Microorganism-derived sucrose synthase and use thereof

Abstract

The present invention relates to a modified sucrose synthase (SUS). In particular, the modified SUS of the invention has the activity of catalyzing sucrose decomposition and producing fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP). In addition, compared with SEQ ID NO: 1, the modified SUS provided by the invention has improved activity for catalyzing the reaction and / or improved stability. The invention also relates to polynucleotides encoding the modified SUS of the invention, vectors and host cells expressing the modified SUS of the invention, and methods of producing NDP-glucose using SUS derived from microorganisms, including wild-type SUS and the modified SUS of the invention, and host cells expressing said SUS.

Description

Sucrose synthase from microorganisms and its application Technical Field

[0001] The present invention relates to the field of enzyme engineering, and in particular to a sucrose synthase derived from microorganisms, a modified variant thereof, and an application thereof in a UDP-glucose cycle. Background Art

[0002] Glycosylation modification is a common way of modifying molecules. Natural glycosylation receptor molecules include other natural polymers such as sugars, proteins, lipids, or other natural product small molecules, including antibiotics, pigments, sweeteners, etc.

[0003] The natural glycosylation process involves the transfer of glycosyl residues from an activated glycosyl donor to an acceptor molecule via catalysts such as glycosyltransferases (GTs). Nucleoside diphosphate (NDP) sugars are the most common type of glycosyl donor, such as uridine diphosphate (UDP) glucose and adenosine diphosphate (ADP) glucose. These substances circulate and are consumed in the body as glycosylation transglycosyl intermediates. This means that the ongoing transglycosylation process requires the regeneration of these nucleoside diphosphate sugars.

[0004] Currently, the regeneration of nucleoside diphosphate sugars is divided into synthetic regeneration and decomposition regeneration. For synthetic regeneration, the corresponding sugar is phosphorylated by kinase to obtain a phosphorylated sugar activated at a specific position. The phosphorylated sugar reacts with UDP or ADP under the catalytic action of the enzyme to generate the corresponding nucleoside diphosphate sugar. The synthetic regeneration process consumes energy, as well as ATP or UTP. Decomposition regeneration regenerates nucleoside diphosphate sugars by degrading certain existing glycosidic bonds through the reverse reaction of the enzyme-catalyzed glycosyl transfer. For example, sucrose synthase (SUS) catalyzes the following reaction:

[0005] Sucrose + NDP—> fructose + NDP-glucose.

[0006] This reaction consumes relatively inexpensive sucrose and produces an equimolar amount of NDP-glucose.

[0007] The sucrose synthases that have been widely characterized and used so far are mainly from plants, such as AtSUS1 from Arabidopsis thaliana and BvSUS1 from Beta vulgaris (see K. et al. “Sucrose synthase: A unique glycosyltransferase for biocatalytic glycosylation process development”, Biotechnology Advances 34 (2016) 88–111). These plant-derived genes are not expressed at ideal levels in common bacterial hosts such as Escherichia coli and are generally unstable. Very few microbial-derived SUSs have been reported and characterized, with the only few examples including sucrose synthases from Nitrosomonas Europaea, Acidithiobacillus caldus, Denitrovibrio acetiphilus, and Melioribacter roseus (M. Diricks et al., “Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes”, Appl Microbiol Biotechnol (2015) 99: 8465–8474).

[0008] Therefore, there is a need to identify SUS with improved activity and / or stability, including wild-type SUS and variants thereof.

