. In some instances, more than one PCR verified isolate, i. e. , “sister” isolates, are indicated by letters following the strain number. For example, strain 5-3 has three sister isolates, strains 5- 3a, 5-3b, and 5-3c.PT-1891-WO-PCTTable 10.PT-1891-WO-PCTExample 7: Small Scale Fermentation Assays

[0128] Strains outlined in the Tables below were run in an Ambrl5 to assay ethanol production and DP sugar profiles.

[0129] Strains were struck on a ScD-ura plate and incubated at 30°C until single colonies were visible (2-3 days). Cells from the ScD-ura plate were scraped into sterile medium and the optical density (OD600) is measured. Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific). An Ambrl5 reaction vessel is inoculated with the cell slurry to reach an initial OD600 of 0.2. The fermentation medium consisted of 295g partially hydrolyzed com starch, 90g filtered sterilized (0.2 pm) light steep water, 79g sterile water, and 36g 500g / L sterile glucose. With continuous stirring, 12mL of fermentation media was added to each bioreactor. Strains were incubated at 30 °C, 450rpm of agitation. Air was supplied at 2.4 smlpm from the time of inoculation to 14 hours, then reduced to 0 smlpm for the remainder of the fermentation. Nitrogen gas was supplied at 0.01 smlpm from 14 hours until the end of fermentation. Samples were taken at 48 or 62 hours and analyzed for ethanol and DP sugar concentrations by HPLC.

[0130] Results of the Ambrl5 assays are reported in Tables 11-16. Alpha-amylase and pullulanase enzymes with a strong promoter are listed in bold. As is shown in the data, there is some sister-to-sister variability in the results. While strains are PCR verified to contain the desired polynucleotide sequence, said sequence may not be integrated at the correct loci, it may have had multiple copies of the sequence integrated into its genome, or a frameshift or other mutation caused an individual sister to vary from the others. The results here suggest that similar transformation occurrences are present in these sisters, but the results do demonstrate the effectiveness of the expression of the alpha-amylase and pullulanase enzymes.Table 11.PT-1891-WO-PCT

[0131] Results in Table 11 show that the combined expression of the alpha-amylase of SEQ ID NO:35 or 37 with the pullulanase of SEQ ID NO:59 produces more ethanol than the comparison strains lacking these enzymes, strains 5-0 and 5-21.Table 12.PT-1891-WO-PCT

[0132] The results in Table 12 show expression of the alpha-amylase of SEQ ID NO:35, 36, or 39 together with the pullulanase of SEQ ID NO:58 generally produces more ethanol than strains lacking these enzymes, strains 5-0 and 5-21. Also, expression of the alpha-amylase of SEQ ID NO:39 together with the pullulanase of SEQ ID NO:60 generally produces more ethanol than strains 5-0 and 5-21.Table 13.PT-1891-WO-PCT[01331 The results in Table 13 show expression of the alpha-amylase of SEQ ID NO:37 or 36 together with the pullulanase of SEQ ID NO:59 generally produces more ethanol than strains lacking the enzyme expression, 5-0 and 5-21. While there is some sister-to-sister variability, this is likely due to artifacts of the transformation as noted above.Table 14.PT-1891-WO-PCT

[0134] The results in Table 14 show strains that express a combination of the alpha-amylase of SEQ ID NO:35 and the pullulanase of SEQ ID NO:58 or 59 generally produce more ethanol than the parent strain 1-7.Table 15.PT-1891-WO-PCT

[0135] Results in table 15 show that strains expressing a combination of the alpha-amylase of SEQ ID NO:35 and the pullulanase of SEQ ID NO:58 or 59 produced ethanol at the same level or above the parent strain 1-7. The combination of the alpha-amylase of SEQ ID NO:39 and the pullulanase of SEQ ID NO: 59 in strains 5-24k and 5-24k showed increased ethanol relative to the parent strain 1-7.Table 16.PT-1891-WO-PCT

