Genetically modified organisms for the expression and secretion of phosphorylase enzymes

Abstract

Described herein are recombinant proteins comprising a sequence at least 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:1, 2, or 3; and a secretion signal sequence heterologous thereto. The disclosure also provides genetically engineered microorganisms and fermentation methods for the production of said recombinant proteins.

Description

PT-2176-WO-PCTGENETICALLY MODIFIED ORGANISMS FOR THE EXPRESSION AND SECRETION OF PHOSPHORYLASE ENZYMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 703.263, filed October 4, 2024 and U.S. Provisional Application No. 63 / 724,939, filed November 26, 2024, each of 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-2176-WO-PCT.xml” which is 362,741 bytes in size created on October 3, 2025 and electronically submitted vis Patent Center herewith the application is incorporated by reference in its entirety .BACKGROUND

[0003] Oligo- and polysaccharides are widely used in the food industry. Hydrolyzed plantbased starches are a source of glucose containing oligo- and polysaccharides. Hydrolyzed starches include maltodextrins and glucose syrups. Maltodextrins, sucrose, and glucose syrups may be used as a texturizer or as a full calorie ingredient providing bulking functionality. Maltodextrins, sucrose, and glucose syrups may also be used as a coating and for encapsulation. Maltodextrins, sucrose, and glucose syrups are easily digested and are fully and rapidly absorbed by the gastrointestinal tract. The fast digestibility of the maltodextrins, sucrose, and glucose results in a strong rise in blood glucose levels after consumption. Maltodextrins, sucrose, and glucose syrups are therefore products with a high glycemic index. Further, maltodextrins, sucrose and glucose syrups are full calorie carbohydrates.

[0004] There is an ongoing need in the food industry7for bulking ingredients and carbohydrates that have a lower or slower digestibility and that are lower in calories. There is also an ongoing need in the food industry for bulking ingredients and carbohydrates that decrease the glycemic impact of the oligo- or polysaccharides used for this purpose. One suggestion has been to replace the existing polysaccharides and carbohydrates with naturally occurring alternatives with a lower calorie content. Another alternative is to modify existing oligo- and polysaccharides.

[0005] Processes for modifying oligo- or polysaccharides such as maltodextrins or glucose syrups may be earned out by means of an enzymatic process, however a need exists for efficientPT-2176-WO-PCT and effective methods for producing said enzymes to reliably produce the low or slow digestible carbohydrates.SUMMARY

[0006] The present disclosure provides a recombinant protein comprising a 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 SEQ ID NO: 1; and a secretion signal sequence heterologous thereto. The present disclosure also provides a recombinant protein comprising a 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:2 and 3; and a secretion signal sequence heterologous thereto. The secretion signal sequence may be 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, 10, 11, 28, 29, and 49, preferably SEQ ID NOs:28, 29, and 49, most preferably SEQ ID NO:29. The recombinant protein 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: 12-17, 42-45, 50-51, and 70-75, preferably SEQ ID NOs:42-45, 50-51, and 73-75, most preferably SEQ ID NOs:43 and 45.

[0007] The disclosure also provides a genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding the recombinant proteins described herein. The genetically engineered microorganism cell may be a bacterial cell, for example an Escherichia coli cell, or a yeast cell, for example a Komagataella phaffii cell or a Kluyveromyces marxianus cell. The heterologous polynucleotide sequence may be operably linked to a heterologous promoter and / or a heterologous terminator. The heterologous promoter may be selected from the group consisting of the IPTG inducible promoter of pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3- phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFp); phosphoglucomutase 1 promoter (PGM Ip); 3-phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter. The heterologous terminator may be selected form the group consisting of GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminatorPT-2176-WO-PCT(PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); TEF1; iso- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator. The promoter may be an inducible promoter, for example the IPTG inducible promoter of SEQ ID NO: 8, the A0X1 promoter of SEQ ID NO: 26, and the inducible synthetic promoter of SEQ ID NO:48.

[0008] The disclosure further provides methods for producing the recombinant proteins described herein comprising contacting a substrate comprising dextrose and / or glycerol with a genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding a recombinant protein described herein for a time and under conditions sufficient to express the recombinant protein. When the microorganism cell is aK. phaffii or K. marxianus cell and the substrate is contacted with the microorganism cell at a temperature between 20 °C to 40 °C, 25 °C to 35 °C, or 28 °C to 32 °C, preferably about 30 °C. The genetically engineered microorganism cell may produce at least 50 pg / mL. at least 75 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 115 pg / mL, at least 125 pg / mL, at least 130 pg / mL, at least 150 pg / mL, or at least 175 pg / mL.

[0009] The disclosure also provides a genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding a maltose phosphorylase enzyme at least 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 NOT; and / or a heterologous polynucleotide sequence encoding a kojibiose phosphorylase enzy me at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 100% identical to at least one of SEQ ID NO:2 or 3. The genetically engineered microorganism cell may be used in a method for producing a recombinant protein by contacting a substrate comprising dextrose and / or glycerol with the genetically engineered microorganism cell for a time and under conditions sufficient to express the recombinant protein.BRIEF DESCRIPTION OF THE FIGURES

[0010] This patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and the payment of the necessary fee.

[0011] The drawings illustrate generally, by way of example, but not by way of limitation, various aspects discussed herein.PT-2176-WO-PCT

[0012] FIG. 1 shows a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 2-3, 1-1, l-2a-e, l-3a-e, l-4a-e, and l-5a-e as outlined in Example 1.

[0013] FIG. 2 shows a photograph of an SDS-PAGE gel with samples from the soluble lysate fractions from strains 2-3, 1-1, l-2a-e, l-3a-e, l-4a-e, and l-5a-e as outlined in Example 1.

[0014] FIG. 3 shows a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 2-2, 1 -1 , l -6a-e, l-7a-e, l -8a-e, and l -9a-e as outlined in Example 1.

[0015] FIG. 4 shows a photograph of an SDS-PAGE gel with samples from the soluble lysate fractions from strains 2-2, 1-1, l-6a-e, l-7a-e, l-8a-e, and l-9a-e as outlined in Example 1.

[0016] FIG. 5 shows photographs of SDS-PAGE gels with samples from the fermentation broth of strains 3-0, 3-2a-f, 3-6a-f, 3-5a-f, and 3-la-f as outlined in Example 2.

[0017] FIG. 6 shows a photograph of an SDS-PAGE gel with samples from cell pellet resuspension of strains 3-3a-f and 3-4a-f as outlined in Example 2.

[0018] FIG. 7 shows photographs of SDS-PAGE gels with samples from the fermentation broth of strains 3-8a-f. 3-12a-f, 3-7a-f. and 3-l la-f as outlined in Example 2.

[0019] FIG. 8 shows photographs of SDS-PAGE gels with samples from cell pellet resuspensions of strains 3-9a-f and 3-10a-f as outlined in Example 2.