[0009] In a first aspect, the present invention provides a modified sucrose synthase (SUS) polypeptide comprising an amino acid substitution at one or more positions selected from the group consisting of positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788, and 790, compared to a wild-type SUS polypeptide thereof, wherein the positions are referenced to SEQ ID NO: 1, wherein the amino acid at position 4 is substituted with V, the amino acid at position 24 is substituted with L, the amino acid at position 41 is substituted with D, E or W, the amino acid at position 108 is substituted with C or M, the amino acid at position 114 is substituted with E, the amino acid at position 133 is substituted with K or R, the amino acid at position 136 is substituted with M, T or K, the amino acid at position 161 is substituted with D, the amino acid at position 300 is substituted with M, G or A, the amino acid at position 433 is substituted with C or R, the amino acid at position 473 is substituted with G, and the amino acid at position 474 is substituted with the amino acid at position 476 is substituted with C, V or H, the amino acid at position 479 is substituted with A, R or N, the amino acid at position 482 is substituted with S, T or V, the amino acid at position 483 is substituted with N, H or G, the amino acid at position 513 is substituted with K, Q or I, the amino acid at position 515 is substituted with R or S, the amino acid at position 518 is substituted with Y, V, I, L, F or T, the amino acid at position 529 is substituted with V, T or H, the amino acid at position 533 is substituted with F or L, and the amino acid at position 534 is substituted with F, W or Y , the amino acid at position 543 is substituted with L, the amino acid at position 544 is substituted with I, the amino acid at position 585 is substituted with K or R, the amino acid at position 629 is substituted with D, the amino acid at position 630 is substituted with N, H or S, the amino acid at position 640 is substituted with P, the amino acid at position 641 is substituted with R or K, the amino acid at position 644 is substituted with A, V or S, the amino acid at position 664 is substituted with Y, the amino acid at position 676 is substituted with K, the amino acid at position 697 is substituted with V, I or K, the amino acid at position 713 is substituted with L or M, the amino acid at position The amino acid at position 715 is substituted with L or V, the amino acid at position 726 is substituted with D or E, the amino acid at position 729 is substituted with D, the amino acid at position 741 is substituted with C or G, the amino acid at position 768 is substituted with P, E or A, the amino acid at position 769 is substituted with P or Q, the amino acid at position 773 is substituted with L, the amino acid at position 788 is substituted with R, and the amino acid at position 790 is substituted with K or R, wherein the modified SUS has the activity of catalyzing the decomposition of sucrose and producing fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP).

[0010] The present invention also provides a modified SUS polypeptide comprising SEQ ID NO: 2-208, or the modified SUS comprises 1-10 amino acid substitutions at positions other than positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790 compared to one of 2-208, wherein the modified SUS has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP).

[0011] In a second aspect, the present invention provides polynucleotides, expression vectors, and host cells encoding the modified SUS polypeptides of the present invention.

[0012] In a third aspect, the present invention provides a method for producing NDP glucose, comprising contacting a SUS polypeptide derived from a microorganism or a host cell comprising the SUS polypeptide with sucrose in the presence of NDP, wherein the SUS polypeptide is a wild-type SUS polypeptide, such as the SUS polypeptide of SEQ ID NO: 1, or a modified SUS polypeptide of the present invention.

[0013] The present invention mainly relates to a modified SUS for catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP. Unless otherwise specified, the terms used herein have the meanings generally understood by those skilled in the art.

[0014] 1. Sucrose synthase from microorganisms and its modified variants

[0015] As used herein, the term "sucrose synthase" refers to an enzyme that catalyzes the reversible reaction of glucose and fructose to produce sucrose. That is, sucrose synthase can also catalyze the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphates (NDPs, such as UDP and ADP).

[0016] As used herein, the term "peptide" refers to a chain of at least two amino acids linked by peptide bonds. The term "polypeptide" is used interchangeably herein with the term "protein" to refer to a chain of ten or more amino acid residues. All peptide and polypeptide formulas or sequences herein are written from left to right, indicating the direction from the amino terminus to the carboxyl terminus.

[0017] The term "amino acid" includes naturally occurring amino acids and non-natural amino acids in proteins. The single-letter and three-letter nomenclature for naturally occurring amino acids in proteins adopts the names commonly used in the art and can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0018] As used herein, the term "modification" includes any chemical modification of a polypeptide, and also includes modifications of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids.

[0019] The inventors unexpectedly discovered that sucrose synthase EsSUS (SEQ ID NO: 1, NCBI Accession No. WP_063464253) from Ectothiorhodospira sp. BSL-9 and its mutants have high activity and high stability, and are suitable for catalyzing the decomposition of sucrose to produce NDP-glucose in the presence of NDP (such as UDP or ADP).

[0020] In some embodiments, the modified SUS polypeptide of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid substitutions compared to its wild-type SUS, wherein the modified SUS polypeptide has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.

