# Patent

## Title
Glucosyltransferase enzymes for production of glucan polymers

## Abstract

Reaction solutions are disclosed herein comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme can synthesize insoluble glucan polymer having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. Further disclosed are methods of using such glucosyltransferase enzymes to produce insoluble poly alpha-1,3-glucan.

## Background
This application is a divisional of pending application Ser. No. 14/036,049, filed Sep. 25, 2013, which claims the benefit of U.S. Provisional Application Nos. 61/705,177; 61/705,178; 61/705,179; 61/705,180 and 61/705,181, each filed Sep. 25, 2012. All of these prior applications are incorporated herein by reference in their entirety.


FIELD OF INVENTION
The invention is in the field of enzyme catalysis. Specifically, this invention pertains to producing high molecular weight, insoluble poly alpha-1,3-glucan using a glucosyltransferase enzyme.
BACKGROUND
Driven by a desire to find new structural polysaccharides using enzymatic syntheses or genetic engineering of microorganisms or plant hosts, researchers have discovered 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). Films prepared from poly alpha-1,3-glucan tolerate temperatures up to 150° C. and provide an advantage over polymers obtained from beta-1,4-linked polysaccharides (Ogawa et al., Fiber Differentiation Methods 47:353-362, 1980).
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. Continuos, strong, cotton-like fibers were obtained from this solution that could be spun and used in textile applications.
Not all 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.
SUMMARY OF INVENTION
In one embodiment, the invention concerns a reaction solution comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34.
In a second embodiment, the glucosyltransferase enzyme in the reaction solution synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. In a third embodiment, the glucosyltransferase synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. In a fourth embodiment, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 250.
In a fifth embodiment, the reaction solution comprises a primer. In a sixth embodiment, this primer can be dextran or hydrolyzed glucan.
In a seventh embodiment, the invention concerns a method for producing poly alpha-1,3-glucan comprising the step of contacting at least water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. The poly alpha-1,3-glucan produced in this method can optionally be isolated.
In an eighth embodiment, the glucosyltransferase enzyme used in the method synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. In a ninth embodiment, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. In a tenth embodiment, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 250.
In an eleventh embodiment, the contacting step of the method further comprises contacting a primer with the water, sucrose, and glucosyltransferase enzyme. In a twelfth embodiment, this primer can be dextran or hydrolyzed glucan.
BRIEF DESCRIPTION OF THE SEQUENCES







TABLE 1







Summary of Nucleic Acid and Protein SEQ ID Numbers










Nucleic acid
Protein


Description
SEQ ID NO.
SEQ ID NO.












“0874 gtf”, Streptococcus sobrinus. DNA codon-
1
 2


optimized for expression in E. coli. The first 156

(1435 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 450874, which discloses


“glucosyltransferase-I”.


“6855 gtf”, Streptococcus salivarius SK126. DNA
3
 4


codon-optimized for expression in E. coli. The first

(1341 aa)


178 amino acids of the protein are deleted compared


to GENBANK Identification No. 228476855, which


discloses “glucosyltransferase-SI”.


“2379 gtf”, Streptococcus salivarius. DNA codon-
5
 6


optimized for expression in E. coli. The first 203

(1247 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 662379, which discloses


“glucosyltransferase”.


“7527” or “gtfJ”, Streptococcus salivarius. DNA
7
 8


codon-optimized for expression in E. coli. The first 42

(1477 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 47527, which discloses


“glucosyltransferase-I”.


“1724 gtf”, Streptococcus downei. DNA codon-
9
10


optimized for expression in E. coli. The first 162

(1436 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 121724, which discloses


“glucosyltransferase-I”.


“0544 gtf”, Streptococcus mutans. DNA codon-
11
12


optimized for expression in E. coli. The first 164

(1313 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 290580544, which


discloses “glucosyltransferase-I”.


“5926 gtf”, Streptococcus dentirousetti. DNA codon-
13
14


optimized for expression in E. coli. The first 144

(1323 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 167735926, which


discloses “glucosyltransferase-I”.


“4297 gtf”, Streptococcus oralis. DNA codon-
15
16


optimized for expression in E. coli. The first 228

(1348 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 7684297, which


discloses “glucosyltransferase”.


“5618 gtf”, Streptococcus sanguinis. DNA codon-
17
18


optimized for expression in E. coli. The first 223

(1348 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 328945618, which


discloses “glucosyltransferase-S”.


“2765 gtf”, unknown Streptococcus sp. C150. DNA
19
20


codon-optimized for expression in E. coli. The first

(1340 aa)


193 amino acids of the protein are deleted compared


to GENBANK Identification No. 322372765, which


discloses “glucosyltransferase-S”.


“4700 gtf”, Leuconostoc mesenteroides. DNA codon-
21
22


optimized for expression in E. coli. The first 36 amino

(1492 aa)


acids of the protein are deleted compared to


GENBANK Identification No. 21654700, which


discloses “dextransucrase DsrD”.


“1366 gtf”, Streptococcus criceti. DNA codon-
23
24


optimized for expression in E. coli. The first 139

(1323 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 146741366, which


discloses “glucosyltransferase”.


“0427 gtf”, Streptococcus sobrinus. DNA codon-
25
26


optimized for expression in E. coli. The first 156

(1435 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 940427, which discloses


“GTF-I”.


“2919 gtf”, Streptococcus salivarius PS4. DNA
27
28


codon-optimized for expression in E. coli. The first 92

(1340 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 383282919, which


discloses “putative glucosyltransferase”.


“2678 gtf”, Streptococcus salivarius K12. DNA codon-
29
30


optimized for expression in E. coli. The first 188

(1341 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 400182678, which


discloses “dextransucrase-S”.


“2381 gtf”, Streptococcus salivarius. DNA codon-
31
32


optimized for expression in E. coli. The first 273

(1305 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 662381, which discloses


“glucosyltransferase”.


“3929 gtf”, Streptococcus salivarius JIM8777. DNA
33
34


codon-optimized for expression in E. coli. The first

(1341 aa)


178 amino acids of the protein are deleted compared


to GENBANK Identification No. 387783929, which


discloses “glucosyltransferase-S precursor (GTF-S)


(Dextransucrase) (Sucrose 6-glucosyltransferase)”.