[0020] FIG. 9 shows a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 4-1, 4-2a-b. 4-3a-b. 4-6a-b, 4-7a-b and the GFP control and samples from cell pellet resuspensions of strains 4-1, 4-4a-, 4-5a-b, 4-8a-b, 4-9a-b and the GFP control as outlined in Example 3.

[0021] FIG. 10 shows a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 4-1, 4-10a-b, 4-l la-b. 4-14a-b, 4-15a-b, and the GFP control and samples from cell pellet resuspensions of strains 4-1, 4-12a-b, 4-13a-b, 4-16a-b, 4-17a-b, and the GFP control as outlined in Example 3.

[0022] FIG. 11 show s a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 4-1. 4-18a-b, 4-19a-b and the GFP control from fermentations in both YPD medium and DMul medium as outlined in Example 3.

[0023] FIG. 12 shows a photograph of an SDS-PAGE gel with samples form the fermentation broth of strains 4-1, 4-2a-b, 4-3a-b, 4-6a-b, 4-7a-b and the GFP control and samples from cell pellet resuspensions of strains 4-1, 4-4a-, 4-5a-b, 4-8a-b, 4-9a-b and the GFP control as outlined in Example 3.

[0024] FIG. 13 shows a photograph of an SDS-PAGE gel with samples from the fermentation broth of strains 4-1, 4-10a-b, 4-l la-b, 4-14a-b, 4-15a-b, and the GFP control and samples fromPT-2176-WO-PCT cell pellet resuspensions of strains 4-1, 4-12a-b, 4-13a-b, 4-16a-b, 4-17a-b, and the GFP control as outlined in Example 3.DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Values 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%” or “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.

[0028] The beta symbol “|3” is commonly used in the art in place of the word beta and they have the same meaning herein. The alpha symbol “a” is commonly used in the art in place of the word alpha and they have the same meaning herein. The delta symbol “A” is commonly used in the art in place of the word delta and they have the same meaning herein.

[0029] 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.PT-2176-WO-PCT

[0030] This disclosure relates to recombinant protein constructs and genetically engineered microorganisms for the production and secretion of maltose phosphorylase and kojibiose phosphorylase enzymes. The recombinant protein constructs may include a secretion signal for secretion of the enzy me from the cell. The polynucleotides encoding the recombinant protein constructs may be under the control of suitable promoters and terminators for expression of the enzymes. The polynucleotides encoding the recombinant protein constructs may be included in a microorganism cell, such that when the cell is contacted under suitable conditions with a substrate comprising dextrose and / or glycerol, the cell expresses the recombinant protein construct.

[0031] 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 enzy me” refer to a protein that catalyzes a chemical reaction. The recitation of any particular enzyme, 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-2176-WO-PCT

[0032] Variants or sequences having substantial identity or homology with the polypeptides described herein can be utilized in the practice of the disclosed 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 ability to exhibit the desired activity. Generally, the variant or modified sequence may include greater than about 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.

[0033] As used herein, the phrases “% sequence identity,’’ “% identity,” and “percent identity,” are used interchangeably and refer to the percentage of residue matches betw een at least tw o 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 identity include global alignments and local alignments w hich 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 default parameters: Max target sequences: 100; Short queries: Automatically adjust parameters for short input sequences; Expect threshold: 10; Word size: 6; Max matches in a query 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: LowPT-2176-WO-PCT 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.

[0034] 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.

[0035] 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.

[0036] 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 the 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.

[0037] As used herein, terms “polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,” and “nucleic acid,” are used interchangeably and refer to a sequence of nucleotides orPT-2176-WO-PCT 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.

[0038] 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.

[0039] 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 naturally occurring 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.

[0040] The polypeptides described herein may be encoded in an expression 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.

[0041] The genetically modified cells described herein include a heterologous nucleic acid sequence encoding i) a heterologous maltose phosphory lase enzyme; ii) a heterologous kojibiosePT-2176-WO-PCT phosphorylase enzyme; or hi) combinations thereof. The maltose phosphorylase enzyme may be any suitable enzy me with maltose phosphorylase activity. The exogenous polynucleotide may be an exogenous maltose phosphory lase gene. The kojibiose phosphorylase enzy me may be any suitable enzyme with kojibiose phosphorylase activity. The exogenous polynucleotide may be an exogenous kojibiose phosphorylase gene.

[0042] As used herein "maltose phosphorylase gene” refers to any gene or polynucleotide that encodes a polypeptide with maltose phosphorylase activity'. As used herein, “maltose phosphorylase enzyme” refers to a polypeptide with maltose phosphorylase activity. As used herein, “maltose phosphorylase activity” refers to the ability to catalyze the reaction of maltose and inorganic phosphate to glucose and beta-D-glucose- 1 -phosphate (0-D-glucose-l -phosphate). The maltose phosphorylase enzy me sequence may be from any suitable source or may be synthetic. Suitable maltose phosphory lase enzymes may be included under the Enzyme Classification EC 2.4. 1.8. The maltose phosphorylase enzyme may be the maltose phosphorylase enzyme from Lactobacillus acidophilus (LaMP). The maltose phosphorylase enzyme may have a 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 SEQ ID NO: 1. The heterologous nucleic acid sequence may encode a maltose phosphorylase 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 SEQ ID NO: 1.

[0043] As used herein “kojibiose phosphorylase gene” refers to any gene or polynucleotide that encodes a polypeptide with kojibiose phosphorylase activity7. As used herein, “kojibiose phosphorylase enzy me” refers to a polypeptide with kojibiose phosphorylase activity7. As used herein, “kojibiose phosphorylase activity” refers to the ability7to catalyze the reversible phosphorolysis of kojibiose (a-D-glucopyranosyl-(1^2)-D-glucopyranose) to P-D-glucose-1- phosphate (0-G1P or beta-GlP) and D-glucose (Glc), with an inversion of the anomeric configuration. The kojibiose phosphorylase enzy me sequence may be from any suitable source or may be synthetic. Suitable kojibiose phosphorylase enzymes may include under the Enzyme Classification EC 2.4.1.230 and classified into glycoside hydrolase family 65 (GH65). The kojibiose phosphorylase enzyme may' be the kojibiose phosphorylase enzyme from Caldicellulosiruptor saccharolyticus (CsKP) or Thermococcus barophilus (TbarKP). The kojibiose phosphorylase enzy me may have a 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:2 and 3. The heterologous nucleic acid sequence may encode a kojibiose phosphorylasePT-2176-WO-PCT 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:2 and 3.