[0136] Results in Table 16 show that strains expressing the alpha-amylase of SEQ ID NO:38 together with the pullulanase of either SEQ ID NO:59 (5-6a, 1, and m) or 58 (5-7a, c, and e) produced more ethanol than control stain 5-0 or control strain 5-21. Further, these strains (5-6a, 1, m and 5-7a, c, e) resulted in less DP4+ sugars in the fermentation broth than control strain 5-12, which expressed the same glycerol reduction combination of the GapN of SEQ ID NO: 17 and the ADH of SEQ ID NO:22. Strains that express the alpha-amylase of SEQ ID NO:38 together with the pullulanase of SEQ ID NOs:58 or 59 produce more ethanol and less DP4+ sugars than corresponding strains lacking these enzymes.PT-1891-WO-PCTExample 8: Exogenous Enzvme Dosing

[0137] Alpha-amylase and pullulanase enzymes are often added to an ethanol fermentation broth exogenously during fermentation as part of an exogenous enzyme cocktail. In this example, dosing of exogenous enzymes into fermentations using strain 5-21 was compared to the rate, titer, andyield of ethanol production in strains 5-10c, 5-6m. 5-7e. 5-14c, 5-20c, 5- 15b, 5-19a, 5-8c, and 5-9b, which express these enzymes, against a control strain 5-21 lacking these enzymes.

[0138] The normal dose of exogenous enzyme is 0.15 g / kg and is added 14 hours after inoculation. To test the feasibility of replacing exogenous enzy me cocktails with the integrated expression of the alpha-amylase and pullulanase, fermentation assays were dosed with 0. 0. 10. or 0.15 g / kg of the enzyme cocktail sold under the tradename DISTILL ASE® DXT by DUPONT™.

[0139] Strains were struck on a ScD-ura or YPD plate and incubated at 30°C until single colonies were visible (2-3 days). Cells from the ScD-ura or YPD plate were scraped into sterile medium and the optical density (OD600) is measured. Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific). An Ambrl5 reaction vessel is inoculated with the cell slurry to reach an initial OD600 of 0.2. The fermentation medium consisted of 295g partially hydrolyzed com starch, 90g filtered sterilized (0.2 pm) light steep water, 79g sterile water, and 36g 500g / L sterile glucose. With continuous stirring, 12mL of fermentation media was added to each bioreactor. Strains were incubated at 30 °C, 450rpm of agitation. Air was supplied at 2.4 smlpm from the time of inoculation to 14 hours, then reduced to 0 smlpm for the remainder of the fermentation. Nitrogen gas w as supplied at 0.01 smlpm from 14 hours until the end of fermentation. 0, 0.10, or 0.15 g / kg of the enzyme cocktail sold under the tradename DISTILLASE® DXT by DUPONT™ was added after 14 hours. Samples of fermentation broth at 62 hours w ere analyzed by HPLC for the titer of ethanol, total glucose equivalents (TGE), and concentration of degree of polymerization 2, 3, and 4+ (DP2, DP3, DP4+) sugars.

[0140] Results of the dosing assay are reported in Tables 17 and 18. The results show that expression of the alpha-amylase and the pullulanase enzymes can replace at least a part, if not all, of the exogenously added enzymes in fermentation. For example, the ethanol titers of strains 5- 15b, 5-7e, 5-10c, and 5-20c, without any added exogenous enzy mes, were higher than the ethanol titer of the control strain 5-21 when 0.1 g / kg exogenous enzymes was added. Likewise, when exogenous enzymes are dosed into fermentations using the strains that express an alpha-amylase and a pullulanase the ethanol titers are even higher, often higher than the equivalent dosage in the control strain 5-21. This demonstrates that the expression on these alpha-amylase and pullulanasePT-1891-WO-PCT enzymes can reduce fermentation costs by reducing the amount of exogenous enzyme that needs to be added in the reaction.Table 17.Table 18.PT-1891-WO-PCTExample 9: Saccharomyces cerevisiae Strain EngineeringStrain 6-1