“6907 gtf”, Streptococcus salivarius SK126. DNA
35
36


codon-optimized for expression in E. coli. The first

(1331 aa)


161 amino acids of the protein are deleted compared


to GENBANK Identification No. 228476907, which


discloses “glucosyltransferase-SI”.


“6661 gtf”, Streptococcus salivarius SK126. DNA
37
38


codon-optimized for expression in E. coli. The first

(1305 aa)


265 amino acids of the protein are deleted compared


to GENBANK Identification No. 228476661, which


discloses “glucosyltransferase-SI”.


“0339 gtf”, Streptococcus gallolyticus ATCC 43143.
39
40


DNA codon-optimized for expression in E. coli. The

(1310 aa)


first 213 amino acids of the protein are deleted


compared to GENBANK Identification No. 334280339,


which discloses “glucosyltransferase”.


“0088 gtf”, Streptococcus mutans. DNA codon-
41
42


optimized for expression in E. coli. The first 189

(1267 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 3130088, which


discloses “glucosyltransferase-SI”.


“9358 gtf”, Streptococcus mutans UA159. DNA
43
44


codon-optimized for expression in E. coli. The first

(1287 aa)


176 amino acids of the protein are deleted compared


to GENBANK Identification No. 24379358, which


discloses “glucosyltransferase-S”.


“8242 gtf”, Streptococcus gallolyticus ATCC BAA-
45
46


2069. DNA codon-optimized for expression in E. coli.

(1355 aa)


The first 191 amino acids of the protein are deleted


compared to GENBANK Identification No. 325978242,


which discloses “glucosyltransferase-I”.


“3442 gtf”, Streptococcus sanguinis SK405. DNA
47
48


codon-optimized for expression in E. coli. The first

(1348 aa)


228 amino acids of the protein are deleted compared


to GENBANK Identification No. 324993442, which


discloses a “ . . . signal domain protein”.


“7528 gtf”, Streptococcus salivarius. DNA codon-
49
50


optimized for expression in E. coli. The first 173

(1427 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 47528, which discloses


“glucosyltransferase S”.


“3279 gtf”, Streptococcus sp. C150. DNA codon-
51
52


optimized for expression in E. coli. The first 178

(1393 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 322373279, which


discloses “glucosyltransferase S”.


“6491 gtf”, Leuconostoc citreum KM20. DNA codon-
53
54


optimized for expression in E. coli. The first 244

(1262 aa)


amino acids of the protein are deleted compared to


GENBANK Identification No. 170016491, which


discloses “glucosyltransferase”.


“6889 gtf”, Streptococcus salivarius SK126. DNA
55
56


codon-optimized for expression in E. coli. The first

(1427 aa)


173 amino acids of the protein are deleted compared


to GENBANK Identification No. 228476889, which


discloses “glucosyltransferase-I”.


“4154 gtf”, Lactobacillus reuteri. DNA codon-
57
58


optimized for expression in E. coli. The first 38 amino

(1735 aa)


acids of the protein are deleted compared to


GENBANK Identification No. 51574154, which


discloses “glucansucrase”.


“3298 gtf”, Streptococcus sp. C150. The first 209

59


amino acids of the protein are deleted compared to

(1242 aa)


GENBANK Identification No. 322373298, which


discloses “glucosyltransferase-S”.


“Wild type gtfJ”, Streptococcus salivarius. GENBANK

60


Identification No. 47527.

(1518 aa)


Wild type gtf corresponding to 2678 gtf, Streptococcus

61


salivarius K12. GENBANK Identification No.

(1528 aa)


400182678, which discloses “dextransucrase-S”.


Wild type gtf corresponding to 6855 gtf, Streptococcus

62


salivarius SK126. GENBANK Identification No.

(1518 aa)


228476855, which discloses “glucosyltransferase-SI”.


Wild type gtf corresponding to 2919 gtf, Streptococcus

63


salivarius PS4. GENBANK Identification No.

(1431 aa)


383282919, which discloses “putative


glucosyltransferase”.


Wild type gtf corresponding to 2765 gtf, Streptococcus

64


sp. C150. GENBANK Identification No. 322372765,

(1532 aa)


which discloses “glucosyltransferase-S”.











DETAILED DESCRIPTION OF THE INVENTION
The disclosures of all cited patent and non-patent literature are incorporated herein by reference in their entirety.
As used herein, the term “invention” or “disclosed invention” is not meant to be limiting, but applies generally to any of the inventions defined in the claims or described herein. These terms are used interchangeably herein.
The terms “poly alpha-1,3-glucan”, “alpha-1,3-glucan polymer” and “glucan polymer” are used interchangeably herein. 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 is a type of polysaccharide. The structure of poly alpha-1,3-glucan can be illustrated as follows:




The terms “glycosidic linkage” and “glycosidic bond” are used interchangeably herein and refer to the type of covalent bond that joins a carbohydrate (sugar) molecule to another group such as another carbohydrate. The term “alpha-1,3-glycosidic linkage” as used herein 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. This linkage is illustrated in the poly alpha-1,3-glucan structure provided above. Herein, “alpha-D-glucose” will be referred to as “glucose”.
The term “sucrose” herein 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.
The “molecular weight” of the poly alpha-1,3-glucan herein can be represented as number-average molecular weight (Mn) or as weight-average molecular weight (Mw). Alternatively, molecular weight can be represented as Daltons, grams/mole, DPw (weight average degree of polymerization), or DPn (number average degree of polymerization). Various means are known in the art for calculating these molecular weight measurements such as with high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).
The terms “glucosyltransferase enzyme”, “gtf enzyme”, “gtf enzyme catalyst”, “gtf”, and “glucansucrase” are used interchangeably herein. 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 (where glucose is hydrolyzed from the glucosyl-gtf enzyme intermediate complex), various soluble oligosaccharides (DP2-DP7), and leucrose (where glucose of the glucosyl-gtf enzyme intermediate complex is linked to fructose). Leucrose is a disaccharide composed of glucose and fructose linked by an alpha-1,5 linkage. Wild type forms of glucosyltransferase 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. A 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).
The terms “reaction” and “enzymatic reaction” are used interchangeably herein and refer to a reaction that is performed by a glucosyltransferase enzyme. A “reaction solution” as used herein generally refers to a solution comprising at least one active glucosyltransferase enzyme in a solution comprising sucrose and water, and optionally other components. It is in the 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. The reaction herein is not naturally occurring.
The terms “percent by volume”, “volume percent”, “vol %” and “v/v %” are used interchangeably herein. The percent by volume of a solute in a solution can be determined using the formula: [(volume of solute)/(volume of solution)]×100%.
The terms “percent by weight”, “weight percentage (wt %)” and “weight-weight percentage (% w/w)” are used interchangeably herein. Percent by weight refers to the percentage of a material on a mass basis as it is comprised in a composition, mixture, or solution.
The terms “increased”, “enhanced” and “improved” are used interchangeably herein. These terms refer to a greater quantity or activity such as a quantity or activity slightly greater than the original quantity or activity, or a quantity or activity in large excess compared to the original quantity or activity, and including all quantities or activities in between. Alternatively, these terms may refer to, for example, a quantity or activity that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20% more than the quantity or activity for which the increased quantity or activity is being compared.
The terms “polynucleotide”, “polynucleotide sequence”, and “nucleic acid sequence” are used interchangeably herein. These terms encompass nucleotide sequences and the like. A 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.
The term “gene” as used herein refers to a polynucleotide sequence that expresses a protein, and which may refer to the coding region alone or may include regulatory sequences upstream and/or downstream to the coding region (e.g., 5′ untranslated regions upstream of the transcription start site of the coding region). A gene that is “native” or “endogenous” refers to a gene as found in nature with its own regulatory sequences; this gene is located in its natural location in the genome of an organism. “Chimeric gene” refers to any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. A “foreign” or “heterologous” gene refers to a gene that is introduced into the host organism by gene transfer. Foreign 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. The polynucleotide sequences in certain embodiments disclosed herein are heterologous. A “transgene” is a gene that has been introduced into the genome by a transformation procedure. A “codon-optimized gene” is a gene having its frequency of codon usage designed to mimic the frequency of preferred codon usage of the host cell.
A native amino acid sequence or polynucleotide sequence is naturally occurring, whereas a non-native amino acid sequence or polynucleotide sequence does not occur in nature.
“Coding sequence” as used herein refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” as used herein refer to nucleotide sequences located upstream of the coding sequence's transcription start site, 5′ untranslated regions and 3′ non-coding regions, and which may influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, silencers, 5′ untranslated leader sequence, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, stem-loop structures and other elements involved in regulation of gene expression.
The term “recombinant” as used herein refers to an artificial combination of two otherwise separated segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques. The terms “recombinant”, “transgenic”, “transformed”, “engineered” or “modified for exogenous gene expression” are used interchangeably herein.
The term “transformation” as used in certain embodiments refers to the transfer of a nucleic acid molecule into a host organism. The nucleic acid molecule may be a plasmid that replicates autonomously, or it may integrate into the genome of the host organism. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms or “transformants”.
The term “recombinant” or “heterologous” refers to an artificial combination of two otherwise separate segments of sequence, e.g., by chemical synthesis or by the manipulation of isolated segments of nucleic acids by genetic engineering techniques.
The terms “sequence identity” or “identity” as used herein with respect to polynucleotide or polypeptide sequences refer 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. Thus, “percentage of sequence identity” or “percent 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.
The Basic Local Alignment Search Tool (BLAST) algorithm, which is available online at the National Center for Biotechnology Information (NCBI) website, may be used, for example, to measure percent identity between or among two or more of the polynucleotide sequences (BLASTN algorithm) or polypeptide sequences (BLASTP algorithm) disclosed herein. Alternatively, percent identity between sequences may be performed using a Clustal algorithm (e.g., ClustalW or ClustalV). For multiple alignments using a Clustal method of alignment, the default values may correspond to GAP PENALTY=10 and GAP LENGTH PENALTY=10. Default parameters for pairwise alignments and calculation of percent identity of protein sequences using a Clustal method may be KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids, these parameters may be KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. Alternatively still, percent identity between sequences may be performed using an EMBOSS algorithm (e.g., needle) with parameters such as GAP OPEN=10, GAP EXTEND=0.5, END GAP PENALTY=false, END GAP OPEN=10, END GAP EXTEND=0.5 using a BLOSUM matrix (e.g., BLOSUM62).
Various polypeptide amino acid sequences and polynucleotide sequences are disclosed herein as features of certain embodiments of the disclosed invention. 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. Alternatively, 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 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the function/activity of the disclosed sequence.
The term “isolated” as used in certain embodiments refers to any cellular component that has been completely or partially purified from its native source (e.g., an isolated polynucleotide or polypeptide molecule). In some instances, an isolated polynucleotide or polypeptide molecule is part of a greater composition, buffer system or reagent mix. For example, the isolated polynucleotide or polypeptide molecule can be comprised within a cell or organism in a heterologous manner. Another example is an isolated glucosyltransferase enzyme.
Embodiments of the disclosed invention concern a reaction solution comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. Significantly, these glucosyltransferase enzymes can synthesize poly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100. Such glucan is suitable for use in spinning fibers and in other industrial applications.
The molecular weight of the poly alpha-1,3-glucan produced by the glucosyltransferase enzymes herein can be measured as DPn (number average degree of polymerization). Alternatively, the molecular weight of the poly alpha-1,3-glucan can be measured in terms of Daltons, grams/mole, or as DPw (weight average degree of polymerization). The poly alpha-1,3-glucan in certain embodiments of the invention can have a molecular weight in DPn or DPw of at least about 100. The molecular weight of the poly alpha-1,3-glucan can alternatively be at least about 250 DPn or DPw. Alternatively still, the DPn or DPw of the poly alpha-1,3-glucan 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 the poly alpha-1,3-glucan herein can be measured using any of several means known in the art. For example, glucan polymer molecular weight can be measured using high-pressure liquid chromatography (HPLC), size exclusion chromatography (SEC), or gel permeation chromatography (GPC).
The poly alpha-1,3-glucan herein is preferably linear/unbranched. The percentage of glycosidic linkages between the glucose monomer units of the poly alpha-1,3-glucan that are alpha-1,3 is at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In such embodiments, accordingly, the poly alpha-1,3-glucan has less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, or 0% of glycosidic linkages that are not alpha-1,3.
It is understood that the higher the percentage of alpha-1,3-glycosidic linkages present in the 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. In certain embodiments, the poly alpha-1,3-glucan has no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer. Examples of branch points include alpha-1,6 branch points, such as those that are present in mutan polymer.
The glycosidic linkage profile of the poly alpha-1,3-glucan can be determined using any method known in the art. For example, the linkage profile can be determined using methods that use nuclear magnetic resonance (NMR) spectroscopy (e.g., 13C NMR or 1H NMR). These and other methods that can be used are disclosed in Food Carbohydrates: Chemistry, Physical Properties, and Applications (S. W. Cui, Ed., Chapter 3, S. W. Cui, Structural Analysis of Polysaccharides, Taylor & Francis Group LLC, Boca Raton, Fla., 2005), which is incorporated herein by reference.
The poly alpha-1,3-glucan herein may be characterized by any combination of the aforementioned percentages of alpha-1,3 linkages and molecular weights. For example, the poly alpha-1,3-glucan produced in a reaction solution herein can have at least 50% alpha-1,3 glycosidic linkages and a DPn or DPw of at least 100. As another example, the poly alpha-1,3-glucan can have 100% alpha-1,3 glycosidic linkages and a DPn or DPw of at least 100. The poly alpha-1,3-glucan in still another example can have 100% alpha-1,3 glycosidic linkages and a DPn or DPw of at least 250.
The glucosyltransferase enzyme in certain embodiments of the invention may be derived from a Streptococcus species, Leuconostoc species or Lactobacillus species, for example. Examples of Streptococcus species from which the glucosyltransferase may be derived include S. salivarius, S. sobrinus, S. dentirousetti, S. downei, S. mutans, S. oralis, S. gallolyticus and S. sanguinis. Examples of Leuconostoc species from which the glucosyltransferase may be derived include L. mesenteroides, L. amelibiosum, L. argentinum, L. carnosum, L. citreum, L. cremoris, L. dextranicum and L. fructosum. Examples of Lactobacillus species from which the glucosyltransferase may be derived 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. 
The glucosyltransferase enzyme herein can comprise, or consist of, an amino acid sequence that is at least 90% identical to the amino acid sequence provided in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, wherein the glucosyltransferase enzyme has activity. Alternatively, the glucosyltransferase enzyme can comprise, or consist of, an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34, wherein the glucosyltransferase enzyme has activity.
All the amino acid residues disclosed herein at each amino acid position of the glucosyltransferase enzyme sequences 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 in the glucosyltransferase enzyme sequences can be as provided in the disclosed sequences or substituted with a conserved amino acid residue (“conservative amino acid substitution”) as follows:

    
    
        1. The following small aliphatic, nonpolar or slightly polar residues can substitute for each other: Ala (A), Ser (S), Thr (T), Pro (P), Gly (G);
        2. The following polar, negatively charged residues and their amides can substitute for each other: Asp (D), Asn (N), Glu (E), Gln (Q);
        3. The following polar, positively charged residues can substitute for each other: His (H), Arg (R), Lys (K);
        4. The following aliphatic, nonpolar residues can substitute for each other: Ala (A), Leu (L), Ile (I), Val (V), Cys (C), Met (M); and
        5. The following large aromatic residues can substitute for each other: Phe (F), Tyr (Y), Trp (W).
    
    


Examples of glucosyltransferase enzymes may be any of the amino acid sequences disclosed herein and that further include 1-300 (or any integer there between) 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 another sequence such as an epitope tag (at either N- or C-terminus) or a heterologous signal peptide (at N-terminus), for example. Thus, examples of glucosyltransferase enzymes include SEQ ID NOs:61, 62, 63 and 64, which represent the wild type sequences from which SEQ ID NOs:30, 4, 28 and 20 are derived, respectively.
The glucosyltransferase enzyme can be encoded by the polynucleotide sequence provided in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33, for example. Alternatively, the glucosyltransferase enzyme can be encoded by a polynucleotide sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, or SEQ ID NO:33.
The glucosyltransferase enzyme in certain embodiments synthesizes poly alpha-1,3-glucan in which at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any integer between 50% and 100%) of the constituent glycosidic linkages are alpha-1,3 linkages. In such embodiments, accordingly, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan in which there is less than about 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of glycosidic linkages that are not alpha-1,3.
In other aspects, the glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan with no branch points or less than about 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% branch points as a percent of the glycosidic linkages in the polymer. Examples of branch points include alpha-1,6 branch points, such as those that are present in mutan polymer.
The glucosyltransferase enzyme can synthesize poly alpha-1,3-glucan having a molecular weight in DPn or DPw of at least about 100. Alternatively, the glucosyltransferase enzyme can synthesize poly alpha-1,3-glucan having a molecular weight in DPn or DPw of at least about 400. Alternatively still, the glucosyltransferase enzyme can synthesize poly alpha-1,3-glucan having a molecular weight in DPn or DPw 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).
One or more different glucosyltransferase enzymes may be used in the disclosed invention. The glucosyltransferase enzyme preferably does not have, or has very little (less than 1%), dextransucrase, reuteransucrase, or alternansucrase activity. The glucosyltransferase in certain embodiments does not comprise amino acid residues 2-1477 of SEQ ID NO:8 or amino acid residues 138-1477 of SEQ ID NO:8, which are derived from the glucosyltransferase identified in GENBANK under GI number 47527 (SEQ ID NO:60).
The glucosyltransferase enzyme herein can be primer-independent or primer-dependent. Primer-independent glucosyltransferase enzymes do not require the presence of a primer to perform glucan synthesis. A primer-dependent glucosyltransferase enzyme requires the presence of an initiating molecule in the reaction solution to act as a primer for the enzyme during glucan polymer synthesis. The term “primer” as used herein refers to any molecule that can act as the initiator for a glucosyltransferase enzyme. Oligosaccharides and polysaccharides can serve a primers herein, for example. Primers that can be used in certain embodiments include dextran and other carbohydrate-based primers, such as hydrolyzed glucan, for example. Hydrolyzed glucan can be prepared by acid hydrolysis of a glucan such as poly alpha-glucan. International Appl. Publ. No. WO2013/036918, which is incorporated herein by reference, discloses such preparation of hydrolyzed glucan using poly alpha-1,3-glucan as the starting material. Dextran for use as a primer herein can be dextran T10 (i.e., dextran having a molecular weight of 10 kD). Alternatively, the dextran can have a molecular weight of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 25 kD, for example.
The glucosyltransferase enzyme used herein may be produced by any means known in the art (e.g., U.S. Pat. No. 7,000,000, which is incorporated herein by reference). For example, the glucosyltransferase enzyme may be produced recombinantly in any bacterial (e.g., E. coli such as TOP10, Bacillus sp.) or eukaryotic (e.g., yeasts such as Pichia sp. and Saccharomyces sp.) heterologous gene expression system. Any of the above-listed nucleic acid sequences can be used for this purpose, for example.
The glucosyltransferase enzyme used herein may be purified and/or isolated prior to its use, or may be used in the form of a cell lysate, for example. A cell lysate or extract may be prepared from a bacteria (e.g., E. coli) used to heterologously express the enzyme. For example, the bacteria may be subjected to disruption using a French pressure cell (French press). The glucosyltransferase enzyme is soluble in these type of preparations. The lysate or extract may be used at about 0.15-0.3% (v/v) in a reaction solution for producing poly alpha-1,3-glucan from sucrose. In certain embodiments, a bacterial cell lysate is first cleared of insoluble material by means such as centrifugation or filtration.
In certain embodiments, the heterologous gene expression system may be one that is designed for protein secretion. The glucosyltransferase enzyme comprises a signal peptide (signal sequence) in such embodiments. The signal peptide may be either its native signal peptide or a heterologous signal peptide.
The activity of the glucosyltransferase enzyme can be determined using any method known in the art. For example, 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 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 OD480 nm for five minutes.
The temperature of the reaction solution herein can be controlled, if desired. In certain embodiments, the solution has a temperature between about 5° C. to about 50° C. The temperature of the solution in certain other embodiments is between about 20° C. to about 40° C. Alternatively, the temperature of the solution may be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40° C.
The temperature of the reaction solution may be maintained using various means known in the art. For example, the temperature of reaction solution can be maintained by placing the vessel containing the reaction solution in an air or water bath incubator set at the desired temperature.