[0044] The maltose phosphorylase and kojibiose phosphory lase enzy mes described herein may be expressed with an added secretion signal or polypeptide tag. The polypeptide tag may be a tag suitable for separation and purification of the expressed enzyme, for example, a poly -histidine tag such as SEQ ID NO:4; a solubility tag, for example, the glutathione S-transferase (GST), the small ubiquitin-like modifier (SUMO), or the maltose-binding protein (MBP) solubility tags; a protease cleavage site; combinations thereof; and the like. For example, the enzymes described herein may include a poly -histidine tag and have a 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:5- 7. The leader sequence may be a secretion signal sequence for secretion of the enzyme from the cell using the cell’s native secretion mechanisms. Suitable secretion signals for use with the enzymes described herein include the E. coli outer membrane protein secretion signal (OmpA, SEQ ID NO:9); the secretion signal from E. coli pectate lyase B (pelB, SEQ ID NOTO); the secretion signal sequence from E. coli maltose binding protein encoded by the malE gene (malE, SEQ ID NO: 11); the a-mating factor (Mfa) secretion signal sequence from Saccharomyces cerevisiae (SEQ ID NO:28); ahybrid sequence of the oligosaccharyl transferase complex 1 (Ostl) signal sequence from S. cerevisiae and the Mfa secretion signal sequence from <S’. cerevisiae (SEQ ID NO: 29); inulinase 1 secretion sequence from l. marxianus (INU1; SEQ ID NO:49); and the secretion signal from the UTH1 protein of Komagataella phaffii (SEQ ID NO:79). A given polypeptide sequence may include more than one leader sequence. For example, a polypeptide sequence may include a polypeptide tag and a secretion signal attached to the enzyme of interest such that the enzyme is secreted from the cell and tagged for separation and purification.

[0045] The maltose phosphorylase enzymes described herein may include the OmpA secretion signal and have a 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 SEQ ID NO: 12. The maltose phosphorylase enzymes described herein may include the pelB secretion signal and have a 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 SEQ ID NO: 13. The maltose phosphory lase enzy mes described herein may include the malE secretion signal and have a 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 SEQ ID NO: 14. The maltose phosphorylase enzymes described herein may include the Mfa secretion signal and have a 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 SEQPT-2176-WO-PCTID NO:42. The maltose phosphorylase enzymes described herein may include the Ostl / Mfa secretion signal and have a 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 SEQ ID NO:43. The maltose phosphorylase enzymes described herein may include the INU1 secretion signal and have a 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 SEQ ID NO:50.

[0046] The kojibiose phosphorylase enz mes described herein may include the OmpA section signal and have a 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: 15 and 70. The kojibiose phosphorylase enzymes described herein may include the pelB section signal and have a 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: 16 and 71. The kojibiose phosphory lase enzymes described herein may include the malE section signal and have a 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: 17 and 72. The kojibiose phosphorylase enzymes described herein may include the Mfa section signal and have a 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:44 and 73. The kojibiose phosphorylase enzymes described herein may include the Ostl / Mfa section signal and have a 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:45 and 74. The kojibiose phosphorylase enzymes described herein may include the INU1 section signal and have a 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:51 and 75.

[0047] As used herein, “recombinant protein” refers to a polypeptide sequence that contains one or more heterologous elements / components, for example a secretion signal sequence, a protein of interest, an enzyme of interest, and a polypeptide tag encoded by a polynucleotide construct engineered into the cell. For example, a recombinant protein as described herein may include a secretion signal and the LaMP, CsKP, or TbarKP enzyme. In another example, a recombinant protein as described herein may include a secretion signal, the LaMP, CsKP, or TbarKP enzyme, and a polypeptide tag.

[0048] Any of the enzymes and / or recombinant proteins described herein may be encoded by a polynucleotide construct. Any of the exogenous polynucleotide sequences described herein, including those polynucleotide constructs encoding an enzyme and / or recombinant proteinPT-2176-WO-PCT described herein, may be under the control of a promoter. The polynucleotide sequence may be operably linked to a heterologous or artificial promoter. Suitable promoters are known and described in the art. Suitable promoters include, but are not limited to pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADEIlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKl p); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3 -phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFp); phosphoglucomutase 1 promoter (PGMlp): 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENO Ip); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter. For example, promoters may include the IPTG inducible promoter of SEQ ID NO: 8, the A0X1 promoter of SEQ ID NO:26, the PDC1 promoter of SEQ ID NO:46, the inducible synthetic promoter of SEQ ID NO:48. and the pGAP promoter of SEQ ID NO:27. The promoter may be an inducible promoter or a constitutive promoter.

[0049] Any of the exogenous polynucleotide sequences described herein, including those polynucleotide constructs encoding an enzyme and / or recombinant protein described herein, may be under the control of a terminator. The polynucleotide sequence may be operably linked to a heterologous or artificial terminator. Suitable terminators are known and described in the art. Suitable terminators include, but are not limited to, GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); TEF1; iso- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator. For example, the terminator may include the ENO1 terminator of SEQ ID NO:47 or the AOX1 terminator of SEQ ID NO: 80.

[0050] Cells including any of the polynucleotides, constructs, or vectors described herein are also provided. The cell may be a prokaryotic cell or a eukaryotic cell. Suitable prokaryotic cells include bacteria cells, for example, Escherichia coli. Suitable eukary otic cells include yeast and fungal cells, for example. Komagataella phaffli Kluyveromyces marxianus. Aspergillus niger. Trichoderma reesei. and Bacillus subtilis. Komagataella phaffli is also referred to in the art at Pichia pastoris.PT-2176-WO-PCT

[0051] Also provided herein are methods for production of a recombinant protein described herein by fermentation of a genetically engineered microorganism cell described herein. The fermentation methods include the step of fermenting a substrate using the genetically engineered microorganisms described herein to product the recombinant protein. 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 recombinant protein from the fermentation broth and / or the genetically engineered microorganism.

[0052] The fermentation method can be run using a suitable fermentation substrate. The substrate of the fermentation method can include glucose, sucrose, molasses, fructose, lactose, glycerol, methanol, hydrolysates of starch, lignocellulosic hydrolysates, or a combination thereof. One skilled in the art will recognize what fermentation substrate is suitable for a given fermentation organism and system.

[0053] The initial dextrose concentration of the fermentation may be at least 10, 25, 50, 75 100, 200, 250, 300, 350, or at least 400 g / L dextrose. The initial dextrose concentration may be between 10 to 500, 100 to 400, 150 to 350, or 250 to 325 g / L.

[0054] 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, when the fermentation is carried out in a yeast such as K. phaffii or K. marxianus, the fermentation temperature can be maintained in the range of 20 °C to 40 °C, 25 °C to 35 °C, or 28 °C to 32 °C, preferably about 30 °C. In another example, when the fermentation is carried out in a bacterium such as E. coli, the fermentation temperature can be maintained in the range of 25 °C to 45 °C, 30 °C to 42 °C, or 35 °C to 40 °C, preferably about 37 °C. However, a skilled artisan will recognize that the fermentation temperature is not limited to any specific range or temperature recited herein and may be modified as appropriate.

