US20170002335A1 - Glucosyltransferase amino acid motifs for enzymatic production of linear poly alpha-1,3-glucan - Google Patents

Glucosyltransferase amino acid motifs for enzymatic production of linear poly alpha-1,3-glucan Download PDF

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US20170002335A1
US20170002335A1 US15/182,747 US201615182747A US2017002335A1 US 20170002335 A1 US20170002335 A1 US 20170002335A1 US 201615182747 A US201615182747 A US 201615182747A US 2017002335 A1 US2017002335 A1 US 2017002335A1
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amino acid
motif
glucan
gtf
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Mark S Payne
Yefim Brun
Richard R Bott
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DuPont Industrial Biosciences USA LLC
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EI Du Pont de Nemours and Co
Danisco US Inc
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
    • C08B37/0009—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid alpha-D-Glucans, e.g. polydextrose, alternan, glycogen; (alpha-1,4)(alpha-1,6)-D-Glucans; (alpha-1,3)(alpha-1,4)-D-Glucans, e.g. isolichenan or nigeran; (alpha-1,4)-D-Glucans; (alpha-1,3)-D-Glucans, e.g. pseudonigeran; Derivatives thereof
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/102—Mutagenizing nucleic acids
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
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    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01027—DNA beta-glucosyltransferase (2.4.1.27)

Definitions

  • the present disclosure is in the field of enzyme catalysis.
  • the disclosure pertains to producing linear poly alpha-1,3-glucan using a glucosyltransferase having certain amino acid sequence motifs.
  • sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named 20160615_CL6452USNPSequenceListing_ST25_ExtraLinesRemoved.txt created on Jun. 14, 2016, and having a size of 704 kilobytes and is filed concurrently with the specification.
  • sequence listing contained in this ASCII-formatted document is part of the specification and is herein incorporated by reference in its entirety.
  • polysaccharides that are biodegradable and can be made economically from renewably sourced feedstocks.
  • One such polysaccharide is poly alpha-1,3-glucan, a glucan polymer characterized by having alpha-1,3-glycosidic linkages. This polymer has been isolated by contacting an aqueous solution of sucrose with a glucosyltransferase (GTF) enzyme isolated from Streptococcus salivarius (Simpson et al., Microbiology 141:1451-1460, 1995).
  • GTF glucosyltransferase
  • U.S. Pat. No. 7,000,000 disclosed the preparation of a polysaccharide fiber using an S. salivarius gtfJ enzyme. At least 50% of the hexose units within the polymer of this fiber were linked via alpha-1,3-glycosidic linkages.
  • S. salivarius gtfJ enzyme utilizes sucrose as a substrate in a polymerization reaction producing poly alpha-1,3-glucan and fructose as end-products (Simpson et al., 1995).
  • the disclosed polymer formed a liquid crystalline solution when it was dissolved above a critical concentration in a solvent or in a mixture comprising a solvent. Continuous, strong, cotton-like fibers were obtained from this solution that could be spun and used in textile applications.
  • glucosyltransferase enzymes can produce glucan with a molecular weight and percentage of alpha-1,3 glycosidic linkages suitable for use in spinning fibers. For example, most glucosyltransferase enzymes do not produce glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. Therefore, it is desirable to identify glucosyltransferase enzymes that can convert sucrose to glucan polymers having a high percentage of alpha-1,3 glycosidic linkages and high molecular weight.
  • glucosyltransferase enzymes containing certain amino acid motifs. These enzymes can synthesize high molecular weight, linear alpha-1,3-glucan polymer. Also disclosed are methods for identifying such enzymes.
  • the disclosure concerns a reaction solution comprising water, sucrose, and a glucosyltransferase enzyme, wherein the glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
  • the catalytic domain comprises an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65.
  • the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:78 aligns with amino acid positions 231-243 of SEQ ID NO:65; the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:79 aligns with amino acid positions 396-425 of SEQ ID NO:65; and/or the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:80 aligns with amino acid positions 549-567 of SEQ ID NO:65.
  • motif (i) comprises SEQ ID NO:78
  • motif (ii) comprises SEQ ID NO:79
  • motif (iii) comprises SEQ ID NO:80.
  • the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages.
  • the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having a DP w of at least 400.
  • Another embodiment of the disclosure concerns a method of producing insoluble poly alpha-1,3-glucan.
  • This method comprises: (a) contacting at least water, sucrose, and a glucosyltransferase enzyme, wherein the glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
  • the catalytic domain comprises an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65.
  • the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:78 aligns with amino acid positions 231-243 of SEQ ID NO:65; the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:79 aligns with amino acid positions 396-425 of SEQ ID NO:65; and/or the position of the amino acid sequence that is at least 90% identical to SEQ ID NO:80 aligns with amino acid positions 549-567 of SEQ ID NO:65.
  • motif (i) comprises SEQ ID NO:78
  • motif (ii) comprises SEQ ID NO:79
  • motif (iii) comprises SEQ ID NO:80.
  • the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages.
  • the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having a DP w of at least 400.
  • Another embodiment of the disclosure concerns a method of identifying a glucosyltransferase enzyme.
  • This method comprises detecting the presence of at least one motif in a glucosyltransferase catalytic domain, the at least one motif selected from the group consisting of:
  • the detecting step is performed (a) in silico, (b) with a method comprising a nucleic acid hybridization step, (c) with a method comprising a protein sequencing step, and/or (d) with a method comprising a protein binding step.
  • the detecting step comprises detecting the presence of each of motifs (i), (ii) and (iii) in the catalytic domain.
  • FIG. 1 Comparison of the main chain tertiary fold of Lactobacillus reuteri GTF (gray) and Streptococcus mutans GTF (black).
  • the structure of the L. reuteri GTF includes a fifth domain (Domain V) that was truncated from the structure of S. mutans GTF.
  • the active site is also indicated and is formed by a cavity in the central domains (the so-called A and B domains); this location is based on spatial similarity with similar domains in alpha amylases.
  • the amino acid sequence of the S. mutans 3AIE GTF structure is SEQ ID NO:66
  • the amino acid sequence of the L. reuteri 3KLK GTF structure is SEQ ID NO:67.
  • FIGS. 2A-O Alignment of twenty-four GTF sequences with sequences of portions of GTFs from S. mutans (3AIE, SEQ ID NO:66) and L. reuteri (3KLK, SEQ ID NO:67) for which crystallographic structures are known; single-letter amino acid code is used.
  • GTF amino acid sequences that produced glucan with 100% alpha-1,3 linkages and high molecular weight (DP w of at least 400 under the tested initial sucrose concentrations, see Table 4) are designated “++”.
  • Those GTFs producing glucan with 100% alpha-1,3 linkages and a DP w of at least 100 are designated “+ ⁇ ”.
  • Other GTFs producing glucan with mixed linkages are designated “ ⁇ ”.
  • FIG. 3 The sequence of the tested GTF enzymes in the vicinity of Motifs 1a and 1b.
  • the sequence region of Motifs 1a and 1b along with upstream and downstream flanking reference sequence motifs are shown as boxed regions. Motifs 1a and 1b are located in box labeled “Insertion 1”.
  • the alignment in this figure represents a portion of the alignment in FIGS. 2A-O .
  • FIGS. 4A and 4B Visualization of Motif 1a through comparison of a homology model of GTF 7527 (SEQ ID NO:65) based on the reference crystallographic structures of S. mutans (3AIE, SEQ ID NO:66) ( FIG. 4A ) and L. reuteri (3KLK, SEQ ID NO:67) ( FIG. 4B ).
  • the main chain folding of the homology model in each view is shown with dark lines while the main chain folding of the reference structure is shown with lighter lines.
  • the residues forming the catalytic sites in the reference crystallographic structures are shown as Van der Waals spheres for reference.
  • Motif 1a (between the arrows) is presented in both homology models as an open loop (black) extending into the solvent as a consequence of there being no homologous segment to provide means to position with respect to the remainder of the GTF catalytic domain.
  • FIG. 5 The sequence of the tested GTF enzymes in the vicinity of Motif 2.
  • the sequence region of Motif 2 along with upstream and downstream flanking reference sequence motifs are shown as boxed regions. Motif 2 is located in box labeled “Insertion 2”.
  • the alignment in this figure represents a portion of the alignment in FIGS. 2A-O .
  • FIGS. 6A and 6B Visualization of Motif 2 through comparison of a homology model of GTF 7527 (SEQ ID NO:65) based on the reference crystallographic structures of S. mutans (3AIE, SEQ ID NO:66) ( FIG. 6A ) and L. reuteri (3KLK, SEQ ID NO:67) ( FIG. 6B ).
  • the main chain folding of the homology model in each view is shown with dark lines while the main chain folding of the reference structure is shown with lighter lines.
  • the residues forming the catalytic sites in the reference crystallographic structures are shown as Van der Waals spheres for reference.
  • Motif 2 (between the arrows) is presented in both homology models as an open loop (black) extending into the solvent as a consequence of there being no homologous segment to provide means to position with respect to the remainder of the GTF catalytic domain.
  • FIG. 7 The sequence of the tested GTF enzymes in the vicinity of Motifs 3a and 3b.
  • the sequence region of Motifs 3a and 3b along with upstream and downstream flanking reference sequence motifs are shown as boxed regions. Motifs 3a and 3b are located in box labeled “Insertion 3”.
  • the alignment in this figure represents a portion of the alignment in FIGS. 2A-O .
  • FIGS. 8A and 8B Visualization of Motif 3a through comparison of a homology model of GTF 7527 (SEQ ID NO:65) based on the reference crystallographic structures of S. mutans (3AIE, SEQ ID NO:66) ( FIG. 8A ) and L. reuteri (3KLK, SEQ ID NO:67) ( FIG. 8BA ).
  • the main chain folding of the homology model in each view is shown with dark lines while the main chain folding of the reference structure is shown with lighter lines.
  • the residues forming the catalytic sites in the reference crystallographic structures are shown as Van der Waals spheres for reference.
  • Motif 3a (between the arrows) is presented in both homology models as an open loop (black) extending into the solvent as a consequence of there being no homologous segment to provide means to position with respect to the remainder of the GTF catalytic domain.
  • the first 162 amino acids of the protein are deleted compared to GENBANK Identification No. 121724; a start methionine is included.