The initial concentration of the sucrose in the solution can be about 20 g/L to about 400 g/L, for example. Alternatively, the initial concentration of the sucrose can be about 75 g/L to about 175 g/L, or from about 50 g/L to about 150 g/L. Alternatively still, the initial concentration of the sucrose can be about 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 160 g/L (or any integer between 40 and 160 g/L), for example. The “initial concentration of sucrose” refers to the sucrose concentration in the solution just after all the reaction solution components have been added (water, sucrose, gtf enzyme).
Sucrose used in the reaction solution can be highly pure (≧99.5%) or be of any other purity or grade. For example, the sucrose can have a purity of at least 99.0%, or be reagent grade sucrose. The sucrose may be derived from any renewable sugar source such as sugar cane, sugar beets, cassava, sweet sorghum, or corn. The sucrose can be provided in any form such as crystalline form or non-crystalline form (e.g., syrup or cane juice).
The pH of the reaction solution herein 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. In certain embodiments, the pH of a solution containing water and sucrose may be set before adding the glucosyltransferase enzyme. The pH of the reaction solution 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. The concentration of the buffer can be from 0 mM to about 100 mM, or about 10, 20, or 50 mM, for example. A suitable amount of DTT (dithiothreitol, e.g., about 1.0 mM) can optionally be added to the reaction solution.
The disclosed invention also concerns a method for producing poly alpha-1,3-glucan comprising the step of contacting at least water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan. The glucosyltransferase enzyme can comprise an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, or SEQ ID NO:34. The poly alpha-1,3-glucan produced in this method can optionally be isolated.
Water, sucrose, and a glucosyltransferase enzyme as described herein are contacted in a reaction solution. Thus, the method can comprise providing a reaction solution comprising water, sucrose and a glucosyltransferase enzyme as described herein. 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. For example, the desired amount of 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 the glucosyltransferase enzyme. The solution may be kept still, or agitated via stirring or orbital shaking, for example. The reaction can be, and typically is, cell-free.
The glucosyltransferase enzyme can optionally be added to water or an aqueous solution (e.g., sucrose in water) that does not contain salt or buffer when initially preparing the reaction solution. The pH of such a preparation can then be modified as desired, such as to pH 5-6 for example. The reaction can be carried out to completion without any added buffer, if desired.
Completion of the reaction in certain embodiments can be determined visually (no more accumulation of precipitated 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. Typically, 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 of the disclosed process is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Alternatively, the percent sucrose consumption may be >90% or >95%.
The yield of the poly alpha-1,3-glucan produced in the disclosed invention 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 the sucrose used in the reaction solution.
The poly alpha-1,3-glucan produced in the disclosed method may optionally be isolated. For example, insoluble poly alpha-1,3-glucan may be separated by centrifugation or filtration. In doing so, the poly alpha-1,3-glucan is separated from the rest 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.
Poly alpha-1,3 glucan is a potentially low cost polymer which can be enzymatically produced from renewable resources containing sucrose using glucosyltransferase enzymes. It has been shown that this polymer can form ordered liquid crystalline solutions when the polymer is dissolved in a solvent under certain conditions (U.S. Pat. No. 7,000,000). Such solutions can be spun into continuous, high strength, cotton-like fibers. The poly alpha-1,3-glucan produced using the disclosed invention has comparable utilities.
EXAMPLES
The disclosed invention is further defined in the following Examples. It should be understood that these Examples, while indicating certain preferred aspects of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
Abbreviations
The meanings of some of the abbreviations used herein are as follows: “g” means gram(s), “h” means hour(s), “mL” means milliliter(s), “psi” means pound(s) per square inch, “wt %” means weight percentage, “μm” means micrometer(s), “° C.” means degrees Celsius, “mg” means milligram(s), “mm” means millimeter(s), “μL” 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.
General Methods
Preparation of Crude Extracts of Glucosyltransferase (Gtf) Enzymes
Gtf enzymes were prepared as follows. E. coli TOP10® cells (Invitrogen, Carlsbad Calif.) were transformed with a pJexpress404®-based construct containing a particular gtf-encoding DNA sequence. Each sequence was codon-optimized to express the gtf enzyme in E. coli. Individual E. coli strains expressing a particular gtf enzyme were grown in LB (Luria broth) medium (Becton, Dickinson and Company, Franklin Lakes, N.J.) with ampicillin (100 μg/mL) at 37° C. with shaking to OD600=0.4-0.5, at which time IPTG (isopropyl beta-D-1-thiogalactopyranoside, Cat. No. I6758, Sigma-Aldrich, St. Louis, Mo.) was added to a final concentration of 0.5 mM. The cultures were incubated for 2-4 hours at 37° C. following IPTG induction. Cells were harvested by centrifugation at 5,000×g for 15 minutes and resuspended (20% w/v) in 50 mM phosphate buffer pH 7.0 supplemented with dithiothreitol (DTT, 1.0 mM). Resuspended cells were passed through a French Pressure Cell (SLM Instruments, Rochester, N.Y.) twice to ensure >95% cell lysis. Lysed cells were centrifuged for 30 minutes at 12,000×g at 4° C. 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.
Determination of Gtf Enzymatic Activity
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. Supernatant (0.01 mL) was added to a mixture containing 1 N NaOH and 0.1% triphenyltetrazolium chloride (Sigma-Aldrich). The mixture was incubated for five minutes after which its OD480 nm was determined using an ULTROSPEC spectrophotometer (Pharmacia LKB, New York, N.Y.) to gauge the presence of the reducing sugars fructose and glucose.
Determination of Glycosidic Linkages
Glycosidic linkages in the glucan product synthesized by a gtf enzyme were determined by 13C 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 13C 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.
Determination of Number Average Degree of Polymerization (DPn)