[0055] The fermentation process may be run with agitation. The fermentation may be carried out with various levels of agitation in order to supply the correct amount of oxygen to the fermentation microorganisms. One skilled in the art will recognize the various levels of oxygen needed in the fermentation process and how to change the agitation to accommodate said level of oxygen. For example, the fermentation process may include agitation between 50-500 rotations per minute (rpm), 100-400 rpm, 150-350 rpm, or 200-300 rpm, preferably about 250 rpm. One ofPT-2176-WO-PCT skill in the art will recognize that other methods may be used to achieve the desired oxygen level in the fermentation, for example mixing, aeration, and the like.

[0056] 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 microorganism and / or genetic modification of the microorganism used in the fermentation process. These characteristics can be affected by adjusting the fermentation process conditions. These characteristics can be adjusted via a combination of microorganism selection or modification and the selection of fermentation process conditions.

[0057] The fermentation process can be run as a fed batch, for example when one or more substrates are fed to the reaction at various time points. Further, the fermentation process can be a batch process, continuous process, or semi-continuous process, as would be understood by a person skilled in the art.

[0058] The fermentation process can produce the recombinant protein at a final concentration of at least 50 pg / mL, at least 75 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 115 pg / mL, at least 125 pg / mL, at least 130 pg / mL, at least 150 pg / mL, or at least 175 pg / mL.EXAMPLES

[0059] 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.

[0060] Throughout the application and Example, strain numbering and sequence identification numbers are used consistently.Example 1 - Escherichia coli strains, plasmids, and expression

[0061] Escherichia coli strain B834(DE3), referred to herein as strain 1-1, was used to express the kojibiose phosphorylase enzyme from Caldicellulosiruptor saccharolyticus (SEQ ID NO:2; CsKP) and the maltose phosphorylase enzyme from Lactobacillus acidophilus (SEQ ID NO: 1; LaMP). The CsKP and LaMP enzymes were previously expressed in E. coli strain BL21(DE3), however this strain requires antibiotic resistance markers for strain engineering. E. coli strainPT-2176-WO-PCTB834(DE3) is a methionine auxotroph and can be genetically engineered without the use of antibiotic markers.

[0062] For this Example, BL21(DE3) strains expressing the CsKP and LaMP enzymes were used as a positive control. The gene encoding the C. saccharolyticus kojibiose phosphorylase was codon optimized for E. coll and synthesized. The gene encoding the L. acidophilus maltose phosphorylase was codon optimized for E. coll and synthesized. Codon optimized sequences were subsequently subcloned into a pET21 vector at the Nhel and Xhol restriction sites, introducing a C-terminal His6-tag. The plasmid was transformed in E. coll BL21(DE3) electrocompetent cells (strain 2-1), thereby creating strain 2-2, including the pET21 vector encoding the CsKP of SEQ ID NO:6, and strain 2-3, including the pET21 vector encoding the LaMP of SEQ ID NO:5.

[0063] Table 2 describes the E. coll B834(DE3) strains used in this Example. The indicated parent strain was transformed with the indicated transformation fragment sequence, positive transformants were selected on Neidhardt’s minimal media without methionine plates (Table 3, 15 g / L agar) and individual isolated colonies designated the strain number indicated in Table 2. In some instances, more than one isolate, e g., “sister” isolates, are indicated by letters following the strain number. For example, strain 1-2 has 5 sister isolates, strains l-2a, l-2b, l-2c, l-2d, and l-2e.

[0064] Each transformation fragment included a polynucleotide encoding a methionine synthase enzyme, metE, for selection of positive transformants of the methionine auxotroph B834(DE3) parent strain 1-1. The plasmids used for the E. coll transformation included a T7 isopropyl [3-d- 1 -thiogalactopyranoside (IPTG) inducible promoter (SEQ ID NO:8). For some strains, the plasmids also included a polynucleotide sequence encoding a secretion signal that, when expressed, resulted in the secretion signal linked to the enzyme of interest. The secretion signals used in this example include the E. coll outer membrane protein secretion signal (OmpA, SEQ ID NO:9); the secretion signal from the E. coll pectate lyase B (pelB, SEQ ID NOTO); and the signal sequence from E. coll maltose binding protein encoded by the malE gene (malE, SEQ ID NO: 11). The polypeptide sequence including the LaMP enzyme and the secretion signals OmpA, pelB, and malE are SEQ ID NOs: 12, 13, and 14, respectively. The polypeptide sequence including the CsKP enzy me and the secretion signals OmpA, pelB, and malE are SEQ ID NOs: 15, 16, and 17, respectively.PT-2176-WO-PCTTable 2.Table 3: Neidhardt’s MediaPT-2176-WO-PCT

[0065] Strains 1-1 through 1-9. 2-2, and 2-3 were assayed for expression of the LaMP or CsKP enzyme containing recombinant proteins. A colony from each strain was selected and suspended in 50 pL of LB (5 g / L yeast extract, 10 g / L peptone, and 10 g / L NaCl) and 5-10 pL of each cell suspension was used to inoculate 2 mL of auto induction media from the autoinduction expression system sold under the tradename OVERNIGHT EXPRESS™ Autoinduction System 1 by Novagen. Cultures were incubated at 37 °C with agitation at 250 rpm for 20-24 hours. Following incubation, the fermentation broth was separated from the cell biomass. The resulting cell biomass was lysed, and the soluble fraction separated from the remaining insoluble material. Samples of the fermentation broth and soluble lysis fraction for each strain was analyzed by sodium dodecylsulfate polyacrylamide gel electrophoreses (SDS-PAGE) using Coomassie staining. Total protein content of each fermentation broth and soluble fraction sample was analyzed using the BradfordPT-2176-WO-PCT assay. Gel images are shown in FIG. 1 (LaMP fermentation broth), FIG. 2 (LaMP soluble lysis fraction), FIG. 3 (CsKP fermentation broth), and FIG. 4 (CsKP soluble lysis fraction) and the protein quantification is reported in Table 3 (LaMP) and Table 4 (CsKP).