  • the first 164 amino acids of the protein are deleted compared to GENBANK Identification No. 290580544; a start methionine is included.
  • the first 144 amino acids of the protein are deleted compared to GENBANK Identification No.
  • DNA 19 20 (1340 aa) codon-optimized for expression in E. coli .
  • the first 193 amino acids of the protein are deleted compared to GENBANK Identification No. 322372765; a start methionine is included.
  • the first 36 amino acids of the protein are deleted compared to GENBANK Identification No. 21654700; a start methionine is included.
  • “1366 GTF” Streptococcus criceti .
  • DNA codon- 23 24 (1323 aa) optimized for expression in E. coli .
  • the first 139 amino acids of the protein are deleted compared to GENBANK Identification No. 146741366; a start methionine is included.
  • the first 156 amino acids of the protein are deleted compared to GENBANK Identification No. 940427; a start methionine is included.
  • the first 92 amino acids of the protein are deleted compared to GENBANK Identification No. 383282919; a start methionine is included.
  • the first 178 amino acids of the protein are deleted compared to GENBANK Identification No. 387783929; a start methionine is included.
  • “6907 GTF”, Streptococcus salivarius SK126. DNA 35 36 (1331 aa) codon-optimized for expression in E. coli .
  • the first 161 amino acids of the protein are deleted compared to GENBANK Identification No. 228476907; a start methionine is included.
  • “6661 GTF” Streptococcus salivarius SK126.
  • the first 265 amino acids of the protein are deleted compared to GENBANK Identification No.
  • DNA 43 44 (1287 aa) codon-optimized for expression in E. coli The first 176 amino acids of the protein are deleted compared to GENBANK Identification No. 24379358; a start methionine is included. “8242 GTF”, Streptococcus gallolyticus ATCC BAA- 45 46 (1355 aa) 2069. DNA codon-optimized for expression in E. coli . The first 191 amino acids of the protein are deleted compared to GENBANK Identification No. 325978242; a start methionine is included. “3442 GTF”, Streptococcus sanguinis SK405. DNA 47 48 (1348 aa) codon-optimized for expression in E. coli .
  • the first 228 amino acids of the protein are deleted compared to GENBANK Identification No. 324993442; a start methionine is included.
  • the first 173 amino acids of the protein are deleted compared to GENBANK Identification No. 47528; a start methionine is included.
  • DNA codon- 51 52 (1393 aa) optimized for expression in E. coli .
  • the first 178 amino acids of the protein are deleted compared to GENBANK Identification No. 322373279; a start methionine is included.
  • DNA codon- 57 58 (1735 aa) optimized for expression in E. coli The first 38 amino acids of the protein are deleted compared to GENBANK Identification No. 51574154. “3298 GTF”, Streptococcus sp. C150. The first 209 59 (1242 aa) amino acids of the protein are deleted compared to GENBANK Identification No. 322373298; a start methionine is included. Wild type GTFJ, Streptococcus salivarius . GENBANK 60 (1518 aa) Identification No. 47527. Wild type GTF corresponding to 2678 GTF, 61 (1528 aa) Streptococcus salivarius K12.
  • 3AIE portion of a GTF from Streptococcus mutans 66 (844 aa) as annotated in the Protein Data Bank under pdb entry no. 3AIE.
  • 3KLK portion of a GTF from Lactobacillus reuteri as 67 (1039 aa) annotated in the Protein Data Bank under pdb entry no. 3KLK.
  • Catalytic active site motif FDxxRxDAxDNV 68 (12 aa) Catalytic active site motif ExWxxxDxxY 69 (10 aa) Catalytic active site motif FxRAHD 70 (6 aa) Catalytic active site motif IxNGYAF 71 (7 aa) Motif SxxRxxN upstream of Motifs 1a and 1b 72 (7 aa) Motif GGxxxLLxNDxDxSNPxVQAExLN downstream 73 (24 aa) of Motifs 1a and 1b Motif WxxxDxxY upstream of Motif 2 74 (8 aa) Motif YxFxRAHD downstream of Motif 2 75 (8 aa) Motif YxxGGQ upstream of Motifs 3a and 3b 76 (6 aa) Motif VRxG downstream of Motifs 3a and 3b 77 (4 aa) Motif 1a: D/N-K-S-I/
  • DNA codon- 84 85 (1325 aa) optimized for expression in E. coli .
  • DNA codon- 86 (1311 aa) optimized for expression in E. coli .
  • DNA codon- 88 (1319 aa) optimized for expression in E. coli .
  • 7527-NT-dlS1a, 2 GTF lacking Motifs 1a and 2.
  • 90 91 (1295 aa) DNA codon-optimized for expression in E. coli .
  • the terms “a” and “an” as used herein are intended to encompass one or more (i.e., at least one) of a referenced feature.
  • Poly alpha-1,3-glucan is a polymer comprising glucose monomeric units linked together by glycosidic linkages, wherein at least about 50% of the glycosidic linkages are alpha-1,3-glycosidic linkages.
  • Poly alpha-1,3-glucan in certain embodiments comprises at least 95% alpha-1,3-glycosidic linkages.
  • glycosidic linkage refers to the covalent bond that joins a carbohydrate (sugar) molecule to another group such as another carbohydrate.
  • alpha-1,3-glycosidic linkage refers to the type of covalent bond that joins alpha-D-glucose molecules to each other through carbons 1 and 3 on adjacent alpha-D-glucose rings.
  • the glycosidic linkages of an alpha-1,3-glucan herein can also be referred to as “glucosidic linkages”.
  • alpha-D-glucose will be referred to as “glucose”.
  • Intrinsic viscosity refers to a measure of the contribution of a glucan polymer (e.g., branched alpha-glucan) to the viscosity of a liquid (e.g., solution) comprising the glucan polymer. Intrinsic viscosity can be measured, for example using the methodology disclosed in the Examples below, or as disclosed by Weaver et al. ( J. Appl. Polym. Sci. 35:1631-1637) and Chun and Park ( Macromol. Chem. Phys. 195:701-711), for example.
  • branching index can be denoted as g′
  • branching ratio can be denoted as g′
  • branching index can be measured, for example using the methodology disclosed in the Examples below, or as disclosed by Zdunek et al. ( Food Bioprocess Technol. 7:3525-3535) and Herget et al. ( BMC Struct. Biol. 8:35).
  • sucrose refers to a non-reducing disaccharide composed of an alpha-D-glucose molecule and a beta-D-fructose molecule linked by an alpha-1,2-glycosidic bond. Sucrose is known commonly as table sugar.
  • GTF enzyme The activity of a GTF enzyme herein catalyzes the reaction of the substrate sucrose to make the products poly alpha-1,3-glucan and fructose.
  • Other products (byproducts) of a GTF reaction can include glucose, various soluble gluco-oligosaccharides (DP2-DP7), and leucrose.
  • Wild type forms of GTF enzymes generally contain (in the N-terminal to C-terminal direction) a signal peptide, a variable domain, a catalytic domain, and a glucan-binding domain.
  • GTF herein is classified under the glycoside hydrolase family 70 (GH70) according to the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009).
  • CAZy Carbohydrate-Active EnZymes
  • glucosyltransferase catalytic domain refers to the domain of a glucosyltransferase enzyme that provides poly alpha-1,3-glucan-synthesizing activity to a glucosyltransferase enzyme.
  • a glucosyltransferase catalytic domain preferably does not require the presence of any other domains to have this activity.
  • reaction solution generally refers to a solution comprising sucrose, water, at least one active glucosyltransferase enzyme, and optionally other components.
  • a reaction solution can alternatively be referred to herein as a “glucan synthesis reaction”, “glucan reaction”, “GTF reaction”, or “reaction composition”, for example.
  • Other components that can be in a glucan synthesis reaction include fructose, glucose, leucrose, and soluble gluco-oligosaccharides (e.g., DP2-DP7).
  • glucan products such as poly alpha-1,3-glucan with a degree of polymerization (DP) of at least 8 or 9, are water-insoluble and thus are not dissolved in a glucan synthesis reaction, but rather may be present out of solution. It is in a reaction solution where the step of contacting water, sucrose and a glucosyltransferase enzyme is performed.
  • the term “under suitable reaction conditions” as used herein refers to reaction conditions that support conversion of sucrose to poly alpha-1,3-glucan via glucosyltransferase enzyme activity.
  • a reaction solution as claimed herein is not believed to be naturally occurring.
  • the “percent dry solids” of a reaction solution refers to the wt % of all the sugars in the glucan synthesis reaction.
  • the percent dry solids of a reaction solution can be calculated, for example, based on the amount of sucrose used to prepare the reaction.
  • the “yield” of poly alpha-1,3-glucan by a reaction solution herein represents the weight of poly alpha-1,3-glucan product expressed as a percentage of the weight of sucrose substrate that is converted in the reaction. For example, if 100 g of sucrose in a reaction solution is converted to products, and 10 g of the products is poly alpha-1,3-glucan, the yield of the poly alpha-1,3-glucan would be 10%. This yield calculation can be considered as a measure of selectivity of the reaction toward poly alpha-1,3-glucan.
  • motif herein refers to a distinctive and recurring structural unit, such as within an amino acid sequence. By “recurring” it is meant that a motif occurs in multiple related polypeptides, for example.
  • motif (i) refers to an amino acid sequence comprising a sequence that is at least 90% identical to SEQ ID NO:78 (Motif 1a, Table 1).
  • motif (ii) refers to an amino acid sequence comprising a sequence that is at least 90% identical to SEQ ID NO:79 (Motif 2, Table 1).
  • motif (iii) refers to an amino acid sequence comprising a sequence that is at least 90% identical to SEQ ID NO:80 (Motif 3a, Table 1).
  • percent by volume percent by volume of a solute in a solution
  • percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)] ⁇ 100%.
  • Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.
  • polynucleotide may be a polymer of DNA or RNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • Nucleotides can be referred to by a single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate (for RNA or DNA, respectively), “G” for guanylate or deoxyguanylate (for RNA or DNA, respectively), “U” for uridylate (for RNA), “T” for deoxythymidylate (for DNA), “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, “W” for A or T, and “N” for any nucleotide (e.g., N can be A, C, T, or G, if referring to a DNA sequence; N can be A, C, U, or
  • RNA refers to a DNA polynucleotide sequence that expresses an RNA (RNA is transcribed from the DNA polynucleotide sequence) from a coding region, which RNA can be a messenger RNA (encoding a protein) or a non-protein-coding RNA.