The DPn 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. The 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. The columns used for SEC were four styrene-divinyl benzene columns from Shodex (Japan) and two linear KD-806M, KD-802 and KD-801 columns to improve resolution at the low molecular weight region of a polymer distribution. The mobile phase was DMAc with 0.11% LiCl. The chromatographic conditions used were 50° C. in the column and detector compartments, 40° C. in the sample and injector compartment, a flow rate of 0.5 mL/min, and an injection volume of 100 μL. The software packages used for data reduction were EmpowerTM version 3 from Waters (calibration with broad glucan polymer standard) and Astra® version 6 from Wyatt (triple detection method with column calibration).
Example 1
Production of Gtf Enzyme 0874 (SEQ ID NO:2)
This Example describes preparing an N-terminally truncated version of a Streptococcus sobrinus gtf enzyme identified in GENBANK under GI number 450874 (SEQ ID NO:2, encoded by SEQ ID NO:1; herein referred to as “0874”).
A nucleotide sequence encoding gtf 0874 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc., Menlo Park, Calif.). The nucleic acid product (SEQ ID NO:1), encoding gtf 0874 (SEQ ID NO:2), was subcloned into pJexpress404® (DNA2.0, Inc.) to generate the plasmid construct identified as pMP57. This plasmid construct was used to transform E. coli TOP10 cells (Invitrogen, Carlsbad, Calif.) to generate the strain identified as TOP10/pMP57.
Production of gtf 0874 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 0874 is shown in Table 2 (see Example 18 below).
Example 2
Production of Gtf Enzyme 6855 (SEQ ID NO:4)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 228476855 (SEQ ID NO:4, encoded by SEQ ID NO:3; herein referred to as “6855”).
A nucleotide sequence encoding gtf 6855 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:3), encoding gtf 6855 (SEQ ID NO:4), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP53. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP53.
Production of gtf 6855 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 6855 is shown in Table 2 (see Example 18 below).
Example 3
Production of Gtf Enzyme 2379 (SEQ ID NO:6)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 662379 (SEQ ID NO:6, encoded by SEQ ID NO:5; herein referred to as “2379”).
A nucleotide sequence encoding gtf 2379 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:5), encoding gtf 2379 (SEQ ID NO:6), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP66. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP66.
Production of gtf 2379 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 2379 is shown in Table 2 (see Example 18 below).
Example 4
Production of Gtf Enzyme 7527 (GtfJ, SEQ ID NO:8)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 47527 (SEQ ID NO:8, encoded by SEQ ID NO:7; herein referred to as “7527” or “GtfJ”).
A nucleotide sequence encoding gtf 7527 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:7), encoding gtf 7527 (SEQ ID NO:8), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP65. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP65.
Production of gtf 7527 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 7527 is shown in Table 2 (see Example 18 below).
Example 5
Production of Gtf Enzyme 1724 (SEQ ID NO:10)
This Example describes preparing an N-terminally truncated version of a Streptococcus downei gtf enzyme identified in GENBANK under GI number 121724 (SEQ ID NO:10, encoded by SEQ ID NO:9; herein referred to as “1724”).
A nucleotide sequence encoding gtf 1724 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:9), encoding gtf 1724 (SEQ ID NO:10), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP52. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP52.
Production of gtf 1724 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 1724 is shown in Table 2 (see Example 18 below).
Example 6
Production of Gtf Enzyme 0544 (SEQ ID NO:12)
This Example describes preparing an N-terminally truncated version of a Streptococcus mutans gtf enzyme identified in GENBANK under GI number 290580544 (SEQ ID NO:12, encoded by SEQ ID NO:11; herein referred to as “0544”).
A nucleotide sequence encoding gtf 0544 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:11), encoding gtf 0544 (SEQ ID NO:12), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP55. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP55.
Production of gtf 0544 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 0544 is shown in Table 2 (see Example 18 below).
Example 7
Production of Gtf Enzyme 5926 (SEQ ID NO:14)
This Example describes preparing an N-terminally truncated version of a Streptococcus dentirousetti gtf enzyme identified in GENBANK under GI number 167735926 (SEQ ID NO:14, encoded by SEQ ID NO:13; herein referred to as “5926”).
A nucleotide sequence encoding gtf 5926 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:13), encoding gtf 5926 (SEQ ID NO:14), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP67. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP67.
Production of gtf 5926 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 5926 is shown in Table 2 (see Example 18 below).
Example 8
Production of Gtf Enzyme 4297 (SEQ ID NO:16)
This Example describes preparing an N-terminally truncated version of a Streptococcus oralis gtf enzyme identified in GENBANK under GI number 7684297 (SEQ ID NO:16, encoded by SEQ ID NO:15; herein referred to as “4297”).
A nucleotide sequence encoding gtf 4297 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:15), encoding gtf 4297 (SEQ ID NO:16), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP62. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP62.
Production of gtf 4297 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 4297 is shown in Table 2 (see Example 18 below).
Example 9
Production of Gtf Enzyme 5618 (SEQ ID NO:18)
This Example describes preparing an N-terminally truncated version of a Streptococcus sanguinis gtf enzyme identified in GENBANK under GI number 328945618 (SEQ ID NO:18, encoded by SEQ ID NO:17; herein referred to as “5618”).
A nucleotide sequence encoding gtf 5618 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:17), encoding gtf 5618 (SEQ ID NO:18), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP56. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP56.
Production of gtf 5618 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 5618 is shown in Table 2 (see Example 18 below).
Example 10
Production of Gtf Enzyme 2765 (SEQ ID NO:20)
This Example describes preparing an N-terminally truncated version of a Streptococcus sp. gtf enzyme identified in GENBANK under GI number 322372765 (SEQ ID NO:20, encoded by SEQ ID NO:19; herein referred to as “2765”).
A nucleotide sequence encoding gtf 2765 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:19), encoding gtf 2765 (SEQ ID NO:20), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP73. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP73.