[0066] The results demonstrate that strains containing the LaMP and CsKP expression constructs lacking the leader secretion signal had the highest expression levels, with enzyme highly expressed both intracellularly (as shown by the protein levels in the soluble lysis fraction shown in FIGs. 2 and 4 and Tables 4 and 5) and some extracellular excreted protein (as shown by the protein levels in the fermentation broth samples in FIGs. 1 and 3 and Tables 4 and 5). While the strains containing the LaMP and CsKP expression constructs with an OmpA, pelB, or malE secretion signal did express the enzymes of interest at lower total recombinant protein levels, most of the expressed recombinant protein was retained with in the cell (as shown by the protein levels in the whole cell lysate soluble fraction shown in FIGs. 2 and 4 and Tables 4 and 5) rather than being secreted by the cell (as shown by the protein levels in the fermentation broth samples in FIGs. 1 and 3 and Tables 4 and 5).Table 4.PT-2176-WO-PCTTable 5.PT-2176-WO-PCT

[0067] Enzyme activity was assayed for LaMP and CsKP enzy mes taken from the soluble lysis fraction of strains l-5a-e and l-9a-e, respectively. The reaction mixtures are outlined in Table 6 and reactions were incubated at 55 °C for 14 minutes beginning when the enzyme was added as the final component of the mixture. Samples were removed every 2 minutes during the reaction and immediately inactivated using 0.2 M citric-phosphate buffer at pH 2.6. The glucose concentration of each collected sample, along with the concentration of glucose in the starting reaction mixture prior to the addition of enzyme, was measured using the glucose assay kit sold under the tradename Megazyme HK Glucose Kit by Neogen. The assay kit glucose concentration via a coupled hexokinase and glucose-6-phosphate dehydrogenase reaction converting the glucose to NADPH and gluconate-6-phosphate. The concentration of NADPH can be monitored by the UV absorbance at 340 nm and converted to the initial glucose concentration of the assay reaction after running a glucose standard curve. Measured enzyme activity is reported in Table 7 as both the reaction units (U) per mL and U / mg by converting using the initial enzyme concentration.

[0068] These results show that all lysate soluble fraction samples showed activity indicating that functional enzy mes were produced in all cases.Table 6.Table 7.PT-2176-WO-PCTExample 2 - Komagataella phaffii strains, plasmids, and expression

[0069] Expression of the LaMP and CsKP enzymes was tested in Komagataella phaffii. The Pichia pastoris strain 9011, which is an alcohol oxidase 1 knockout mutant (aoxlA) of wild-ty pe Pichia pastoris and is available from ATUM, was designated strain 3-0. Komagataella phaffii is the updated taxonomic designation for Pichia pastoris and they are both used interchangeably in the art.

[0070] Strain 3-0 was transformed with the transformation fragments as outlined in Table 8. Positive transformants were selected on YPD + zeocin plates and individual isolated and PCR verified colonies were designated with the strain number in Table 7. In some instances, more than one isolate, e.g., “sister” isolates, are indicated by letters following the strain number. For example, strain 3-1 has 6 sister isolates, strains 3-la, 3-lb, 3-lc, 3-ld, 3-le, and 3-lf.

[0071] Each transformation fragment included an expression construct for the recombinant protein of interest under the control of a methanol inducible promoter (P. pastoris alcohol oxidase 1 promoter (pAOXl); SEQ ID NO:26) or a constitutive promoter (pGAP; SEQ ID NO:27). Each recombinant protein construct contained (i) an optional secretion signal; (ii) a polynucleotide sequence encoding the LaMP enzyme or the CsKP enzyme; and (iii) an optional 6x-histidine tag (SEQ ID NO: 4) for purification. The secretion signals used in this example include a-mating factor (Mfa) sequence from Saccharomyces cerevisiae (SEQ ID NO:28) and a hybrid sequence of the oligosaccharyl transferase complex 1 (Ostl) signal sequence from S. cerevisiae and the Mfa sequence from S. cerevisiae (SEQ ID NO: 29).Table 8.PT-2176-WO-PCT

[0072] Strains 3-1 through 3-12 were assayed for expression of the LaMP or CsKP enzyme containing recombinant proteins.

[0073] Starter cultures of strains 3-1, 3-2, 3-3. 3-4, 3-7, 3-8, 3-9, and 3-10 were prepared by inoculating 25 mL of media (lOg / L yeast extract, 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0.1M phosphate buffer (pH 6), 0.4mg Biotin, 20g / L glycerol) with an isolated colony from the selected strain and incubating overnight (16-20 hours) at 30 °C. Cultures were standardized to 50 mL at an optical density at 600 nm (OD600) of 1.0, then spun down and resuspended in 10 mL induction medium (lOg / L yeast extract. 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0.1M phosphate buffer (pH 6), 0.4mg Biotin, 20g / L methanol) and incubated at 29 °C for 48 hours. An additional 0.5 wt% methanol was added between 20-24 hours and 26-30 hours to further induce expression.

[0074] Starter cultures of strains 3-5, 3-6, 3-11, and 3-12 were prepared by inoculating 25 mL of media (lOg / L yeast extract, 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0. IM phosphate buffer (pH 6), 0.4mg Biotin, 20g / L dextrose) with an isolated colony from the selected strain and incubating overnight (16-20 hours) at 30 °C. Cultures were standardized to 50 mL at an optical density at 600 nm (OD600) of 1.0, then concentrated to 10 mL. Strains 3-5, 3-6, 3-11, and 3-12 were resuspended in 10 mL of the starter culture media (lOg / L yeast extract, 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0. IM phosphate buffer (pH 6), 0.4mg Biotin, 20g / L dextrose) and incubated at 29 °C for 48 hours.

[0075] Following incubation, samples were collected for analysis. For strains containing a construct encoding a recombinant protein with a secretion signal (strains 3-1, 3-2. 3-5, 3-6, 3-7, 3-8, 3-11, and 3-12), the fermentation broth was separated from the cell biomass and a fermentation broth sample was analyzed by SDS-PAGE using Coomassie straining. For strainsPT-2176-WO-PCT containing a construct encoding a recombinant protein without a secretion signal (strains 3-3, 3- 4, 3-9, and 3-10) 500 pL of the culture was taken and the fermentation broth separated from the cell pellet, with a 500 pL phosphate-buffered saline (PBS) resuspension of the cell pellet used for analysis by reducing 30 pL of the whole cell lysate with 10 pL sample buffer, boiling, and run on SDS-PAGE gel. Total protein content in the fermentation broth from strains 3-1. 3-2, 3-5, 3-6. 3- 7, 3-8, 3-11 , and 3-12 was analyzed by Bradford assay. To correct for differences in cell biomass and growth rate, the total protein concentrations were corrected for the OD600 absorbance before separate of the fermentation broth. Gel images are shown in FIG. 5 (LaMP expressing strains 3- 1, 3-2, 3-5, and 3-6), FIG. 6 (LaMP expressing strains 3-3 and 3-4), FIG. 7 (CsKP expressing strains 3-7, 3-8, 3-11, and 3-12), and FIG. 7 (CsKP expressing strains 3-9 and 3-10) and the total protein quantification is reported in Table 9 (LaMP) and TablelO (CsKP).