  • a gene may refer to the coding region alone, or may include regulatory sequences upstream and/or downstream to the coding region (e.g., promoters, 5′-untranslated regions, 3′-transcription terminator regions).
  • a coding region encoding a protein can alternatively be referred to herein as an “open reading frame” (ORF).
  • a gene that is “native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; such a gene is located in its natural location in the genome of a host cell.
  • a “chimeric” gene refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature (i.e., the regulatory and coding regions are heterologous with each other). Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature.
  • a “foreign” or “heterologous” gene refers to a gene that is introduced into the host organism by gene transfer.
  • Foreign/heterologous genes can comprise native genes inserted into a non-native organism, native genes introduced into a new location within the native host, or chimeric genes.
  • Polynucleotide sequences in certain embodiments herein are heterologous.
  • a “transgene” is a gene that has been introduced into the genome by a gene delivery procedure (e.g., transformation).
  • a “codon-optimized” open reading frame has its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
  • a “non-native” amino acid sequence or polynucleotide sequence herein comprised in a cell or organism herein does not occur in a native (natural) counterpart of such cell or organism.
  • regulatory sequences refer to nucleotide sequences located upstream of a gene's transcription start site (e.g., promoter), 5′ untranslated regions, introns, and 3′ non-coding regions, and which may influence the transcription, processing or stability, and/or translation of an RNA transcribed from the gene. Regulatory sequences herein may include promoters, enhancers, silencers, 5′ untranslated leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures, and other elements involved in regulation of gene expression. One or more regulatory elements herein may be heterologous to a coding region herein.
  • a “promoter” as used herein refers to a DNA sequence capable of controlling the transcription of RNA from a gene.
  • a promoter sequence is upstream of the transcription start site of a gene. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. Promoters that cause a gene to be expressed in a cell at most times under all circumstances are commonly referred to as “constitutive promoters”. One or more promoters herein may be heterologous to a coding region herein.
  • a “strong promoter” as used herein refers to a promoter that can direct a relatively large number of productive initiations per unit time, and/or is a promoter driving a higher level of gene transcription than the average transcription level of the genes in a cell.
  • 3′ non-coding sequence refers to DNA sequences located downstream of a coding sequence. This includes polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression.
  • a first nucleic acid sequence is “hybridizable” to a second nucleic acid sequence when a single-stranded form of the first nucleic acid sequence can anneal to the second nucleic acid sequence under suitable annealing conditions (e.g., temperature, solution ionic strength).
  • suitable annealing conditions e.g., temperature, solution ionic strength.
  • DNA manipulation technique refers to any technique in which the sequence of a DNA polynucleotide sequence is modified. Although the DNA polynucleotide sequence being modified can be used as a substrate itself for modification, it does not have to be physically in hand for certain techniques (e.g., a sequence stored in a computer can be used as the basis for the manipulation technique). A DNA manipulation technique can be used to delete and/or mutate one or more DNA sequences in a longer sequence. Examples of a DNA manipulation technique include recombinant DNA techniques (restriction and ligation, molecular cloning), polymerase chain reaction (PCR), and synthetic DNA methods (e.g., oligonucleotide synthesis and ligation).
  • a DNA manipulation technique can entail observing a DNA polynucleotide in silico, determining desired modifications (e.g., one or more deletions) of the DNA polynucleotide, and synthesizing a DNA polynucleotide that contains the desired modifications.
  • in silico means in or on an information storage and/or processing device such as a computer; done or produced using computer software or simulation, i.e., virtual reality.
  • a cassette can refer to a promoter operably linked to a DNA sequence encoding a protein-coding RNA.
  • a cassette may optionally be operably linked to a 3′ non-coding sequence.
  • the structure of a cassette herein can optionally be represented by the simple notation system of “X::Y::Z”. Specifically, X describes a promoter, Y describes a coding sequence, and Z describes a terminator (optional); X is operably linked to Y, and Y is operably linked to Z.
  • upstream and downstream as used herein with respect to polynucleotides refer to “5′ of” and “3′ of”, respectively.
  • RNA e.g., mRNA or a non-protein-coding RNA
  • expression of a coding region of a polynucleotide sequence can be up-regulated or down-regulated in certain embodiments.
  • operably linked refers to the association of two or more nucleic acid sequences such that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence. That is, the coding sequence is under the transcriptional control of the promoter.
  • a coding sequence can be operably linked to one (e.g., promoter) or more (e.g., promoter and terminator) regulatory sequences, for example.
  • recombinant when used herein to characterize a DNA sequence such as a plasmid, vector, or construct refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis and/or by manipulation of isolated segments of nucleic acids by genetic engineering techniques. Methods for preparing recombinant constructs/vectors herein can follow standard recombinant DNA and molecular cloning techniques as described by J. Sambrook and D. Russell ( Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); T. J. Silhavy et al.
  • transformation refers to the transfer of a nucleic acid molecule into a host organism or host cell by any method.
  • a nucleic acid molecule that has been transformed into an organism/cell may be one that replicates autonomously in the organism/cell, or that integrates into the genome of the organism/cell, or that exists transiently in the cell without replicating or integrating.
  • Non-limiting examples of nucleic acid molecules suitable for transformation are disclosed herein, such as plasmids and linear DNA molecules.
  • Host organisms/cells herein containing a transforming nucleic acid sequence can be referred to as “transgenic”, “recombinant”, “transformed”, “engineered”, as a “transformant”, and/or as being “modified for exogenous gene expression”, for example.
  • sequence identity refers to the nucleic acid bases or amino acid residues in two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the results by 100 to yield the percentage of sequence identity. It would be understood that, when calculating sequence identity between a DNA sequence and an RNA sequence, T residues of the DNA sequence align with, and can be considered “identical” with, U residues of the RNA sequence.
  • first and second polynucleotides For purposes of determining “percent complementarity” of first and second polynucleotides, one can obtain this by determining (i) the percent identity between the first polynucleotide and the complement sequence of the second polynucleotide (or vice versa), for example, and/or (ii) the percentage of bases between the first and second polynucleotides that would create canonical Watson and Crick base pairs.
  • BLAST Basic Local Alignment Search Tool
  • NCBI National Center for Biotechnology Information
  • polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments. Variants of these sequences that are at least about 70-85%, 85-90%, or 90%-95% identical to the sequences disclosed herein can be used or referenced.
  • a variant amino acid sequence or polynucleotide sequence can have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a sequence disclosed herein.
  • the variant amino acid sequence or polynucleotide sequence has the same function/activity of the disclosed sequence, or at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.
  • Any polypeptide amino acid sequence disclosed herein not beginning with a methionine can typically further comprise at least a start-methionine at the N-terminus of the amino acid sequence.
  • amino acid residues at each amino acid position of the proteins disclosed herein are examples. Given that certain amino acids share similar structural and/or charge features with each other (i.e., conserved), the amino acid at each position of a protein herein can be as provided in the disclosed sequences or substituted with a conserved amino acid residue (“conservative amino acid substitution”) as follows:
  • isolated refers to a polynucleotide or polypeptide molecule that has been completely or partially purified from its native source.
  • the isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix.
  • the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. “Isolated” herein can also characterize embodiments that are synthetic/man-made, and/or have properties that are not naturally occurring.
  • the term “increased” as used herein can refer to a quantity or activity that is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 50%, 100%, or 200% more than the quantity or activity for which the increased quantity or activity is being compared.
  • the terms “increased”, “elevated”, “enhanced”, “greater than”, “improved” and the like are used interchangeably herein.
  • Glucosyltransferase enzymes that can synthesize high molecular weight, linear alpha-1,3-glucan polymer are sought after.
  • some embodiments disclosed herein concern a reaction solution comprising water, sucrose, and a glucosyltransferase enzyme, wherein the glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
  • the molecular weight of poly alpha-1,3-glucan produced by glucosyltransferase enzymes herein can be measured as DP w (weight average degree of polymerization) or DP n (number average degree of polymerization).
  • the molecular weight of poly alpha-1,3-glucan herein can be measured as number-average molecular weight (M n ) or as weight-average molecular weight (M w ).
  • M n number-average molecular weight
  • M w weight-average molecular weight
  • molecular weight can be measured in terms of Daltons or grams/mole.
  • Poly alpha-1,3-glucan in certain embodiments can have a molecular weight in DP w or DP n of at least about 100.
  • the molecular weight can be at least about 400 DP w or DP n .
  • DP w or DP n in still another embodiment can be at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 (or any integer between 100 and 1000).
  • the molecular weight of poly alpha-1,3-glucan can be measured using any of several means known in the art.
  • glucan polymer molecular weight can be measured using high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).
  • Poly alpha-1,3-glucan in certain embodiments has at least about 95%, 96%, 97%, 98%, 99%, or 100° A alpha-1,3 glycosidic linkages.
  • poly alpha-1,3-glucan has less than about 5%, 4%, 3%, 2%, 1° A, or 0% of glycosidic linkages that are not alpha-1,3. It should be understood that the higher the percentage of alpha-1,3-glycosidic linkages present in poly alpha-1,3-glucan, the greater the probability that the poly alpha-1,3-glucan is linear, since there are lower occurrences of certain glycosidic linkages forming branch points in the polymer.
  • poly alpha-1,3-glucan with 100% alpha-1,3 glycosidic linkages is completely linear.
  • poly alpha-1,3-glucan has no branch points or less than about 5%, 4%, 3%, 2%, or 1° A branch points as a percent of the glycosidic linkages in the polymer.
  • branch points include alpha-1,6 branch points.
  • the glycosidic linkage profile of poly alpha-1,3-glucan herein can be determined using any method known in the art.
  • the linkage profile can be determined using methods that use nuclear magnetic resonance (NMR) spectroscopy (e.g., 13 C NMR or 1 H NMR).
  • NMR nuclear magnetic resonance
  • 13 C NMR 13 C NMR or 1 H NMR
  • Poly alpha-1,3-glucan produced by a glucosyltransferase herein is typically insoluble in most aqueous systems.
  • the solubility of a glucan polymer in an aqueous systems is related to its linkage type, molecular weight and/or degree of branching.
  • Poly alpha-1,3-glucan is generally insoluble at a DP w of 8 and above in aqueous (or mostly aqueous) liquids at 20° C.