[0021] In some embodiments, the modified SUS polypeptide of the present invention comprises an amino acid substitution at one or more positions selected from positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788, and 790 compared to its wild-type SUS, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the modified SUS has activity in catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP). Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid at position 24 is substituted with L. Preferably, the amino acid at position 41 is substituted with D, E, or W. Preferably, the amino acid at position 108 is substituted with C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid at position 133 is substituted with K or R. Preferably, the amino acid at position 136 is substituted with M, T, or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid at position 300 is substituted with M, G, or A. Preferably, the amino acid at position 433 is substituted with C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid at position 474 is substituted with C, V, or H. Preferably, the amino acid at position 476 is substituted with V or C. Preferably, the amino acid at position 479 is substituted with A, R, or N. Preferably, the amino acid at position 482 is substituted with S, T, or V. Preferably, the amino acid at position 483 is substituted with N, H, or G. Preferably, the amino acid at position 513 is substituted with K, Q, or I. Preferably, the amino acid at position 515 is substituted with R or S. Preferably, the amino acid at position 518 is substituted with Y, V, I, L, F, or T. Preferably, the amino acid at position 529 is substituted with V, T, or H. Preferably, the amino acid at position 533 is substituted with F or L. Preferably, the amino acid at position 534 is substituted with F, W, or Y. Preferably, the amino acid at position 543 is substituted with L. Preferably, the amino acid at position 544 is substituted with I. Preferably, the amino acid at position 585 is substituted with K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid at position 630 is substituted with N, H, or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid at position 641 is substituted with R or K. Preferably, the amino acid at position 644 is substituted with A, V, or S. Preferably, the amino acid at position 664 is substituted with Y. Preferably, the amino acid at position 676 is substituted with K. Preferably, the amino acid at position 697 is substituted with V, I or K. Preferably, the amino acid at position 713 is substituted with L or M.Preferably, the amino acid at position 715 is substituted with L or V. Preferably, the amino acid at position 726 is substituted with D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid at position 741 is substituted with C or G. Preferably, the amino acid at position 768 is substituted with P, E or A. Preferably, the amino acid at position 769 is substituted with P or Q. Preferably, the amino acid at position 773 is substituted with L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid at position 790 is substituted with K or R.

[0022] In some embodiments, the modified SUS polypeptide of the present invention comprises amino acid substitutions at positions 133 and 135, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529.

[0023] In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally at one or more positions selected from positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.

[0024] In some embodiments, the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a natural variant thereof.

[0025] In some embodiments, the modified SUS polypeptide of the present invention comprises an amino acid sequence of one of SEQ ID NOs: 2-208, or a sequence consisting of SEQ ID NOs: NO: 2-208, or the modified SUS comprises 1-10 amino acid substitutions at positions other than positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790 compared to one of 2-208, wherein the modified SUS polypeptide has the activity of catalyzing the decomposition of sucrose and producing fructose and NDP-glucose in the presence of NDP.

[0026] In some embodiments, the mutation in the modified SUS polypeptide of the present invention is an amino acid substitution or a combination of amino acid substitutions selected from the following, compared to its wild type (positions are numbered with reference to SEQ ID NO: 1):

[0027] -A4V;

[0028] -Q24L;

[0029] -S108C;

[0030] -S108M;

[0031] -A114E;

[0032] -N133K;

[0033] -N133R;

[0034] -N136M;

[0035] -N136T;

[0036] -N136K;

[0037] -G161D;

[0038] -T479N;

[0039] -A482S;

[0040] -A482T;

[0041] -A482V;

[0042] -G529V;