[0076] The results show7that strains encoding a recombinant protein with a secretion signal leader signal resulted in visible protein expression at the expected molecular weight (strains 3-1, 3-2, 3-5. 3-6, 3-7. 3-8, 3-11 and 3-12). In contrast, strains with encoded recombinant proteins designed for intracellular expression (strains 3-3, 3-4, 3-9, and 3-10) resulted in no visible protein expression for either CsKP or LaMP. Based on both the SDS-PAGE and total protein content analysis, the recombinant protein constructs containing the Ostl / Mfa hybrid secretion signal and the methanol inducible promoter for both CsKP and LaMP had the highest expression levels. In general, protein expression was higher in strains with a construct including a methanol inducible promoter (pAOXl) than in the strains with constructs under the control of a constitutive promoter.Table 9.PT-2176-WO-PCTTable 10.PT-2176-WO-PCT

[0077] Enzyme activity was assayed for LaMP and CsKP enzymes taken from the soluble lysis fraction of strains 3-1, 3-2, 3-5, 3-6, 3-7, 3-8, 3-11, and 3-12. The reaction mixtures are outlined in Table 5 and reactions were incubated at 55 °C for 14 minutes beginning when the enzyme was added as the final component of the mixture. Samples were removed every 2 minutes during the reaction and immediately inactivated using 0.2 M citric-phosphate buffer at pH 2.6. The glucose concentration of each collected sample, along with the concentration of glucose in the starting reaction mixture prior to the addition of enzyme, was measured using the glucose assay kit sold under the tradename Megazyme HK Glucose Kit by Neogen. The assay kit glucose concentration via a coupled hexokinase and glucose-6-phosphate dehydrogenase reaction converting the glucose to NADPH and gluconate-6-phosphate. The concentration of NADPH can be monitored by the UV absorbance at 340 nm and converted to the initial glucose concentration of the assay reaction after running a glucose standard curve. Measured enzyme activity is reported in Table 11 as both the reaction units (U) per mL and U / mg by converting using the initial enzyme concentration.

[0078] These results show that all lysate soluble fraction samples showed activity indicating that functional enzymes were produced in all cases.PT-2176-WO-PCTTable 11.Example 3 - Klu veromyces marxianus strains, plasmids, and expression

[0079] Expression of the LaMP and CsKP enzymes was done in Kluyveromyces marxianus. The yeast cell Kluyveromyces marxianus CD21, deposited under Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain (BCCM MUCL) designation 58456, is designated strain 4-0 herein. Strain 4-1 is an uracil auxotroph derivative of strain 4-0 with a deletion of the URA3 locus.

[0080] Strain 4-1 was transformed according to Table 12 using the indicated transformation fragments. Resulting transformants were streaked for single colony isolation on ScD-ura plates and single colonies were selected. Selected colonies were evaluated by colony PCR for the integration of the indicated sequence. PCR verified isolates were designated as outlined in TablePT-2176-WO-PCT12. In some instances, more than one isolate, e.g., “sister’ isolates, are indicated by letters following the strain number. For example, strain 4-2 has 2 sister isolates, strains 4-2a and 4-2b.

[0081] Each transformation fragment included an expression construct for the recombinant protein of interest under the control of the pyruvate decarboxylate 1 promoter from Saccharomyces cerevisiae (pPDCl, SEQ ID NO:46) or the synthetic promoter of SEQ ID NO:48. Each recombinant protein construct contained (i) an optional secretion signal; (ii) a polynucleotide sequence encoding the LaMP enzyme or the CsKP enzyme: and (iii) an optional 6x-histidine tag (SEQ ID NO: 4) for purification. The secretion signals used in this example include a-mating factor (Mfa) sequence from Saccharomyces cerevisiae (SEQ ID NO:28), a hybrid sequence of the oligosaccharyl transferase complex 1 (Ostl) signal sequence from S. cerevisiae and the Mfa sequence from S. cerevisiae (SEQ ID NO:29), and inulinase 1 secretion sequence from K. marxianus (INU1; SEQ ID NO:49).Table 12.PT-2176-WO-PCT

[0082] Strains 4-1 through 4-19 were assayed for expression of the LaMP or CsKP enzyme containing recombinant proteins. A single colony was incubated overnight in 20 mL of YPD (bacteriological peptone 20g / L, yeast extract 10 g / L, and glucose 20 g / L) medium at 30 °C, 250 rpm, and 70% humidity. The production culture of either 20 mL YPD medium or 20 mL DMul medium (Table 13) was inoculated to an optical density at 600 nm (OD600) of 1 and incubated for 72 hours at 31 °C, 250 rpm, and 70% humidity.PT-2176-WO-PCTTable 13: DMul Production Medium - Maltodextrin*GA added just prior to inoculationTable 14: lOOOx trace elementsTable 15: 1000X DM1 vitamin solutionPT-2176-WO-PCT

[0083] Following incubation, the fermentation broth was separated from the cell biomass, and both the fermentation broth and the cell biomass were saved for analysis. The separated cell biomass was resuspended in water to an equal volume of the original fermentation product. Fermentation broth samples and whole cell resuspension samples were analyzed by SDS-PAGE with Coomassie straining. The fermentation broth sample was used to analyze expression from strains expressing a recombinant protein with a secretion signal (strains 4-2, 4-3, 4-6, 4-7, 4-10, 4-11, 4-14, 4-15, 4-18 and 4-19) and the whole cell suspension sample was used to analyze expression from strains expressing a recombinant protein without a secretion signal (strains 4-4, 4-5, 4-8, 4-9, 4-12, 4-13, 4-16, and 4-17). Total protein content in the fermentation broth was analyzed by Bradford assay. Gel images are shown in FIG. 9 (strains 4-1 through 4-9 and GFP control expressed in YPD medium), FIG. 10 (strains 4-1 and 4-10 through 4-17 and GFP control expressed in YPD medium), FIG. 11 (strains 4-1, 4-18, 4-19 and GFP control in YPD and DMul medium, fermentation broth samples analyzed), FIG. 12 (strains 4-1 through 4-9 and GFP control expressed in DMul medium), FIG. 13 (strains 4-1 and 4-10 through 4-17 and GFP control expressed in DMul medium), and the total protein quantification is reported in Table 16 (LaMP) and Table 17 (CsKP).