  • a glucosyltransferase enzyme herein can produce poly alpha-1,3-glucan as presently disclosed.
  • a glucosyltransferase enzyme in certain embodiments further comprises a glucosyltransferase catalytic domain comprising an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65, and have glucosyltransferase activity.
  • a glucosyltransferase catalytic domain can comprise an amino acid sequence that is, for example, at least 91° A, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% (but not 100° A) identical to amino acid positions 54-957 of SEQ ID NO:65, and have glucosyltransferase activity.
  • SEQ ID NOs:65 (GTF 7527), 30 (GTF 2678), 4 (GTF 6855), 28 (GTF 2919), and 20 (GTF 2765) each represent a glucosyltransferase that, compared to its respective wild type counterpart, lacks the signal peptide domain and all or a substantial portion of the variable domain.
  • each of these glucosyltransferase enzymes has a catalytic domain followed by a glucan-binding domain.
  • catalytic domain sequences in these enzymes is as follows: 7527 (residues 54-957 of SEQ ID NO:65), 2678 (residues 55-960 of SEQ ID NO:30), 6855 (residues 55-960 of SEQ ID NO:4), 2919 (residues 55-960 of SEQ ID NO:28), 2765 (residues 55-960 of SEQ ID NO:20).
  • the amino acid sequences of catalytic domains of GTFs 2678, 6855, 2919 and 2765 have about 94.9%, 99.0%, 95.5% and 96.4% identity, respectively, with a catalytic domain sequence of 7527 (i.e., amino acids 54-957 of SEQ ID NO:65) (Table 4).
  • These particular glucosyltransferase enzymes can produce poly alpha-1,3-glucan with 100° A alpha-1,3 linkages and a DP w of at least 400 (Table 4).
  • a glucosyltransferase enzyme in certain embodiments can comprise, or consist of, a glucosyltransferase catalytic domain that is at least 90%, 91° A, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% (but not 100° A) identical to the amino acid sequence of a catalytic domain of GTF 2678, 6855, 2919, or 2765.
  • a glucosyltransferase catalytic domain sequence does not comprise residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:4, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
  • Amino acid positions 54-957 of SEQ ID NO:65 represent, approximately, a catalytic domain sequence of the glucosyltransferase identified in GENBANK under GI number 47527 (SEQ ID NO:60).
  • SEQ ID NO:65 generally represents the catalytic domain and glucan-binding domain of SEQ ID NO:60; the signal peptide and variable domains are missing from SEQ ID NO:65.
  • a catalytic domain sequence of SEQ ID NO:65 was able to catalyze the production of poly alpha-1,3-glucan.
  • Example 14 also shows that a catalytic domain sequence of SEQ ID NO:14 (residues 57-906 of SEQ ID NO:14 [GTF 5926]) was able to catalyze production of poly alpha-1,3-glucan.
  • the molecular weight of poly alpha-1,3-glucan produced by each of these catalytic domain sequences generally corresponded with the molecular weight of the product produced by their enzyme counterparts containing both the catalytic domain and glucan binding domain (refer to activity of SEQ ID NOs:65 and 14 in Table 4, DP w 150).
  • a catalytic domain sequence herein is an important structural component for a glucosyltransferase enzyme to be capable of producing poly alpha-1,3-glucan.
  • a glucosyltransferase enzyme herein need only have a catalytic domain sequence, such as one comprising an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65 (or positions 55-960 of SEQ ID NO:30, positions 55-960 of SEQ ID NO:4, positions 55-960 of SEQ ID NO:28, or positions 55-960 of SEQ ID NO:20), the glucosyltransferase enzyme can be comprised within a larger amino acid sequence.
  • the catalytic domain may be linked at its C-terminus to a glucan-binding domain, and/or linked at its N-terminus to a variable domain and/or signal peptide.
  • glucosyltransferase enzymes can be any as disclosed herein and that include 1-300 (or any integer there between [e.g., 10, 15, 20, 25, 30, 35, 40, 45, or 50]) residues on the N-terminus and/or C-terminus. Such additional residues may be from a corresponding wild type sequence from which the glucosyltransferase enzyme is derived, or may be a heterologous sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N-terminus), for example.
  • a glucosyltransferase enzyme herein typically lacks an N-terminal signal peptide.
  • An expression system for producing a glucosyltransferase enzyme herein may employ an enzyme-encoding polynucleotide that further comprises sequence encoding an N-terminal signal peptide to direct extra-cellular secretion, if desired.
  • the signal peptide in such embodiments is cleaved from the enzyme during the secretion process.
  • the signal peptide may either be native or heterologous to the glucosyltransferase.
  • An example of a signal peptide useful herein is one from a bacterial (e.g., a Bacillus species such as B. subtilis ) or fungal species.
  • a bacterial signal peptide is an aprE signal peptide, such as one from Bacillus (e.g., B. subtilis , see Vogtentanz et al., Protein Expr. Purif. 55:40-52, which is incorporated herein by reference).
  • Bacillus e.g., B. subtilis , see Vogtentanz et al., Protein Expr. Purif. 55:40-52, which is incorporated herein by reference.
  • FIGS. 2A-O show that a catalytic domain sequence of GTF 7527 (residues 54-957 of SEQ ID NO:65) aligns with catalytic domain sequences of several other glucosyltransferase enzymes, with several regions showing complete conservation across all the sequences (residues with dark background).
  • the dark background residues in FIGS. 2A-O visually map out the catalytic domain of each sequence, indicating their length to be about 850 to 900 amino acid residues long.
  • the catalytic domain of the glucosyltransferase enzyme can be about 800-950 (or any integer between 800 and 950) amino acid residues long, for example.
  • FIGS. 2A-O Certain of the conserved regions in FIGS. 2A-O include catalytic active site motifs SEQ ID NOs:68, 69, 70, and 71 (refer to Example 3).
  • a catalytic domain sequence of a glucosyltransferase enzyme in some aspects can contain one or more of SEQ ID NOs:68, 69, 70, and 71 in alignment, respectively, with SEQ ID NOs:68, 69, 70, and 71 as present in amino acids 54-957 of SEQ ID NO:65.
  • Other conserved regions in FIGS. 2A-O include SEQ ID NOs:72, 73, 74, 75, 76 and 77 (refer to Example 4).
  • a catalytic domain sequence of a glucosyltransferase enzyme in some aspects can contain one or more of SEQ ID NOs:72, 73, 74, 75, 76 and 77 in alignment, respectively, with SEQ ID NOs:72, 73, 74, 75, 76 and 77 as present in amino acids 54-957 of SEQ ID NO:65.
  • the catalytic domain of a glucosyltransferase enzyme herein can have activity as exhibited by a catalytic domain of a glucosyltransferase classified under the glycoside hydrolase family 70 (GH70).
  • GH70 glycoside hydrolase family 70
  • Such a GH70 glucosyltransferase may be found in the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009), for example.
  • a glucosyltransferase enzyme herein can comprise a glucosyltransferase catalytic domain comprising the following three motifs:
  • Motif (i) corresponds with “Motif 1a” ( FIG. 3 ). Motif (ii) corresponds with “Motif 2” ( FIG. 5 ). Motif (iii) corresponds with “Motif 3a” ( FIG. 7 ).
  • Motif (i) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:78. Motif (ii) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:79. Motif (iii) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:80.
  • motif (i) can comprise SEQ ID NO:78
  • motif (ii) can comprise SEQ ID NO:79
  • motif (iii) can comprise SEQ ID NO:80.
  • the first residue of SEQ ID NO:78 can be an aspartate (D) and the fourth residue can be an isoleucine (I).
  • the first residue can be an aspartate (D) and the fourth residue can be a valine (V), or the first residue can be an asparagine (N) and the fourth residue can be an isoleucine (I), or the first residue can be an asparagine (N) and the fourth residue can be a valine (V).
  • the sixth residue of SEQ ID NO:79 can be a lysine (K), the fourteenth residue can be a valine (V), and the sixteenth residue can be a glutamine (Q).
  • the sixth residue can be a lysine (K), the fourteenth residue can be an alanine (A), and the sixteenth residue can be a glutamine (Q); or the sixth residue can be a lysine (K), the fourteenth residue can be an valine (V), and the sixteenth residue can be a lysine (K). Additional examples include where the sixth residue can be a threonine (T).
  • the ninth residue of SEQ ID NO:80 can be an asparagine (N) and the eighteenth residue can be an asparagine (N).
  • the ninth residue can be an asparagine (N) and the eighteenth residue can be a serine (S), or the ninth residue can be a lysine (K) and the eighteenth residue can be an asparagine (N), or the ninth residue can be a lysine (K) and the eighteenth residue can be a serine (S).
  • motif (i) (SEQ ID NO:78), motif (ii) (SEQ ID NO:79) and motif (iii) (SEQ ID NO:80) align with residues 231-243, 396-425 and 549-567, respectively, of the GTF 7527 sequence (SEQ ID NO:65) shown in FIGS. 2A-O .
  • motif (i) (SEQ ID NO:78), motif (ii) (SEQ ID NO:79) and motif (iii) (SEQ ID NO:80) align with residues 231-243, 396-425 and 549-567, respectively, of the GTF 7527 sequence (SEQ ID NO:65) shown in FIGS. 2A-O .
  • motifs (i), (ii) and (iii) in a glucosyltransferase catalytic domain can be determined with reference to certain conserved sequences, namely SEQ ID NOs:72, 73, 74, 75, 76 and 77, if desired.
  • Motif 1a (SEQ ID NO:78) is flanked by upstream and downstream conserved sequences as shown in FIG. 3 .
  • Preceding Motif 1a is the sequence SxxRxxN (SEQ ID NO:72), and following this motif is the sequence GGxxxLLxNDxDxSNPxVQAExLN (SEQ ID NO:73).
  • the position of motif (i) can be located between SEQ ID NOs:72 and 73.
  • SEQ ID NO:72 can be directly adjacent (upstream) to motif (i), or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or 1-15) amino acid residues upstream motif (i).
  • SEQ ID NO:73 can be directly adjacent (downstream) to motif (i), or 1, 2, 3, 4, or 5 (or 1-5) amino acid residues downstream motif (i).