Production of gtf 2765 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 2765 is shown in Table 2 (see Example 18 below).
Example 11
Production of Gtf Enzyme 4700 (SEQ ID NO:22)
This Example describes preparing an N-terminally truncated version of a Leuconostoc mesenteroides gtf enzyme identified in GENBANK under GI number 21654700 (SEQ ID NO:22, encoded by SEQ ID NO:21; herein referred to as “4700”).
A nucleotide sequence encoding gtf 2765 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:21), encoding gtf 4700 (SEQ ID NO:22), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP83. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP83.
Production of gtf 4700 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 4700 is shown in Table 2 (see Example 18 below).
Example 12
Production of Gtf Enzyme 1366 (SEQ ID NO:24)
This Example describes preparing an N-terminally truncated version of a Streptococcus criceti gtf enzyme identified in GENBANK under GI number 146741366 (SEQ ID NO:24, encoded by SEQ ID NO:23; herein referred to as “1366”).
A nucleotide sequence encoding gtf 1366 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:23), encoding gtf 1366 (SEQ ID NO:24), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP86. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP86.
Production of gtf 1366 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 1366 is shown in Table 2 (see Example 18 below).
Example 13
Production of Gtf Enzyme 0427 (SEQ ID NO:26)
This Example describes preparing an N-terminally truncated version of a Streptococcus sobrinus gtf enzyme identified in GENBANK under GI number 940427 (SEQ ID NO:26, encoded by SEQ ID NO:25; herein referred to as “0427”).
A nucleotide sequence encoding gtf 0427 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:25), encoding gtf 0427 (SEQ ID NO:26), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP87. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP87.
Production of gtf 0427 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 0427 is shown in Table 2 (see Example 18 below).
Example 14
Production of Gtf Enzyme 2919 (SEQ ID NO:28)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 383282919 (SEQ ID NO:28, encoded by SEQ ID NO:27; herein referred to as “2919”).
A nucleotide sequence encoding gtf 2919 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:27), encoding gtf 2919 (SEQ ID NO:28), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP88. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP88.
Production of gtf 2919 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 2919 is shown in Table 2 (see Example 18 below).
Example 15
Production of Gtf Enzyme 2678 (SEQ ID NO:30)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 400182678 (SEQ ID NO:30 encoded by SEQ ID NO:29; herein referred to as “2678”).
A nucleotide sequence encoding gtf 2678 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:29), encoding gtf 2678 (SEQ ID NO:30), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP89. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP89.
Production of gtf 2678 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 2678 is shown in Table 2 (see Example 18 below).
Example 16
Production of Gtf Enzyme 2381 (SEQ ID NO:32)
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 662381 (SEQ ID NO:32 encoded by SEQ ID NO:31; herein referred to as “2381”).
A nucleotide sequence encoding gtf 2381 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:31), encoding gtf 2381 (SEQ ID NO:32), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP96. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP96.
Production of gtf 2381 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 2381 is shown in Table 2 (see Example 18 below).
Example 17
Production of Gtf Enzyme 3929 (SEQ ID NO:34) and Additional Gtf Enzymes
This Example describes preparing an N-terminally truncated version of a Streptococcus salivarius gtf enzyme identified in GENBANK under GI number 387783929 (SEQ ID NO:34 encoded by SEQ ID NO:33; herein referred to as “3929”).
A nucleotide sequence encoding gtf 3929 was synthesized using codons optimized for protein expression in E. coli (DNA2.0, Inc.). The nucleic acid product (SEQ ID NO:33), encoding gtf 3929 (SEQ ID NO:34), was subcloned into pJexpress404® to generate the plasmid construct identified as pMP97. This plasmid construct was used to transform E. coli TOP10 cells to generate the strain identified as TOP10/pMP97.
Production of gtf 3929 by bacterial expression and determination of its enzymatic activity were performed following the procedures disclosed in the General Methods section. The enzymatic activity of gtf 3929 is shown in Table 2 (see Example 18 below).
Additional gtf enzymes were produced in a similar manner. Briefly, N-terminally truncated versions of enzymes identified in GENBANK under GI numbers 228476907 (a Streptococcus salivarius gtf, SEQ ID NO:36, herein referred to as “6907”), 228476661 (a Streptococcus salivarius gtf, SEQ ID NO:38, herein referred to as “6661”), 334280339 (a Streptococcus gallolyticus gtf, SEQ ID NO:40, herein referred to as “0339”), 3130088 (a Streptococcus mutans gtf, SEQ ID NO:42, herein referred to as “0088”), 24379358 (a Streptococcus mutans gtf, SEQ ID NO:44, herein referred to as “9358”), 325978242 (a Streptococcus gallolyticus gtf, SEQ ID NO:46, herein referred to as “8242”), 324993442 (a Streptococcus sanguinis gtf, SEQ ID NO:48, herein referred to as “3442”), 47528 (a Streptococcus salivarius gtf, SEQ ID NO:50, herein referred to as “7528”), 322373279 (a Streptococcus sp. gtf, SEQ ID NO:52, herein referred to as “3279”), 170016491 (a Leuconostoc citreum gtf, SEQ ID NO:54, herein referred to as “6491”), 228476889 (a Streptococcus salivarius gtf, SEQ ID NO:56, herein referred to as “6889”), 51574154 (a Lactobacillus reuteri gtf, SEQ ID NO:58, herein referred to as “4154”), and 322373298 (a Streptococcus sp. gtf, SEQ ID NO:59, herein referred to as “3298”) were prepared and tested for enzymatic activity (Table 2, see Example 18 below).
Example 18
Production of Insoluble Glucan Polymer with Gtf Enzymes
This Example describes using the gtf enzymes prepared in the above Examples to synthesize glucan polymer.
Reactions were performed with each of the above gtf enzymes following the procedures disclosed in the General Methods section. Briefly, gtf reaction solutions were prepared comprising sucrose (50 g/L), potassium phosphate buffer (pH 6.5, 50 mM) and a gtf enzyme (2.5% extract by volume). After 24-30 hours at 22-25° C., insoluble glucan polymer product was harvested by centrifugation, washed three times with water, washed once with ethanol, and dried at 50° C. for 24-30 hours.
Following the procedures disclosed in the General Methods section, the glycosidic linkages in the insoluble glucan polymer product from each reaction were determined by 13C NMR, and the DPn for each product was determined by SEC. The results of these analyses are shown in Table 2.