[0043] -G529T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> -G529H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> -G726D;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> -A741C;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> -A741G;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> -L768P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> -L768E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> -L768A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] <h2 style=";text-align:left;direction:ltr"> -T773L;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0052] <h2 style=";text-align:left;direction:ltr"> -A790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0053] <h2 style=";text-align:left;direction:ltr"> -A790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0054] <h2 style=";text-align:left;direction:ltr"> -E769P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0055] <h2 style=";text-align:left;direction:ltr"> -E769Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0056] <h2 style=";text-align:left;direction:ltr"> -L788R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0057] <h2 style=";text-align:left;direction:ltr"> -133R+136M;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0058] <h2 style=";text-align:left;direction:ltr"> -133K+136M;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0059] <h2 style=";text-align:left;direction:ltr"> -133R+136K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0060] <h2 style=";text-align:left;direction:ltr"> -133K+136K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0061] <h2 style=";text-align:left;direction:ltr"> -529T+768E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0062] <h2 style=";text-align:left;direction:ltr"> -529T+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0063] <h2 style=";text-align:left;direction:ltr"> -529H+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0064] <h2 style=";text-align:left;direction:ltr"> -529V+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0065] <h2 style=";text-align:left;direction:ltr"> -529V+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0066] <h2 style=";text-align:left;direction:ltr"> -529H+768A+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0067] <h2 style=";text-align:left;direction:ltr"> -529T+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0068] <h2 style=";text-align:left;direction:ltr"> -529H+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0069] <h2 style=";text-align:left;direction:ltr"> -136M+768A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0070] <h2 style=";text-align:left;direction:ltr"> -136M+529H+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0071] <h2 style=";text-align:left;direction:ltr"> -136M+529T+768A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0072] <h2 style=";text-align:left;direction:ltr"> -136M+529V+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0073] <h2 style=";text-align:left;direction:ltr"> -136M+529V+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0074] <h2 style=";text-align:left;direction:ltr"> -136K+529V+768E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0075] <h2 style=";text-align:left;direction:ltr"> -136K+529T+768E;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0076] <h2 style=";text-align:left;direction:ltr"> -136K+529V+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0077] <h2 style=";text-align:left;direction:ltr"> -136K+529T+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0078] <h2 style=";text-align:left;direction:ltr"> -133R+529V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0079] <h2 style=";text-align:left;direction:ltr"> -133R+529V+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0080] <h2 style=";text-align:left;direction:ltr"> -133K+529V+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0081] <h2 style=";text-align:left;direction:ltr"> -133K+529T+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0082] <h2 style=";text-align:left;direction:ltr"> -133K+529T+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0083] <h2 style=";text-align:left;direction:ltr"> -133K529H+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0084] <h2 style=";text-align:left;direction:ltr"> -133R+136M+529T+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0085] <h2 style=";text-align:left;direction:ltr"> -133R+136M+529T+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0086] <h2 style=";text-align:left;direction:ltr"> -133R+136M+529H+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0087] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529T+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0088] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529T+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0089] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0090] <h2 style=";text-align:left;direction:ltr"> -133R+136K+529V+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0091] <h2 style=";text-align:left;direction:ltr"> -133R+136K+529T+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0092] <h2 style=";text-align:left;direction:ltr"> -133R+136K+529H+768E+790R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0093] <h2 style=";text-align:left;direction:ltr"> -133R+136K+529H+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0094] <h2 style=";text-align:left;direction:ltr"> -133K+136K+529T+768E+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0095] <h2 style=";text-align:left;direction:ltr"> -133K+136K+529T+768A+790K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0096] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+300M;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0097] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+300G;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0098] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+300A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0099] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+433C;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0100] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+433R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0101] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+473G;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0102] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+474C;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0103] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+474V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0104] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+474H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0105] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+476V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0106] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+476C;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0107] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+479A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0108] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+479R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0109] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+479N;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0110] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+483N;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0111] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+483H;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0112] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+483G;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0113] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+513K;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0114] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+513Q;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0115] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+515R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0116] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+543L;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0117] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518Y;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0118] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0119] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518I;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0120] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518F;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0121] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518L;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0122] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518V;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0123] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+518T;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0124] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+640P;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0125] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+641R;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0126] <h2 style=";text-align:left;direction:ltr"> -133K+136M+529H+768E+790K+644A;<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0127] -133K+136M+529H+768E+790K+644V;

[0128] -133K+136M+529H+768E+790K+664Y;

[0129] -133K+136M+529H+768E+790K+676k;

[0130] -133K+136M+529H+768E+790K+741G;

[0131] -133K+136M+529H+768E+790K+513I+515S+518I+641R;

[0132] -133K+136M+529H+768E+790K+513I+515S+518I+641K+644V;

[0133] -133K+136M+529H+768E+790K+513I+515S+518I+641R+644V;

[0134] -133K+136M+529H+768E+790K+513I+515S+518I+641R+644A;

[0135] -133K+136M+529H+768E+790K+513K+515S+518Y+641K+644V;

[0136] -133K+136M+529H+768E+790K+513K+515S+518V;

[0137] -133K+136M+529H+768E+790K+513I+515S+518V+640P+641R+644V;

[0138] -133K+136M+529H+768E+790K+518V+641R+644V;

[0139] -133K+136M+529H+768E+790K+513K+515S+518V+640P+641K;

[0140] -133K+136M+529H+768E+790K+513I+515S+518V+640P+641R;