[0141] Strain 1-22 described by Miller et al. (PCT Patent Application Publication No. WO2017106739, published June 22, 2017, incorporated herein by reference in its entirety) is a Saccharomyces cerevisiae host strain that is ura3 negative (ura3-~) and amdS negative (amdS-) and in which one allele of the GPP1 gene is knocked out and replaced with an expression cassette for i) the C. gleam gapN of SEQ ID NO: 17 with a PGK1 promoter and an ADH terminator and ii) the Candida maltosa ADH of SEQ ID NO: 22 with a TDH3 promoter and a SciCYCl terminator. Herein, Strain 6-1 refers to Strain 1-22 of Miller et al. PCT Patent Application Publication No. WO2017106739.Strains 6-2

[0142] Strain 6-1 was transformed with SEQ ID NOs:83, 88, and 89. Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. The PCR verified isolate was designated strain 6-2.PT-1891-WO-PCTStrains 6-3a-f

[0143] Strain 6-2 was transformed with SEQ ID NO:89, 90, and 91. SEQ ID NO:90 contained a 5’ GPP1 flanking sequence (SEQ ID NO:3); a PGK1 promoter (SEQ ID NO:29); a gene encoding the C. gleum gapN enzyme of SEQ ID NO: 17; an ADH1 terminator (SEQ ID NO:32); a TDH3 promoter (SEQ ID NO:24); a gene encoding the C. maltosa ADH enzyme of SEQ ID NO:22; a CYC1 terminator (SEQ ID NO:30); and a 5’ portion of the Aspergillus nidulans amdS gene under the control of the TEF1 promoter (SEQ ID NO:24). SEQ ID NO:91 contained a 3’ portion of the Aspergillus nidulans amdS gene under the control of the ADH terminator (SEQ ID NO:32) and a sequence encoding the Aspergillus niger alpha-amylase of SEQ ID NO:35 under the control of the TEF1 promoter (SEQ ID NO:24) and TEF1 terminator (SEQ ID NO:33). Transformants were selected on YNB + acetamide plates then struck out for single colony isolation. PCR verified isolates were designated as 6 sister strains 6-3a-f.Strains 6-4a-c

[0144] Strain 6-3d was transformed with SEQ ID NO: 82. Transformants were selected on YPD + 200 mg / L G418 and grown for 2 days at 30°C. Transformants were selected, diluted 1 / 10, and plated on YPD+200 mg / L G418 to obtain single colony isolates. Isolates were screened for loss of the URA3 (ScD-ura plates) and amdS marker (on YNB / acetamide plates). Plates were grown overnight or for 2 days (YNB acetamide) at 30 °C. Strains that had lost the amdS marker were grown overnight in a YPD medium in deep-well plates with no selection to cure the G418 plasmid and plated for singles on YPD agar plates and grown overnight at 30 °C. PCR verified isolates were designated strains 6-4a, 6-4b, and 6-4c.Strains 6-5a-d

[0145] Strains 6-4a-c was transformed with SEQ ID NO:77. Transformants were selected on synthetic complete media lacking uracil (ScD-Ura) then struck out for single colony isolation. Transformation of stain 6-4a resulted in the PCR verified isolate 6-5a, transformation of strain 6- 4b resulted in the PCR verified isolates 6-5b and 6-5c, and transformation of strain 6-4c resulted in the PCR verified isolate 6-5d.Example 10: Ambrl5 Assays

[0146] Strains 6-3a-f, 6-0, 6-2, 5-0, 5-21, and 5-15a-b were run in an Ambrl5 to assay ethanol production and total glucose equivalents in the fermentation broth at the end of fermentation. ThePT-1891-WO-PCT strain 6-0 has the same background as strain 6-1 but has the ScURA3 gene restored via transformation with SEQ ID NO:77.