[0084] The results show that at least one of each sister strain encoding a recombinant protein with a secretion signal leader signal resulted in visible protein expression at the expected molecular weight. Strains with encoded recombinant proteins designed for intracellular expression resulted in some visible protein expression for both CsKP or LaMP, however recombinant protein expression was a small fraction of the total protein visualized. Based on both the SDS-PAGE and total protein content analysis, the recombinant protein constructs containing any of the tested secretion signals and the scPDCl promoter for both CsKP and LaMP had the most abundant recombinant protein when cultured in YPD medium. Consistent expression was not observed when strains were cultured in DMul medium, where constructs containing the INU1 secretion signal showed the most consistent expression across sister strains. In general, protein expression was higher in strains utilizing the scPDC 1 promoter and YPD medium when compared to all other combinations tested.Table 16.PT-2176-WO-PCT

[0085] Enzyme activity was assayed for LaMP and CsKP enzymes taken from the fermentation broth of strains 4-2a-b, 4-3a-b, 4-6a-b, and 4-7a-b. Prior to the reaction, fermentation broth samples were concentrated using a 10K molecular weight cutoff column. The reaction mixtures are outlined in Table 17 and reactions were incubated at 55 °C for 14 minutes beginning when the enzyme was added as the final component of the mixture. Samples were removed every 2 minutes during the reaction and immediately inactivated using 0.2 M citric-phosphate buffer at pH 2.6. The glucose concentration of each collected sample, along with the concentration of glucose inPT-2176-WO-PCT the starting reaction mixture prior to the addition of enzyme, was measured using the glucose assay kit sold under the tradename Megazyme HK Glucose Kit by Neogen. The assay kit glucose concentration via a coupled hexokinase and glucose-6-phosphate dehydrogenase reaction converting the glucose to NADPH and gluconate-6-phosphate. The concentration of NADPH can be monitored by the UV absorbance at 340 nm and converted to the initial glucose concentration of the assay reaction after running a glucose standard curve. Measured enzyme activity is reported in Table 18.

[0086] These results show that fermentation broth samples showed activity indicating that functional enzymes were produced in all cases. For strain 4-7b, the fermentation broth was likely not concentrated sufficiently to get the enzyme concentration to a measurable level, but the activity in the fermentation both of 4-7a confirms that the recombinant protein of SEQ ID NO:45 is active.Table 17.Table 18.Example 4 - Kluyveromyces marxianus strains, plasmids, and expression

[0087] Expression of the TbarKP enzymes will be done in Kluyveromyces marxianus. The yeast cell Kluyveromyces marxianus CD21. deposited under Belgian Coordinated Collections of Micro-organisms / Mycotheque de 1'Universite Catholique de Louvain (BCCM MUCL) designation 58456, is designated strain 4-0 herein. Strain 4-1 is an uracil auxotroph derivative of strain 4-0 with a deletion of the URA3 locus.PT-2176-WO-PCT

[0088] Strain 4- 1 will be transformed according to Table 19 using the indicated transformation fragments. Resulting transformants will be streaked for single colony isolation on ScD-ura plates and single colonies will be selected. Selected colonies will be evaluated by colony PCR for the integration of the indicated sequence. PCR verified isolates will be designated as outlined in Table 12. In some instances, more than one isolate, e.g.. “sister’ isolates, are indicated by letters following the strain number.

[0089] Each transformation fragment included an expression construct for the recombinant protein of interest under the control of the pyruvate decarboxylate 1 promoter from Saccharomyces cerevisiae (pPDCl, SEQ ID NO:46) or the synthetic promoter of SEQ ID NO:48. Each recombinant protein construct contained (i) an optional secretion signal; and (ii) a polynucleotide sequence encoding the TbarKP enzyme. The secretion signals used in this example include a hybrid sequence of the oligosaccharyl transferase complex 1 (Ostl) signal sequence from S. cerevisiae and the Mfa sequence from 5. cerevisiae (SEQ ID NO:29) and the secretion signal from the UTH1 protein of Komagataella phaffti (SEQ ID NO:79).Table 19.

[0090] Strains 5-1 and 5-2 will be assayed for expression of the TbarKP enzyme containing recombinant proteins. A single colony will be incubated overnight in 20 mL of YPD (bacteriological peptone 20g / L, yeast extract 10 g / L, and glucose 20 g / L) medium at 30 °C. 250 rpm, and 70% humidity. The production culture of either 20 mL YPD medium or 20 mL DMulPT-2176-WO-PCT medium (Table 13) will be inoculated to an optical density at 600 nm (OD600) of 1 and incubated for 72 hours at 31 °C, 250 rpm, and 70% humidity.

[0091] Following incubation, the fermentation broth will be separated from the cell biomass, and both the fermentation broth and the cell biomass will be saved for analysis. The separated cell biomass will be resuspended in water to an equal volume of the original fermentation product. Fermentation broth samples and whole cell resuspension samples will be analyzed by SDS-PAGE with Coomassie straining. Total protein content in the fermentation broth will be analyzed by Bradford assay.

[0092] Enzyme activity will be assayed for TbarKP enzyme taken from the fermentation broth of strains 5-1 and 5-2. Prior to the reaction, fermentation broth samples will be concentrated 5x- lOx using a 10K molecular weight cutoff column. The reaction mixtures are outlined in Table 20 and reactions will be incubated at 55 °C for 14 minutes beginning when the enzy me was added as the final component of the mixture. Samples will be removed every 2 minutes during the reaction and immediately inactivated using 0.2 M citric-phosphate buffer at pH 2.6. The glucose concentration of each collected sample, along with the concentration of glucose in the starting reaction mixture prior to the addition of enzyme, will be measured using the glucose assay kit sold under the tradename Megazyme HK Glucose Kit by Neogen. The assay kit glucose concentration via a coupled hexokinase and glucose-6-phosphate dehydrogenase reaction converting the glucose to NADPH and gluconate-6-phosphate. The concentration of NADPH can be monitored by the UV absorbance at 340 nm and converted to the initial glucose concentration of the assay reaction after running a glucose standard curv e.Table 20.Example 5 - Komagataella phaffii strains, plasmids, and expression

[0093] Expression of the TbarKP enzyme will be tested in Komagataella phaffii. The Pichia pastoris strain 9011, which is an alcohol oxidase 1 knockout mutant (aoxl ) of wild-type Pichia pastoris and is available from ATUM, was designated strain 3-0. Komagataella phaffii is the updated taxonomic designation for Pichia pastoris and they are both used interchangeably in the art.PT-2176-WO-PCT

[0094] Strain 3-0 will be transformed with the transformation fragments as outlined in Table 21. Positive transformants will be selected on YPD + zeocin plates and individual isolated and PCR verified colonies were designated with the strain number in Table 21. In some instances, more than one isolate, e.g., "sister" isolates, are indicated by letters following the strain number.

[0095] Each transformation fragment included an expression construct for the recombinant protein of interest under the control of a methanol inducible promoter (P. pastoris alcohol oxidase 1 promoter (pAOXl); SEQ ID NO:26). Each recombinant protein construct contained (i) a secretion signal; and (ii) a polynucleotide sequence encoding the TbarKP enzyme. The secretion signals used in this example include a hybrid sequence of the oligosaccharyl transferase complex 1 (Ostl) signal sequence from S. cerevisiae and the Mfa sequence from S. cerevisiae (SEQ ID NO:29) and the secretion signal from the UTH1 protein of Komagataella phaffii (SEQ ID NO:79).Table 21.