TABLE 2







Linkages and DPn of Glucan Produced by Various Gtf Enzymes














Reducing
Insoluble
Glucan Alpha




SEQ ID
Sugars
Glucan
Linkages













Gtf
NO.
Produced?
Produced?
%1,3
%1,6
DPn
















0874
2
yes
yes
100
0
60


6855
4
yes
yes
100
0
440


2379
6
yes
yes
37
63
310


7527
8
yes
yes
100
0
440


1724
10
yes
yes
100
0
250


0544
12
yes
yes
62
36
980


5926
14
yes
yes
100
0
260


4297
16
yes
yes
31
67
800


5618
18
yes
yes
34
66
1020


2765
20
yes
yes
100
0
280


4700
22
yes
no


1366
24
yes
no


0427
26
yes
yes
100
0
120


2919
28
yes
yes
100
0
250


2678
30
yes
yes
100
0
390


2381
32
yes
no


3929
34
yes
yes
100
0
280


6907
36
yes
no


6661
38
yes
no


0339
40
yes
no


0088
42
yes
no


9358
44
yes
no


8242
46
yes
no


3442
48
yes
no


7528
50
yes
no


3279
52
yes
no


6491
54
yes
no


6889
56
yes
no


4154
58
yes
no


3298
59
yes
no


none
na
no
no









Several gtf enzymes produced insoluble glucan products (Table 2). However, only gtf enzymes 6855 (SEQ ID NO:4), 7527 (gtfJ, 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) produced glucan comprising at least 50% alpha-1,3 linkages and having a DPn of at least 100. These enzymes are therefore suitable for producing glucan polymers for fiber applications.
Only gtfs 6855 (SEQ ID NO:4), 7527 (gtfJ, 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) produced glucan polymer comprising 100% alpha-1,3 linkages and having a DPn of at least 100. These results, in which only nine out of thirty gtfs were able to produce glucan with 100% alpha-1,3 linkages and a DPn of at least 100, indicate that not all gtf enzymes are capable of producing high molecular weight, insoluble glucan with a high level of alpha-1,3 linkages. Fewer gtf enzymes were able to produce glucan polymer comprising 100% alpha-1,3 linkages and having a DPn of at least 250.
Thus, gtf enzymes capable of producing glucan polymer comprising 100% alpha-1,3 linkages and a DPn of at least 100 were identified. These enzymes can be used to produce glucan suitable for producing fibers.

## Claims

1. A reaction solution comprising water, sucrose and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 92% identical to SEQ ID NO:30.

2. The reaction solution of claim 1, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100.

3. The reaction solution of claim 2, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100.

4. The reaction solution of claim 3, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 250.

5. The reaction solution of claim 1, further comprising a primer.

6. The reaction solution of claim 5, wherein the primer is dextran.

7. The reaction solution of claim 5, wherein the primer is hydrolyzed glucan.

8. The reaction solution of claim 1, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 95% identical to SEQ ID NO:30.

9. The reaction solution of claim 8, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 97% identical to SEQ ID NO:30.

10. The reaction solution of claim 9, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 99% identical to SEQ ID NO:30.

11. The reaction solution of claim 1, wherein a heterologous amino acid sequence of 1-300 residues is at the N-terminus and/or C-terminus of said glucosyltransferase enzyme.

12. A method for producing poly alpha-1,3-glucan comprising:
a) contacting at least water, sucrose, and a glucosyltransferase enzyme that synthesizes poly alpha-1,3-glucan, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 92% identical to SEQ ID NO:30;
whereby poly alpha-1,3-glucan is produced; and

b) optionally, isolating the poly alpha-1,3-glucan produced in step (a).

13. The method of claim 12, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having at least 50% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100.

14. The method of claim 13, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 100.

15. The method of claim 14, wherein said glucosyltransferase enzyme synthesizes poly alpha-1,3-glucan having 100% alpha-1,3 glycosidic linkages and a number average degree of polymerization of at least 250.

16. The method of claim 12, wherein step (a) further comprises contacting a primer with the water, sucrose, and glucosyltransferase enzyme.

17. The method of claim 16, wherein the primer is dextran.

18. The method of claim 16, wherein the primer is hydrolyzed glucan.

19. The method of claim 12, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 95% identical to SEQ ID NO:30.

20. The method of claim 19, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 97% identical to SEQ ID NO:30.

21. The method of claim 20, wherein said glucosyltransferase enzyme consists of an amino acid sequence that is at least 99% identical to SEQ ID NO:30.

22. The method of claim 12, wherein a heterologous amino acid sequence of 1-300 residues is at the N-terminus and/or C-terminus of said glucosyltransferase enzyme.