[0147] Strains were struck on a YPD plate and incubated at 30°C until single colonies were visible (2-3 days). Cells from the YPD plate were scraped into sterile medium and the optical density (OD600) is measured. Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific). An Ambrl5 reaction vessel is inoculated with the cell slurry to reach an initial OD600 of 0.2. The fermentation medium consisted of 295g partially hydrolyzed com starch, 90g filtered sterilized (0.2 pm) light steep water, 79g sterile water, and 36g 500g / L sterile glucose. With continuous stirring, 12mL of fermentation media was added to each bioreactor. Strains were incubated at 30 °C, 450rpm of agitation. Air was supplied at 2.4 smlpm from the time of inoculation to 14 hours, then reduced to 0 smlpm for the remainder of the fermentation. Nitrogen gas was supplied at 0.01 smlpm from 14 hours until the end of fermentation. 0.08 or 0.12 g / kg of the enzyme cocktail sold under the tradename DISTILLASE® DXT by DUPONT™ was added after 14 hours. Samples were taken at 63.5 hours and analyzed for ethanol, total glucose equivalents, and DP sugar concentrations by HPLC.

[0148] Results are outlined in Table 19 and show that when the strains expressing an alphaamylase and a pullulanase are used in the fermentation reaction, less exogenous enzyme can be added than in fermentation reactions with strains that do not express these enzymes. For example, when strain 6-0 is fermented with 0.12 g / kg exogenous enzyme the fermentation produces an average of 127.75 g / L ethanol. However, with only 0.08 g / kg exogenous enzy me addition, strains 6-3a, 6-3d, 6-3e, and 6-3f produced more ethanol than the 6-0strain with 0.12 g / kg enzyme.Table 19.PT-1891-WO-PCTExample 11: Ambrl5 Assays

[0149] Strains 6-5a-c, 6-0, and 6-3d were run in an Ambrl5 to assay ethanol production and total glucose equivalents in the fermentation broth at the end of fermentation.

[0150] Strains were struck on a ScD-Ura plate and incubated at 30°C until single colonies were visible (2-3 days). Cells from the ScD-Ura plate were scraped into sterile medium and the optical density (OD600) is measured. Optical density is measured at a wavelength of 600 nm with a 1 cm path length using a model Genesys 20 spectrophotometer (Thermo Scientific). An Ambrl5 reaction vessel is inoculated with the cell slurry to reach an initial OD600 of 0.2. The fermentation medium consisted of 295g partially hydrolyzed com starch, 90g filtered sterilized (0.2 pm) light steep water, 79g sterile water, and 36g 500g / L sterile glucose. With continuous stirring, 12mL of fermentation media was added to each bioreactor. Strains were incubated at 30 °C, 450rpm of agitation. Air was supplied at 2.4 smlpm from the time of inoculation to 14 hours, then reduced to 0 smlpm for the remainder of the fermentation. Nitrogen gas was supplied at 0.01 smlpm fromPT-1891-WO-PCT14 hours until the end of fermentation. 0.08 or 0.12 g / kg of the enzyme cocktail sold under the tradename DISTILLASE® DXT by DUPONT™ was added after 14 hours. Samples were taken at 63.5 hours and analyzed for ethanol, total glucose equivalents, and DP sugar concentrations by HPLC.[01511 Results are outlined in Table 20 and show that when the strains expressing an alphaamylase and a pullulanase are used in the fermentation reaction, less exogenous enzyme can be added than in fermentation reactions with strains that do not express these enzymes. For example, when strain 6-0 is fermented with 0.12 g / kg exogenous enzyme the fermentation produces an average of 128.07 g / L ethanol. However, using only 0.08 g / kg exogenous enzyme, strains 6-3d, 6-5a, 6-5b, and 6-5c produce more ethanol. This shows that expression of these alpha-amylase and pullulanase enzy mes can replace at least a portion or, if not all, exogenous enzyme adding in a fermentation reaction to produce ethanol.Table 20.

Claims

PT-1891-WO-PCTCLAIMSWhat is claimed is:

1. A genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding an alpha-amylase enzyme 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:35-39, preferably at least one of SEQ ID NOs:35, 36, 38, and 39, most preferably SEQ ID NO: 35; and an exogenous polynucleotide encoding a pullulanase enzyme 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:58-60, preferably at least one of SEQ ID NOs:59 and 60, most preferably SEQ ID NO:58.

2. The engineered yeast cell of claim 1, wherein the engineered cell additionally comprises a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme 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: 13-17.