[0141] -133K+136M+529H+768E+790K+513I+515S+518V+640P+641K+644A;

[0142] -133K+136M+529H+768E+790K+513I+515S+518V+641K+644V;

[0143] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V;

[0144] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641K;

[0145] -133K+136M+529H+768E+790K+513I+515S+518V+641K+644S+543L;

[0146] -133K+136M+529H+768E+790K+513I+515S+518F+641R+644V;

[0147] -133K+136M+529H+768E+790K+518F+641R+644V;

[0148] -133K+136M+529H+768E+790K+513I+515S+518F+640P+641K+644A;

[0149] -133K+136M+529H+768E+790K+513K+515S+518Y+641K;

[0150] -133K+136M+529H+768E+790K+513I+515S+518T+640P+641R+644V;

[0151] -133K+136M+529H+768E+790K+513K+515S+518T+641K+644A;

[0152] -133K+136M+529H+768E+790K+513I+515R+640P+641R+644V;

[0153] -133K+136M+529H+768E+790K+513I+515S+518T+640P+641K+644V;

[0154] -133K+136M+529H+768E+790K+513I+515R+640P+641K+644A;

[0155] -133K+136M+529H+768E+790K+513I+515S+518Y+640P+641K+644V;

[0156] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V (synonymous mutation?)+664Y;

[0157] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y;

[0158] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+I483H;

[0159] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41D;

[0160] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41W;

[0161] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+41E;

[0162] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F;

[0163] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L;

[0164] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534F;

[0165] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W;

[0166] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y;

[0167] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I;

[0168] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585K;

[0169] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+585R;

[0170] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+629D;

[0171] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630N;

[0172] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630H;

[0173] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+630S;

[0174] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697V;

[0175] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697I;

[0176] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+697K;

[0177] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713L;

[0178] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+713M;

[0179] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715L;

[0180] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+715V;

[0181] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726D;

[0182] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+726E;

[0183] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+729D;

[0184] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585R+629D+697V+715V;

[0185] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+697I+715V;

[0186] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697K+715L;

[0187] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715L;

[0188] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I;

[0189] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+629D+697V+715V;

[0190] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697K+715V;

[0191] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+715V;

[0192] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585R+629D+697V+715V;

[0193] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585R+629D+697K+715V;

[0194] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697I+715L;

[0195] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+544I+585R+629D+697V;

[0196] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585R+629D+697I+715V;

[0197] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I;

[0198] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585R+697V+715L;

[0199] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+585K+629D+697V+715V;

[0200] -533F+534Y+585K+629D+697I+715V;

[0201] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+697V+715V;

[0202] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585R+629D+697V+715V;

[0203] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585R+629D+697K+715V;

[0204] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+585K+629D+697I+715V;

[0205] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+585K+629D+697K+715L;

[0206] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+544I+585K+629D+697I+715V;

[0207] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534Y+544I+629D+697I+715V;

[0208] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+585K+629D+697I+715V;

[0209] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533 L+534Y+544I+629D+697V+715V;

[0210] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534Y+585K+629D+697K+715V;

[0211] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+585K+629D+697K+715V;

[0212] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+697I+715V;

[0213] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697V+715L;

[0214] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534Y+544I+629D+697I+715L;

[0215] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697I+715V;

[0216] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+544I+585K+629D+715L;

[0217] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585R+629D+697I+715L;

[0218] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697I+715L;

[0219] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715V;

[0220] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+585K+629D+697V+715V;

[0221] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+629D+697I+715V;

[0222] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533L+534W+544I+585K+629D+697V+715V;

[0223] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585R+697V+715V;

[0224] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+544I+585K+697V+715V;

[0225] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715L;

[0226] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585K+697V+715L;

[0227] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533 F+534W+585R+629D+697I+715V;

[0228] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+629D+697K+715L;

[0229] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+629D+697V+715V;

[0230] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+629D+697V+715V;

[0231] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585K+629D+697I+715V;

[0232] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+533F+534W+585R+697V+715V; and

[0233] -133K+136M+529H+768E+790K+513I+515S+518L+640P+641R+644V+664Y+534W+585R+697V+715V.

[0234] As used herein, the term "wild-type SUS" refers to naturally occurring SUS. In some embodiments, the wild-type SUS is a SUS from the genus Ectothiorhodospira, such as SUS from Ectothiorhodospira sp. BSL-9 (EsSUS, SEQ ID NO: 1).