  • Motif 2 (SEQ ID NO:79) is flanked by upstream and downstream conserved sequences as shown in FIG. 5 . Specifically, preceding Motif 2 is the sequence WxxxDxxY (SEQ ID NO:74), and following this motif is the sequence YxFxRAHD (SEQ ID NO:75). Thus, the position of motif (ii) can be located between SEQ ID NOs:74 and 75. SEQ ID NO:74 can be directly adjacent (upstream) to motif (ii), or 1-65 (or any integer between 1 and 65) amino acid residues upstream motif (ii). SEQ ID NO:75 can be directly adjacent (downstream) to motif (ii), or 1, 2, 3, 4, or 5 (or 1-5) amino acid residues downstream motif (ii).
  • Motif 3a (SEQ ID NO:80) is flanked by upstream and downstream conserved sequences as shown in FIG. 7 . Specifically, preceding Motif 3a is the sequence YxxGGQ (SEQ ID NO:76), and following this motif is the sequence VRxG (SEQ ID NO:77). Thus, the position of motif (iii) can be located between SEQ ID NOs:76 and 77. SEQ ID NO:76 can be directly adjacent (upstream) to motif (iii), or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (or 1-11) amino acid residues upstream motif (iii). SEQ ID NO:77 can be directly adjacent (downstream) to motif (iii), or 1, 2, 3, 4, 5, 6, 7, 8, or 9 (or 1-9) amino acid residues downstream motif (iii).
  • SEQ ID NOs:72-77 can be any amino acid (indicated by an “x” in each sequence in Table 1).
  • SEQ ID NOs:72 and 73 are as shown in any of the GTF sequences in FIGS. 2 and 3 at the amino acids of each GTF sequence aligning with positions 214-220 and 245-268, respectively, of SEQ ID NO:65 (GTF 7527).
  • SEQ ID NOs:74 and 75 are as shown in any of the GTF sequences in FIGS. 2 and 5 at the amino acids of each GTF sequence aligning with positions 334-341 and 428-435, respectively, of SEQ ID NO:65 (GTF 7527).
  • SEQ ID NOs:76 and 77 are as shown in any of the GTF sequences in FIGS. 2 and 7 at the amino acids of each GTF sequence aligning with positions 537-542 and 572-575, respectively, of SEQ ID NO:65 (GTF 7527).
  • a glucosyltransferase enzyme herein can be derived from any microbial source, such as a bacteria or fungus.
  • bacterial glucosyltransferase enzymes are those derived from a Streptococcus species, Leuconostoc species or Lactobacillus species.
  • Streptococcus species include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S. sanguinis .
  • Examples of Leuconostoc species include L. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L.
  • Lactobacillus species include L. acidophilus, L. delbrueckii, L. helveticus, L. salivarius, L. casei, L. curvatus, L. plantarum, L. sakei, L. brevis, L. buchneri, L. fermentum and L. reuteri.
  • a glucosyltransferase enzyme in some aspects does not comprise SEQ ID NO:4, SEQ ID NO:20, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:65.
  • a glucosyltransferase enzyme herein does not comprise positions 2-1341 of SEQ ID NO:4, positions 2-1340 of SEQ ID NO:20, positions 2-1340 of SEQ ID NO:28, positions 2-1341 of SEQ ID NO:30, or positions 2-1341 of SEQ ID NO:65.
  • a glucosyltransferase enzyme herein can produce poly alpha-1,3-glucan as presently disclosed, such as is the above disclosure.
  • glucosyltransferase enzymes may be used in certain aspects.
  • the glucosyltransferase enzyme in certain embodiments does not have, or has very little (less than 1%), dextransucrase, reuteransucrase, alternansucrase activity, or mutansucrase activity.
  • a reaction solution herein may contain one, two, or more glucosyltransferase enzymes, for example.
  • a glucosyltransferase enzyme for a glucan synthesis reaction herein may be produced by any means known in the art.
  • a glucosyltransferase enzyme may be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system.
  • heterologous expression systems include bacterial (e.g., E. coli such as TOP10 or MG1655; Bacillus sp.) and eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) expression systems.
  • a heterologous gene expression system may be one that is designed for protein secretion.
  • a glucosyltransferase enzyme typically comprises a signal peptide (signal sequence) in such embodiments.
  • the signal peptide may be either its native signal peptide or a heterologous signal peptide.
  • a glucosyltransferase enzyme described herein may be used in any purification state (e.g., pure or non-pure).
  • a glucosyltransferase enzyme may be purified and/or isolated prior to its use.
  • glucosyltransferase enzymes that are non-pure include those in the form of a cell lysate.
  • a cell lysate or extract may be prepared from a bacteria (e.g., E. coli ) used to heterologously express the enzyme.
  • the bacteria may be subjected to disruption using a French pressure cell.
  • bacteria may be homogenized with a homogenizer (e.g., APV, Rannie, Gaulin).
  • a glucosyltransferase enzyme is typically soluble in these types of preparations.
  • a bacterial cell lysate, extract, or homogenate herein may be used at about 0.15-0.3% (v/v), for example, in a reaction solution for producing poly alpha-1,3-glucan from sucrose.
  • glucosyltransferase enzyme activity can be determined using any method known in the art.
  • glucosyltransferase enzyme activity can be determined by measuring the production of reducing sugars (fructose and glucose) in a reaction solution containing sucrose (50 g/L), dextran T10 (1 mg/mL) and potassium phosphate buffer (pH 6.5, 50 mM), where the solution is held at 22-25° C. for 24-30 hours.
  • the reducing sugars can be measured, for instance, by adding 0.01 mL of the reaction solution to a mixture containing 1 N NaOH and 0.1% triphenyltetrazolium chloride and then monitoring the increase in absorbance at OD 480nm for five minutes.
  • a reaction solution herein refers to a solution comprising at least sucrose, water and an active glucosyltransferase enzyme, and optionally other components.
  • Other components that can be in a glucan synthesis reaction include fructose, glucose, leucrose, soluble oligosaccharides (e.g., DP2-DP7), for example.
  • DP2-DP7 soluble oligosaccharides
  • certain glucan products such as poly alpha-1,3-glucan with a DP of at least 8 or 9 may be water-insoluble and thus are not dissolved in a glucan synthesis reaction, but rather may be present out of solution.
  • a reaction solution herein may be one that, in addition to producing insoluble glucan product, produces byproducts such as leucrose and/or soluble oligosaccharides.
  • the temperature of a reaction solution herein can be controlled, if desired.
  • the temperature of the reaction is between about 5° C. to about 50° C.
  • the temperature in certain other embodiments is between about 20° C. to about 40° C., or about 20° C. to about 30° C. (e.g., about 25° C.).
  • the initial concentration of sucrose in a reaction solution herein can be about 20 g/L to about 400 g/L, for example.
  • the initial concentration of sucrose can be about 75 g/L to about 175 g/L, or from about 50 g/L to about 150 g/L.
  • the initial concentration of sucrose can be about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 g/L (or any integer value between 40 and 160 g/L), for example.
  • “Initial concentration of sucrose” refers to the sucrose concentration in a GTF reaction solution just after all the reaction solution components have been added (e.g., at least water, sucrose, GTF enzyme).
  • sucrose used in a glucan synthesis reaction herein can be highly pure 99.5%) or be of any other purity or grade.
  • sucrose can have a purity of at least 99.0%, or can be reagent grade sucrose.
  • incompletely refined sucrose can be used. Incompletely refined sucrose herein refers to sucrose that has not been processed to white refined sucrose. Thus, incompletely refined sucrose can be completely unrefined or partially refined. Examples of unrefined sucrose are “raw sucrose” (“raw sugar”) and solutions thereof. Examples of partially refined sucrose have not gone through one, two, three, or more crystallization steps.
  • sucrose herein may be derived from any renewable sugar source such as sugar cane, sugar beets, cassava, sweet sorghum, or corn.
  • Suitable forms of sucrose useful herein are crystalline form or non-crystalline form (e.g., syrup, cane juice, beet juice), for example.
  • ICUMSA International Commission for Uniform Methods of Sugar Analysis
  • ICUMSA Methods of Sugar Analysis Official and Tentative Methods Recommended by the International Commission for Uniform Methods of Sugar Analysis (ICUMSA) (Ed. H. C. S. de Whalley, Elsevier Pub. Co., 1964), for example, which is incorporated herein by reference.
  • ICUMSA can be measured, for example, by ICUMSA Method GS1/3-7 as described by R. J. McCowage, R. M. Urquhart and M. L. Burge ( Determination of the Solution Colour of Raw Sugars, Brown Sugars and Coloured Syrups at pH 7.0 —Official , Verlag Dr Albert Bartens, 2011 revision), which is incorporated herein by reference.
  • the pH of a glucan synthesis reaction in certain embodiments can be between about 4.0 to about 8.0. Alternatively, the pH can be about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, or 8.0.
  • the pH can be adjusted or controlled by the addition or incorporation of a suitable buffer, including but not limited to: phosphate, tris, citrate, or a combination thereof.
  • Buffer concentration in a glucan synthesis reaction can be from 0 mM to about 100 mM, or about 10, 20, or 50 mM, for example.
  • the present disclosure also concerns a method for producing insoluble poly alpha-1,3-glucan comprising:
  • glucosyltransferase enzyme comprises a catalytic domain comprising the following three motifs:
  • a glucan synthesis method as presently disclosed comprises contacting at least water, sucrose, and a glucosyltransferase enzyme as described herein. These and optionally other reagents can be added altogether or added in any order as discussed below.
  • This step can comprise providing a reaction solution comprising water, sucrose and a glucosyltransferase enzyme. It will be understood that, as the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan, the reaction solution becomes a reaction mixture given that insoluble poly alpha-1,3-glucan falls out of solution as indicated by clouding of the reaction.
  • the contacting step of the disclosed method can be performed in any number of ways.
  • sucrose can first be dissolved in water (optionally, other components may also be added at this stage of preparation, such as buffer components), followed by the addition of glucosyltransferase enzyme.
  • the solution may be kept still, or agitated via stirring or orbital shaking, for example.
  • a glucan synthesis reaction is cell-free.
  • Completion of a reaction in certain embodiments can be determined visually (no more accumulation of insoluble poly alpha-1,3-glucan) and/or by measuring the amount of sucrose left in the solution (residual sucrose), where a percent sucrose consumption of over about 90% can indicate reaction completion, for example.