[0096] Strains 6-1 and 6-2 will be assayed for expression of the TbarKP enzyme containing recombinant proteins. Starter cultures of strains 6-1 and 6-2 will be prepared by inoculating 25 mL of media (lOg / L yeast extract. 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0.1M phosphate buffer (pH 6), 0.4mg Biotin, 20g / L glycerol) with an isolated colony from the selected strain and incubating overnight (16-20 hours) at 30 °C. Cultures will be standardized to 50 mL at an optical density at 600 nm (OD600) of 1.0, then spun down and resuspended in 10 mL induction medium (lOg / L yeast extract. 20g / L peptone, 13.4g / L yeast nitrogen base (no amino acids), 0. IM phosphate buffer (pH 6). 0.4mg Biotin, 20g / L methanol) and incubated at 29 °C for 48 hours. An additional 0.5 wt% methanol will be added between 20-24 hours and 26-30 hours to further induce expression.PT-2176-WO-PCT

[0097] Following incubation, samples will be collected for analysis. Fermentation broth will be separated from the cell biomass and a fermentation broth sample will be analyzed by SDS- PAGE using Coomassie straining. Total protein content will be analyzed by Bradford assay.

[0098] Enzy me activity' will be assayed for TbarKP enzymes taken from the soluble lysis fraction of strains 6-1 and 6-2. The reaction mixtures are outlined in Table 5 and reactions were incubated at 55 °C for 14 minutes beginning when the enzyme was added as the final component of the mixture. Samples were removed every 2 minutes during the reaction and immediately inactivated using 0.2 M citric-phosphate buffer at pH 2.6. The glucose concentration of each collected sample, along with the concentration of glucose in the starting reaction mixture prior to the addition of enzyme, was measured using the glucose assay kit sold under the tradename Megazyme HK Glucose Kit by Neogen. The assay kit glucose concentration via a coupled hexokinase and glucose-6-phosphate dehydrogenase reaction converting the glucose to NADPH and gluconate-6-phosphate. The concentration of NADPH can be monitored by the UV absorbance at 340 nm and converted to the initial glucose concentration of the assay reaction after running a glucose standard curve.

Claims

PT-2176-WO-PCTCLAIMSWhat is claimed is:

1. A recombinant protein comprising: a sequence at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or 1 0% identical to SEQ ID NO: 1 ; and a secretion signal sequence heterologous thereto.

2. A recombinant protein comprising: a 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 SEQ ID NO:2 or SEQ ID NO:3; and a secretion signal sequence heterologous thereto.

3. The recombinant protein of any preceding claim, wherein the secretion signal sequence is 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, 10, 11, 28, 29, and 49, preferably SEQ ID NOs:28, 29, and 49, most preferably SEQ ID NO:29.

4. The recombinant protein of any preceding claim, wherein the recombinant protein 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: 12-17, 42-45, 50-51, and 70-75, preferably SEQ ID NOs:42-45. 50-51, and 73-75, most preferably SEQ ID NOs:43 and 45.

5. A genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding the recombinant protein of any preceding claim.

6. A method for producing the recombinant protein of any one of claims 2-4, the method comprising contacting a substrate comprising dextrose and / or glycerol with a genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding the recombinant protein of any one of claims 1-4 for a time and under conditions sufficient to express the recombinant protein.PT-2176-WO-PCT7. The microorganism cell or method of claim 5 or claim 6, wherein the genetically engineered microorganism cell is a bacterial cell, for example an Escherichia coli cell, or a yeast cell, for example a Komagataella phaffli cell or a Kluyveromyces marxianus cell.

8. The microorganism cell or method of any one of claims 5-7, wherein the heterologous polynucleotide sequence is operably linked to a heterologous promoter and / or a heterologous terminator.

9. The microorganism cell or method of claim 8, wherein the heterologous promoter is selected from the group consisting of the IPTG inducible promoter of pyruvate decarboxylase promoter (PDCp), translation elongation factor 2 promoter (TEF2p), SED1 promoter, alcohol dehydrogenase 1A promoter (ADHlp), hexokinase 2 promoter (HXK2p), FLO5 promoter, pyruvate kinase 1 promoter (PYKlp); 6-phosphogluconate dehydrogenase promoter (6PGDp); glyceraldehyde-3-phosphate dehydrogenase promoter (TDH3p); translational elongation factor 1 promoter (TEFp); phosphoglucomutase 1 promoter (PGM Ip); 3 -phosphoglycerate kinase promoter (PGKlp); enolase promoter (ENOlp); asparagine synthetase promoter (ASNSp); 50S ribosomal protein LI promoter (RPLAp); RPL16B; and PDC1 promoter, and / or the heterologous terminator is selected form the group consisting of GAL 10 terminator, PDC terminator, transaldolase terminator (TAL) 6PGD terminator (6PGDt); ASNS terminator (ASNSt); ENO1 terminator (ENOlt); hexokinase 1 terminator (HXKlt); PGK1 terminator (PGKlt); PGM1 terminator (PGMlt); PYK1 terminator (PYKlt); RPLA terminator (RPLAt); transaldolase 1 terminator (TALlt); TDH3 terminator (TDH3t); translation elongation factor 2 terminator (TEF2t); triosephosphate isomerase 1 terminator (TPIlt); TEF1; iso- 1 -cytochrome c terminator (CYC1); HXK2 terminator; GPM1 terminator; URA3 terminator; ADH1 terminator; and ScGALlO terminator.

10. The microorganism cell or method of claim 8. wherein the promoter is an inducible promoter, for example the IPTG inducible promoter of SEQ ID NO:8, the AOX1 promoter of SEQ ID NO:26, and the inducible synthetic promoter of SEQ ID NO:48.

11. The method of any one of claims 6-10, wherein the microorganism cell is aX. pha ffli or K. marxianus cell and the substrate is contacted with the microorganism cell at a temperature between 20 °C to 40 °C, 25 °C to 35 °C, or 28 °C to 32 °C, preferably about 30 °C.PT-2176-WO-PCT12. The method of any one of claims 6-11 wherein the substrate comprises dextrose.

13. The method of any one of claims 6-12, wherein the genetically engineered microorganism cell produces at least 50 pg / mL. at least 75 pg / mL, at least 80 pg / mL, at least 90 pg / mL, at least 100 pg / mL, at least 1 15 pg / mL, at least 125 pg / mL, at least 130 pg / mL, at least 150 pg / mL, or at least 175 pg / mL.

14. A genetically engineered microorganism cell comprising a heterologous polynucleotide sequence encoding a maltose phosphorylase enzyme at least 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: 1; and / or a heterologous polynucleotide sequence encoding a kojibiose phosphorylase 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 NO:2 or 3.

15. A method for producing a recombinant comprising contacting a substrate comprising dextrose and / or glycerol with the genetically engineered microorganism cell of claim 14 for a time and under conditions sufficient to express the recombinant protein.

16. The microorganism cell or method of claim 14 or claim 15, wherein the genetically engineered microorganism cell is a yeast cell, for example a Komagataella phaffli cell or a Kluyveromyces marxianus cell.