3. The engineered yeast cell of claim 2, wherein the gapN enzyme is 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: 13, 14, 16, and 17; preferably wherein the gapN enzyme is at least 85% identical to SEQ ID NO: 13 the gapN enzyme is at least 85% identical to SEQ ID NO: 14; the gapN enzyme is at least 85% identical to SEQ ID NO: 16; and / or the gapN enzyme is at least 85% identical to SEQ ID NO: 17.

4. The engineered yeast cell of any preceding claim, wherein the engineered yeast cell additionally comprises an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme 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:22-23.PT-1891-WO-PCT5. The engineered yeast cell of any preceding claim, wherein the engineered yeast cell comprises a deletion or disruption of a native glycerol-3-phosphate phosphatase (GPP) gene.

6. The engineered yeast cell of any preceding claim, wherein the engineered yeast cell comprises a deletion or disruption of a native glycerol-3-phosphate dehydrogenase (GDP) gene.

7. The engineered yeast cell of any preceding claim, wherein the engineered yeast cell additionally comprises an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme.

8. A genetically engineered yeast cell capable of producing ethanol, the engineered yeast cell comprising an exogenous polynucleotide encoding an alpha-amylase enzyme 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:35-39, preferably at least one of SEQ ID NOs:35, 36, 38, and 39, most preferably SEQ ID NO:35; an exogenous polynucleotide encoding a pullulanase enzyme 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:58-60, preferably at least one of SEQ ID NOs:59 and 60, most preferably SEQ ID NO:58; an exogenous polynucleotide encoding a glyceraldehyde-3-phosphate dehydrogenase (gapN) enzyme 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: 13-21; an exogenous polynucleotide sequence encoding an alcohol dehydrogenase (ADH) enzyme 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: 22-23; an exogenous polynucleotide sequence encoding a glucoamylase (GA) enzyme; and a deletion or disruption of at least 1 allele of a native GPP gene.

9. The engineered yeast cell of claim 7 or 8, wherein the encoded GA enzy me is 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:9-12.PT-1891-WO-PCT10. The engineered yeast cell of any preceding claim, wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous or artificial promoter 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-adenosyl methionine transferase 2 (SAM2) promoter; an alcohol dehydrogenase 1 (ADH1) promoter, and a 3-phosphogly cerate kinase (PGK1) promoter; and / or wherein one or more of the exogenous polynucleotide sequences is operably linked to a heterologous or artificial terminator selected from the group consisting of an iso-l-cytophrome c (CYC1) terminator, a URA3 terminator, a PDC terminator, an ADH1 terminator, a TEF1 terminator, or a GAL 10 terminator.

11. The engineered yeast cell of any preceding claim, wherein the yeast cell is selected from the group consisting of Saccharomyces spp., Schizosaccharomyces spp., Pichict spp.. Paffla spp., Kluyveromyces spp., Candida spp., Talaromyces spp., Brettanomyces spp., Pachysolen spp., Debaryomyces spp., and Yarrowia spp..

12. The engineered yeast cell of any preceding claim, wherein the yeast cell is a Saccharomyces cerevisiae cell.

13. The engineered yeast cell of any preceding claim, wherein the engineered yeast cell is capable of producing ethanol at a titer of at least 60. at least 80, at least 100, or at least 120 g / L ethanol after 48 hours and wherein ethanol production by the engineered yeast cell is increased relative to ethanol production in an equivalent yeast cell lacking the alpha-amylase and pullulanase enzy mes.

14. A method for producing ethanol, the method comprising: contacting a substrate with the engineered yeast cell of any preceding claim, where the engineered yeast cell produces at least 60, at least 80, at least 100, or at least 120 g / L ethanol after 48 hours and wherein ethanol production by the engineered yeast cell is increased relative to ethanol production in an equivalent yeast cell lacking the alpha-amylase and pullulanase enzymes.PT-1891-WO-PCT15. The method of claim 14, wherein the substrate comprises starch, glucose, sucrose, cellulosic biomass, or combinations thereof.

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