  • a reaction of the disclosed process will take about 12, 24, 36, 48, 60, 72, 84, or 96 hours to complete, depending on certain parameters such as the amount of sucrose and glucosyltransferase enzyme used in the reaction.
  • the percent sucrose consumption of a reaction in certain embodiments is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sucrose initially contacted with water and a glucosyltransferase enzyme.
  • the percent sucrose consumption may be >90% or >95%.
  • the yield of poly alpha-1,3-glucan produced in some aspects of a glucan synthesis method herein can be at least about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, based on the weight of sucrose converted in the reaction.
  • Poly alpha-1,3-glucan produced in the disclosed method may optionally be isolated.
  • insoluble poly alpha-1,3-glucan may be separated by centrifugation or filtration.
  • poly alpha-1,3-glucan is separated from most of the reaction solution, which may comprise water, fructose and certain byproducts (e.g., leucrose, soluble oligosaccharides DP2-DP7).
  • This solution may also comprise residual sucrose and glucose monomer.
  • Isolation can optionally further comprise washing the poly alpha-1,3-glucan one, two, or more times with water or other aqueous liquid, and/or drying the poly alpha-1,3-glucan.
  • a glucosyltransferase enzyme in certain embodiments of a glucan synthesis method herein can further comprise a glucosyltransferase catalytic domain comprising an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65, and have glucosyltransferase activity.
  • a glucosyltransferase catalytic domain can comprise an amino acid sequence that is, for example, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% (but not 100° A) identical to amino acid positions 54-957 of SEQ ID NO:65, and have glucosyltransferase activity.
  • poly alpha-1,3-glucan synthesis methods are examples. Any other feature disclosed herein can apply to a glucan synthesis method, accordingly.
  • any of the poly alpha-1,3-glucan product, glucosyltransferase enzyme (e.g., the catalytic domain and its motifs i, ii and iii), and reaction solution condition features disclosed herein can be applied as appropriate.
  • the present disclosure also concerns a method of identifying a glucosyltransferase enzyme.
  • This method comprises detecting the presence at least one motif in a glucosyltransferase catalytic domain, the at least one motif selected from the group consisting of:
  • a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:78 (i) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:78, (ii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:79, and (iii) a motif comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:80; thereby identifying a glucosyltransferase enzyme that produces insoluble poly alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages and a weight average degree of polymerization (DP w ) of at least 100. Since the poly alpha-1,3-glucan produced in this a method is mostly or completely linear, this method can optionally be characterized as a method of identifying a glucosyltransferase enzyme that produces linear poly alpha-1,3-glucan.
  • a GTF identification method herein can optionally comprise detecting one of, two of, or all three, of motifs (i), (ii) and/or (iii) in a glucosyltransferase catalytic domain.
  • the detection step in a GTF identification method herein can comprise detecting an isolated amino acid sequence of a glucosyltransferase enzyme having motifs (i), (ii), or (iii).
  • the detecting step can also be performed by detecting an isolated polynucleotide sequence encoding a glucosyltransferase enzyme having motifs (i), (ii), or (iii).
  • the codons used to prepare the isolated polynucleotide sequence in such embodiments optionally are preferred codons for a species (e.g., E. coli or S. cerevisiae ) that may be used to heterologously express the glucosyltransferase enzyme.
  • motifs (i), (ii), or (iii) in the catalytic domain of a glucosyltransferase enzyme can be detected following any means known in the art and/or any procedure described herein. For example, detection can be performed (a) in silico, (b) with a method comprising a nucleic acid hybridization step, (c) with a method comprising a protein sequencing step, and/or (d) with a method comprising a protein binding step.
  • Motifs (i), (ii) and (iii) were identified by in silico detection (see Example 4 below).
  • the amino acid sequences of glucosyltransferase enzymes (and/or nucleotide sequences encoding such glucosyltransferase enzymes) stored in a computer or database e.g., public databases such as GENBANK, EMBL, REFSEQ, GENEPEPT, SWISS-PROT, PIR, PDB
  • a computer or database e.g., public databases such as GENBANK, EMBL, REFSEQ, GENEPEPT, SWISS-PROT, PIR, PDB
  • Such review could comprise using any means known in the art such as through use of an alignment algorithm or software as described above (e.g., BLASTN, BLASTP, ClustalW, ClustalV, EMBOSS).
  • the sequence of the glucosyltransferase catalytic domain being reviewed could be aligned with a catalytic domain sequence of SEQ ID NO:65 (GTF 7527), which comprises Motifs 1a (SEQ ID NO:78), 2 (SEQ ID NO:79) and 3a (SEQ ID NO:80), to detect the presence or absence of motifs (i), (ii), and/or (iii).
  • sequence of the glucosyltransferase catalytic domain being reviewed could be aligned with a catalytic domain sequence of SEQ ID NO:30 (GTF 2678), SEQ ID NO:4 (GTF 6855), SEQ ID NO:28 (GTF 2919), and/or SEQ ID NO:20 (GTF 2765), all of which comprise Motifs 1a (SEQ ID NO:78), 2 (SEQ ID NO:79) and 3a (SEQ ID NO:80), to identify the presence or absence of motifs (i), (ii), and/or (iii).
  • Another in silico means for detecting motifs (i), (ii), and/or (iii) in a glucosyltransferase catalytic domain sequence can comprise comparing the predicted three-dimensional structure (tertiary structure) of a glucosyltransferase catalytic domain sequence with a reference structure.
  • the structures of both the catalytic domain being reviewed and the reference can be visually compared using any means known in the art such as with a computer program that provides a structure based on amino acid sequence input (e.g., software package MOE, Chemical Computing Group, Montreal, Canada).
  • the comparison may detect the presence of motif (i), (ii), and/or (iii) by showing a domain(s) in the structure being reviewed that does not have a corresponding domain in the reference structure. Examples of this type of comparison are shown in FIGS. 4 a , 4 b , 6 a , 6 b , 8 a and 8 b.
  • detecting a glucosyltransferase enzyme having motifs (i), (ii), and (iii) in its catalytic domain can be through using a method comprising a nucleic acid hybridization step.
  • a method can comprise using DNA hybridization (e.g., Southern blot, dot blot), RNA hybridization (e.g., northern blot), or any other method that has a nucleic acid hybridization step (e.g., DNA sequencing, PCR, RT-PCR, all of which may comprise hybridization of an oligonucleotide), for example.
  • an oligonucleotide that would hybridize to a nucleotide sequence encoding Motif 1a (SEQ ID NO:78), 2 (SEQ ID NO:79), or 3a (SEQ ID NO:80) could be used to detect its presence or absence in a polynucleotide sequence encoding the glucosyltransferase catalytic domain being reviewed.
  • the conditions and parameters for carrying out hybridization methods in general are well known and disclosed, for example, in Sambrook J, Fritsch E F and Maniatis T, Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory: Cold Spring Harbor, N.Y.
  • a glucosyltransferase enzyme that comprises motifs (i), (ii), and (iii) in its catalytic domain can be detected using a method comprising a protein sequencing step.
  • a protein sequencing step can comprise one or more procedures such as N-terminal amino acid analysis, C-terminal amino acid analysis, Edman degradation, or mass spectrometry, for example.
  • a glucosyltransferase enzyme that comprises motifs (i), (ii), and (iii) in its catalytic domain can be detected using a method comprising a protein binding step.
  • a protein binding step could be performed using an antibody that specifically binds to one of these motifs, for example.
  • Antibodies for identifying the presence or absence of motif (i) can be specific for an amino acid sequence that is at least 90% identical to SEQ ID NO:78.
  • Antibodies for identifying the presence or absence of motif (ii) can be specific for an amino acid sequence that is at least 90% identical to SEQ ID NO:79.
  • Antibodies for identifying the presence or absence of motif (iii) can be specific for an amino acid sequence that is at least 90% identical to SEQ ID NO:80.
  • Motif (i) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:78. Motif (ii) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:79. Motif (iii) can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO:80.
  • motif (i) can comprise SEQ ID NO:78
  • motif (ii) can comprise SEQ ID NO:79
  • motif (iii) can comprise SEQ ID NO:80.
  • the first residue of SEQ ID NO:78 can be an aspartate (D) and the fourth residue can be an isoleucine (I).
  • the first residue can be an aspartate (D) and the fourth residue can be a valine (V), or the first residue can be an asparagine (N) and the fourth residue can be an isoleucine (I), or the first residue can be an asparagine (N) and the fourth residue can be a valine (V).
  • the sixth residue of SEQ ID NO:79 can be a lysine (K), the fourteenth residue can be a valine (V), and the sixteenth residue can be a glutamine (Q).
  • the sixth residue can be a lysine (K), the fourteenth residue can be an alanine (A), and the sixteenth residue can be a glutamine (Q); or the sixth residue can be a lysine (K), the fourteenth residue can be an valine (V), and the sixteenth residue can be a lysine (K). Additional examples include where the sixth residue can be a threonine (T).
  • the ninth residue of SEQ ID NO:80 can be an asparagine (N) and the eighteenth residue can be an asparagine (N).
  • the ninth residue can be an asparagine (N) and the eighteenth residue can be a serine (S), or the ninth residue can be a lysine (K) and the eighteenth residue can be an asparagine (N), or the ninth residue can be a lysine (K) and the eighteenth residue can be a serine (S).
  • motifs (i), (ii) and (iii) in a glucosyltransferase enzyme catalytic domain sequence can be used appropriately to detect one or more of these motifs.
  • the relative positions of motifs (i) (SEQ ID NO:78), (ii) (SEQ ID NO:79) and (iii) (SEQ ID NO:80) align with residues 231-243, 396-425 and 549-567, respectively, of the GTF 7527 sequence (SEQ ID NO:65) shown in FIGS. 2A-O .
  • the relative position(s) of the amino acid sequence(s) detected in the glucosyltransferase catalytic domain can thus be determined with reference to the above amino acid positions in SEQ ID N0:65.
  • the sequence of a glucosyltransferase catalytic domain can be aligned with SEQ ID N0:65 using any means known in the art and/or as described above.
  • motif (i), (ii), and/or (iii) can be detected based on proximity to certain conserved sequences, namely SEQ ID NOs:72, 73, 74, 75, 76 and 77, as described above.
  • An identification method in some aspects can further comprise detecting a glucosyltransferase catalytic domain as presently disclosed.
  • a glucosyltransferase catalytic domain can be detected that comprises an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65, positions 55-960 of SEQ ID NO:30, positions 55-960 of SEQ ID NO:4, positions 55-960 of SEQ ID NO:28, and/or positions 55-960 of SEQ ID NO:20.
  • a glucosyltransferase catalytic domain can be detected that comprises an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, or 99.5% (but not 100%) identical to any of the foregoing sequences.
  • an identification method does not detect a glucosyltransferase catalytic domain sequence comprising residues 54-957 of SEQ ID NO:65, residues 55-960 of SEQ ID NO:30, residues 55-960 of SEQ ID NO:4, residues 55-960 of SEQ ID NO:28, or residues 55-960 of SEQ ID NO:20.
  • FIGS. 2A-O Certain of the conserved regions in FIGS. 2A-O include catalytic active site motifs SEQ ID NOs:68, 69, 70, and 71 (refer to Example 3).
  • a catalytic domain sequence of a glucosyltransferase enzyme in some aspects can be identified based on having one or more of SEQ ID NOs:68, 69, 70, and 71 in alignment, respectively, with SEQ ID NOs:68, 69, 70, and 71 as present in amino acids 54-957 of SEQ ID NO:65.
  • Other conserved regions in FIGS. 2A-O include SEQ ID NOs:72, 73, 74, 75, 76 and 77 (refer to Example 4).
  • a catalytic domain sequence of a glucosyltransferase enzyme in some aspects can be identified based on having one or more of SEQ ID NOs:72, 73, 74, 75, 76 and 77 in alignment, respectively, with SEQ ID NOs:72, 73, 74, 75, 76 and 77 as present in amino acids 54-957 of SEQ ID NO:65.
  • a glucosyltransferase enzyme herein need only have a catalytic domain sequence, such as one comprising an amino acid sequence that is at least 90% identical to amino acid positions 54-957 of SEQ ID NO:65 (or positions 55-960 of SEQ ID NO:30, positions 55-960 of SEQ ID NO:4, positions 55-960 of SEQ ID NO:28, or positions 55-960 of SEQ ID NO:20), a glucosyltransferase enzyme identified in a method herein is typically comprised within a larger amino acid sequence.
  • the catalytic domain may be linked at its C-terminus to a glucan-binding domain, and/or linked at its N-terminus to a variable domain and/or signal peptide.
  • the catalytic domain of a glucosyltransferase enzyme identified herein can have activity as exhibited by a catalytic domain of a glucosyltransferase classified under the glycoside hydrolase family 70 (GH70).
  • GH70 glycoside hydrolase family 70
  • Such a GH70 glucosyltransferase may be found in the CAZy (Carbohydrate-Active EnZymes) database (Cantarel et al., Nucleic Acids Res. 37:D233-238, 2009), for example.
  • a glucosyltransferase enzyme identified herein can synthesize insoluble poly alpha-1,3-glucan having at least 95% alpha-1,3 glycosidic linkages and DP w of at least 100.
  • an identified GTF enzyme can synthesize poly alpha-1,3-glucan in which at least about 95%, 96%, 97%, 98%, 99%, or 100% of the constituent glycosidic linkages are alpha-1,3 linkages.
  • the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan in which there is less than about 5%, 4%, 3%, 2%, 1%, or 0% of glycosidic linkages that are not alpha-1,3.
  • a glucosyltransferase enzyme identified herein can synthesize poly alpha-1,3-glucan having no branch points or less than about 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer.
  • branch points include alpha-1,6 branch points.
  • a glucosyltransferase enzyme identified herein can synthesize poly alpha-1,3-glucan having a molecular weight in DP w or DP n of at least about 100.
  • the glucosyltransferase enzyme may synthesize poly alpha-1,3-glucan having a molecular weight in DP n or DP w of at least about 400.
  • the glucosyltransferase enzyme may synthesize poly alpha-1,3-glucan having a molecular weight in DP n or DP w of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 (or any integer between 100 and 1000).
  • a glucosyltransferase enzyme identified herein can be further analyzed, if desired. For example, if one or more of motifs i, ii, and/or iii is deleted and/or mutated (such that the one or more motifs are no longer at least 90% identical to SEQ ID NO:78, 79, or 80, respectively) from an identified glucosyltransferase (parent GTF), the modified glucosyltransferase (child GTF) can be expected to produce a branched alpha-glucan polymer.
  • a branched alpha-glucan polymer produced by a child GTF herein can have an intrinsic viscosity and/or branching index that is reduced by at least 30%, for example, compared to the intrinsic viscosity and/or branching index of poly alpha-1,3-glucan synthesized by the corresponding parent GTF.
  • the intrinsic viscosity and/or branching index of an alpha-glucan polymer can be measured by any means known in the art, or as provided in the below Examples.
  • a glucosyltransferase enzyme identified in a method as presently disclosed can optionally be produced. Such production can be by any means known in the art.
  • a glucosyltransferase enzyme can be produced recombinantly in a heterologous expression system, such as a microbial heterologous expression system (e.g., U.S. Pat. No. 7,000,000).
  • heterologous expression systems include bacterial (e.g., E. coli such as TOP10, Bacillus sp.) and eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) expression systems.
  • compositions and methods disclosed herein include:
  • g means gram(s), “h” means hour(s), “mL” means milliliter(s), “psi” means pound(s) per square inch, “wt %” means weight percentage, “ ⁇ m” means micrometer(s), “° C.” means degrees Celsius, “mg” means milligram(s), “mm” means millimeter(s), “ ⁇ L” means microliter(s), “mmol” means millimole(s), “min” means minute(s), “mol %” means mole percent, “M” means molar, “rpm” means revolutions per minute, “MPa” means megaPascals, “IV” means intrinsic viscosity, “g” means branching ratio.
  • the resulting supernatant was analyzed by the BCA (bicinchoninic acid) protein assay (Sigma-Aldrich) and SDS-PAGE to confirm expression of the GTF enzyme, and the supernatant was stored at ⁇ 20° C.
  • BCA bisinchoninic acid
  • GTF enzyme activity was confirmed by measuring the production of reducing sugars (fructose and glucose) in a GTF reaction solution.
  • a reaction solution was prepared by adding a GTF extract (prepared as above) to a mixture containing sucrose (50 or 150 g/L), potassium phosphate buffer (pH 6.5, 50 mM), and optionally dextran (1 mg/mL, dextran T10, Cat. No. D9260, Sigma-Aldrich); the GTF extract was added to 2.5%-5% by volume. The reaction solution was then incubated at 22-25° C. for 24-30 hours, after which it was centrifuged.
  • Glycosidic linkages in the glucan product synthesized by a GTF enzyme were determined by 13 C NMR (nuclear magnetic resonance). Dry glucan polymer (25-30 mg) was dissolved in 1 mL of deuterated dimethyl sulfoxide (DMSO) containing 3% by weight of LiCl with stirring at 50° C. Using a glass pipet, 0.8 mL of the solution was transferred into a 5-mm NMR tube. A quantitative 13 C NMR spectrum was acquired using a Bruker Avance 500-MHz NMR spectrometer (Billerica, Mass.) equipped with a CPDUL cryoprobe at a spectral frequency of 125.76 MHz, using a spectral window of 26041.7 Hz. An inverse gated decoupling pulse sequence using waltz decoupling was used with an acquisition time of 0.629 second, an inter-pulse delay of 5 seconds, and 6000 pulses. The time domain data was transformed using an exponential multiplication of 2.0 Hz.
  • the DP n of a glucan product synthesized by a GTF enzyme was determined by size-exclusion chromatography (SEC). Dry glucan polymer was dissolved at 5 mg/mL in N,N-dimethyl-acetamide (DMAc) and 5% LiCl with overnight shaking at 100° C.
  • SEC system used was an AllianceTM 2695 separation module from Waters Corporation (Milford, Mass.) coupled with three on-line detectors: a differential refractometer 2410 from Waters, a multiangle light scattering photometer HeleosTM 8+ from Wyatt Technologies (Santa Barbara, Calif.), and a differential capillary viscometer ViscoStarTM from Wyatt.
  • Multidetector size exclusion chromatography allowed measurement of molar mass distribution (MMD) using a combination of light scattering (LS) photometer and differential refractometer (DR). Molar mass (M) of the separated fractions across the polymer distribution was measured as a ratio of two detector responses:
  • GTF ID Nucleotide sequences encoding N-terminally truncated versions of GTF enzymes (Table 2, GTF ID) were synthesized using codons optimized for protein expression in E. coli .
  • the nucleic acid products (Table 2, nt SEQ ID NO) encoding the GTF enzymes (Table 2, AA SEQ ID NO) were subcloned into pJexpresss404® (DNA2.0, Menlo Park, Calif.) to generate GTF expression plasmids (Table 2, plasmid ID).
  • the GTF expression plasmids were used to transform E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.) to generate GTF expression strains (Table 2, strain ID). Production of GTF enzymes by bacterial expression and determination of enzymatic activities were performed as described in General Methods.
  • This Example describes using the GTF enzymes prepared in Example 1 to synthesize glucan polymer.
  • the following GTF enzymes produced glucan polymers comprising at least 50% alpha-1,3-linkages and having a DP n of at least 100: 6855 (SEQ ID NO:4), 7527 (SEQ ID NO:8), 1724 (SEQ ID NO:10), 0544 (SEQ ID NO:12), 5926 (SEQ ID NO:14), 2765 (SEQ ID NO:20), 0427 (SEQ ID NO:26), 2919 (SEQ ID NO:28), 2678 (SEQ ID NO:30), and 3929 (SEQ ID NO:34) (refer to Table 3).
  • the following GTF enzymes produced glucan polymers comprising 100% alpha-1,3-linkages, indicating linear polymers: 6855 (SEQ ID NO:4), 7527 (SEQ ID NO:8), 1724 (SEQ ID NO:10), 5926 (SEQ ID NO:14), 2765 (SEQ ID NO:20), 0427 (SEQ ID NO:26), 2919 (SEQ ID NO:28), 2678 (SEQ ID NO:30), and 3929 (SEQ ID NO:34). These results clearly indicate that not all GTF enzymes are capable of producing linear alpha-1,3-glucan polymer.
  • This Example describes aligning the amino acid sequences of several GTF enzymes to determine whether they share any structures.
  • GTF enzymes were evaluated in Example 2 for their ability to produce glucan polymers with a focus on those enzymes that produce glucan with 100% alpha-1,3-linkages.
  • the sequences of several of these enzymes were aligned with three dimensional structures that are formed by certain S. mutans and L. reuteri GTF sequences (3AIE [SEQ ID NO:66] and 3KLK [SEQ ID NO:67], respectively); the S. mutans and L. reuteri GTF sequences were aligned to superpose common tertiary structures using the software package MOE (Chemical Computing Group, Montreal, Canada).
  • FIGS. 2A-O show the alignment.
  • the sequences of the S. mutans and L. reuteri GTFs for which crystallographic structures are known were included in the alignment; S. mutans GTF is abbreviated as “3AIE” (SEQ ID NO:66) and L. reuteri GTF is abbreviated as “3KLK” (SEQ ID NO:67) in FIGS. 2A-O .
  • FIGS. 2A-O The alignment in FIGS. 2A-O indicates that all the aligned GTF sequences maintain numerous invariant regions (shown with dark background). These invariant sequences are located throughout the catalytic domain of each GTF (based on a homology model as opposed to an experimentally determined structure).
  • the catalytic domains in the aligned GTFs are about 900-950 amino acid residues long and begin after position 1 (artificial start methionine) in each of the sequences shown in FIGS. 2A-O .
  • the sequence following the catalytic domain in each GTF represents the glucan-binding domain.
  • the aligned GTF sequences share as little as 40% sequence identity with the sequences of the known GTF structures ( S. mutans 3AIE and L.
  • FIGS. 2A-O the alignment of these sequences in FIGS. 2A-O indicates a distributed pattern of conserved sequence motifs and patterns of specific residues that are conserved in all the aligned sequences (residues with dark background in FIGS. 2A-O ).
  • conserved sequence motifs can be related to important structural features such as the catalytic site described below and can serve as reference points to identify unique or characteristic features that may be associated with specific performance benefits.
  • the catalytic site residues may be found in sequence motifs repeated in all the aligned sequences ( FIGS. 2A-O ). Specifically, with reference to the sequence from GTF 7527 (SEQ ID NO:65) in FIGS. 2A-O , Arg292 and Asp294 are found in the motif FDxxRxDAxDNV (SEQ ID NO:68) corresponding to Arg475 and Asp477 of S. mutans 3AIE GTF and Arg1023 and Asp1025 of L. reuteri 3KLK GTF; Glu332 is found in the sequence motif ExWxxxDxxY (SEQ ID NO:69) corresponding to Glu515 in S. mutans 3AIE GTF and Glu1063 in L.
  • the tested GTF enzymes have catalytic domains comprising several highly conserved regions.
  • the GTF enzymes whose sequences were aligned in FIGS. 2A-O were further evaluated for their ability to produce glucan polymers with a focus on those enzymes that produce glucan with 100% alpha-1,3-linkages (Table 4).
  • DP w 50 and DP w 150 represent, respectively, the DP w of glucan produced by a GTF in a reaction solution having an initial sucrose concentration of 50 g/L or 150 g/L.
  • c SEQ ID NO: 65 is a shorter version of the 7527 GTF of SEQ ID NO: 8.
  • d Percent identity of respective GTF with SEQ ID NO: 65 (per EMBOSS alignment).
  • f Percent identity of catalytic domain region with amino acid residues 54-957 of SEQ ID NO: 65 (per EMBOSS alignment).
  • sequence motifs were found in the amino acid sequences of all five GTF enzymes that produce high molecular weight glucan with 100% alpha-1,3-linkages, and appear as three different “insertions” situated around the catalytic domain of the known GTF structures. Briefly, these sequence motifs are designated as:
  • Motif 1a (SEQ ID NO: 78): D/N-K-S-I/V-L-D-E-Q-S-D-P-N-H Motif 2 (SEQ ID NO: 79): N-K-D-G-S-K/T-A-Y-N-E-D-G-T-V/A-K-Q/K-S-T-I-G-K-Y- N-E-K-Y-G-D-A-S Motif 3a (SEQ ID NO: 80): L-P-T-D-G-K-M-D-N/K-S-D-V-E-L-Y-R-T-N/S-E
  • motifs 1a, 2 and 3a align with residues 231-243, 396-425 and 549-567, respectively, of the 7527 GTF sequence (SEQ ID NO:65) in FIGS. 2A-O . These motifs appear to be conserved among GTF enzymes that synthesize high molecular weight alpha-1,3-glucan.
  • motif 1a is flanked by upstream and downstream sequences as shown in FIG. 3 .
  • preceding motif 1a is the sequence SxxRxxN (SEQ ID NO:72)
  • following motif 1a is the sequence GGxxxLLxNDxDxSNPxVQAExLN (SEQ ID NO:73). Both of these sequences were found in all the aligned GTF sequences and can serve as reference points for identifying motif 1a in other GTF sequences.
  • motif 2 is flanked by upstream and downstream sequences as shown in FIG. 5 .
  • preceding motif 2 by about 50 amino acids is the sequence WxxxDxxY (SEQ ID NO:74) and following motif 2 is the sequence YxFxRAHD (SEQ ID NO:75).
  • the downstream sequence includes two of the active site residues, His587 and Asp588 (numbered with respect to the S. mutans GTF structure, 3AIE). Both of these sequences were found in all the aligned GTF sequences and can serve as reference points for identifying motif 2 in other GTF sequences.
  • motif 3a is flanked by upstream and downstream sequences as shown in FIG. 7 .
  • preceding motif 3a is sequence YxxGGQ (SEQ ID NO:76) and following motif 3a is the sequence VRxG (SEQ ID NO:77). Both of these sequences were found in all the aligned GTF sequences and can serve as reference points for identifying motif 2 in other GTF sequences.
  • GTF enzymes produced low molecular weight glucan having 100% alpha-1,3-linkages (Table 4). Specifically, these enzymes were 5926 (SEQ ID NO: 14), 0427 (SEQ ID NO: 26), 0874 (SEQ ID NO: 2) and 1724 (SEQ ID NO: 10). The sequences for each of these enzymes are indicated with a “+ ⁇ ” in FIGS. 2A-O . Two sequence motifs were found in the amino acid sequences of these GTF enzymes, and appear as two different “insertions” situated around the catalytic domain of the known GTF structures. Briefly, these sequence motifs are designated as:
  • a nucleotide sequence encoding a polypeptide similar to the 7527 GTF of SEQ ID NO:65, but with a deletion of Motif 1a was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park Calif.).
  • Plasmid pMP101 was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP101. It is noted that a GTF catalytic domain sequence is located at amino acid positions 54-941 (approximate) of SEQ ID NO:85.
  • Plasmid pMP103 was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP103. It is noted that a GTF catalytic domain sequence is located at amino acid positions 54-935 (approximate) of SEQ ID NO:89.
  • a nucleotide sequence encoding a polypeptide similar to the 7527 GTF of SEQ ID NO:65, but with deletion of Motifs 1a and 3a was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park Calif.).
  • Plasmid pMP105 was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP105. It is noted that a GTF catalytic domain sequence is located at amino acid positions 54-919 (approximate) of SEQ ID NO:93.
  • a nucleotide sequence encoding a polypeptide similar to the 7527 GTF of SEQ ID NO:65, but with deletion of Motifs 1a, 2 and 3a was synthesized using codons optimized for expression in E. coli (DNA 2.0, Menlo Park Calif.).
  • This Example describes measuring the intrinsic viscosity (IV) and branching (g′) of glucan polymer synthesized by each of the deletion-containing GTF enzymes prepared in Examples 6-12. These measurements were compared to those obtained with glucan polymer produced by the 7527 GTF of SEQ ID NO:65, which does not have any internal deletions of Motifs 1a, 2 and/or 3a.
  • glucan polymer synthesized by 7527 GTF poly alpha-1,3-glucan, has 100% alpha-1,3 linkages and is thus linear (see Table 4, for example).
  • glucan polymer samples produced by deletion-containing versions of 7527 GTF were analyzed as described in the General Methods, and are shown in Table 5 below.
  • Non-deleted 7527 GTF is listed as “7527-NT” in Table 5.
  • GTF amino acid Motifs 1a, 2 and 3a play a role in production of linear poly alpha-1,3-glucan by those GTF enzymes that contain these motifs
  • This Example describes testing catalytic domain sequences of certain GTFs for the ability to produce insoluble poly alpha-1,3-glucan. Specifically, catalytic domain sequences of GTFs 7527 (SEQ ID NO:65) and 5926 (SEQ ID NO:14) were tested for activity.
  • a GTF catalytic domain sequence having amino acid residues 54-957 of SEQ ID NO:65 was prepared using the heterologous expression techniques described above. Briefly, a DNA sequence (codon-optimized for expression in E. coli ) encoding a methionine at the first amino acid position followed by amino acid residues 54-957 of SEQ ID NO:65 was prepared and used to express this catalytic domain sequence.
  • This protein compared to the amino acid sequence identified in GENBANK under GI number 47527 (SEQ ID NO:60), is truncated by 230 amino acids at the N-terminus and 384 amino acids at the C-terminus.
  • a GTF catalytic domain sequence having amino acid residues 57-906 of SEQ ID NO:14 was prepared using the heterologous expression techniques described above. Briefly, a DNA sequence (codon-optimized for expression in E. coli ) encoding a methionine at the first amino acid position followed by amino acid residues 57-906 of SEQ ID NO:14 was prepared and used to express this catalytic domain sequence.
  • This protein compared to the amino acid sequence identified in GENBANK under GI number 167735926 (SEQ ID NO:83), is truncated by 199 amino acids at the N-terminus and 417 amino acids at the C-terminus.
  • catalytic domain sequences of GTF 7527 (residues 54-957 of SEQ ID NO:65) and GTF 5926 (residues 57-906 of SEQ ID NO:14) were able to catalyze production of poly alpha-1,3-glucan.
  • the molecular weight of the poly alpha-1,3-glucan produced by each of these catalytic domain sequences generally corresponded with the molecular weight of the product produced by their counterparts containing both the catalytic domain and glucan binding domain (refer to activity of SEQ ID NOs:65 and 14 in Table 4, DP w 150).
  • the catalytic domain of a glucosyltransferase enzyme can be used to produce insoluble poly alpha-1,3-glucan in a reaction solution.

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