[0235] For the present invention, in order to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for the purpose of optimal comparison (for example, a gap may be introduced in the first amino acid or nucleic acid sequence for optimal alignment with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percentage of identity = number of identical positions / total number of positions (i.e., overlapping positions) × 100). Preferably, the two sequences are of the same length.

[0236] Those skilled in the art are aware that various computer programs can be used to determine the identity between two sequences.

[0237] "Percent amino acid identity" or "percent amino acid sequence identity" means that when the amino acids of two polypeptides are compared, the two polypeptides, when optimally aligned, have approximately the specified percentage of identical amino acids. For example, "95% amino acid identity" means that when the amino acids of two polypeptides are compared, the two polypeptides, when optimally aligned, have 95% of the same amino acids.

[0238] In some embodiments, the wild-type SUS has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, and particularly preferably at least 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:1.

[0239] In some embodiments, the modified SUS polypeptide of the present invention comprises an amino acid substitution at one or more positions selected from positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788, and 790 compared to its wild-type SUS, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the modified SUS has activity in catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP). Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid at position 24 is substituted with L. Preferably, the amino acid at position 41 is substituted with D, E, or W. Preferably, the amino acid at position 108 is substituted with C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid at position 133 is substituted with K or R. Preferably, the amino acid at position 136 is substituted with M, T, or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid at position 300 is substituted with M, G, or A. Preferably, the amino acid at position 433 is substituted with C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid at position 474 is substituted with C, V, or H. Preferably, the amino acid at position 476 is substituted with V or C. Preferably, the amino acid at position 479 is substituted with A, R, or N. Preferably, the amino acid at position 482 is substituted with S, T, or V. Preferably, the amino acid at position 483 is substituted with N, H, or G. Preferably, the amino acid at position 513 is substituted with K, Q, or I. Preferably, the amino acid at position 515 is substituted with R or S. Preferably, the amino acid at position 518 is substituted with Y, V, I, L, F, or T. Preferably, the amino acid at position 529 is substituted with V, T, or H. Preferably, the amino acid at position 533 is substituted with F or L. Preferably, the amino acid at position 534 is substituted with F, W, or Y. Preferably, the amino acid at position 543 is substituted with L. Preferably, the amino acid at position 544 is substituted with I. Preferably, the amino acid at position 585 is substituted with K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid at position 630 is substituted with N, H, or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid at position 641 is substituted with R or K. Preferably, the amino acid at position 644 is substituted with A, V, or S. Preferably, the amino acid at position 664 is substituted with Y. Preferably, the amino acid at position 676 is substituted with K. Preferably, the amino acid at position 697 is substituted with V, I or K. Preferably, the amino acid at position 713 is substituted with L or M.Preferably, the amino acid at position 715 is substituted with L or V. Preferably, the amino acid at position 726 is substituted with D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid at position 741 is substituted with C or G. Preferably, the amino acid at position 768 is substituted with P, E or A. Preferably, the amino acid at position 769 is substituted with P or Q. Preferably, the amino acid at position 773 is substituted with L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid at position 790 is substituted with K or R. In some embodiments, the modified SUS polypeptide of the present invention comprises amino acid substitutions at positions 133 and 135, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally at one or more positions selected from positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790. Preferably, the wild-type SUS has at least 65%, preferably at least 70%, 75% or 80%, more preferably at least 85%, 90% or 95%, particularly preferably at least 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:1.

[0240] In some embodiments, the modified SUS polypeptide of the present invention comprises an amino acid substitution at one or more positions selected from positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788, and 790 compared to its wild-type SUS, wherein the positions are numbered with reference to SEQ ID NO: 1, and the modified SUS is different from SEQ ID NO: NO: 1 has at least 90%, 91%, 92%, 93%, 94%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99% or 99.5% sequence identity, wherein the modified SUS has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP). Preferably, the amino acid at position 4 is substituted with V. Preferably, the amino acid at position 24 is substituted with L. Preferably, the amino acid at position 41 is substituted with D, E or W. Preferably, the amino acid at position 108 is substituted with C or M. Preferably, the amino acid at position 114 is substituted with E. Preferably, the amino acid at position 133 is substituted with K or R. Preferably, the amino acid at position 136 is substituted with M, T or K. Preferably, the amino acid at position 161 is substituted with D. Preferably, the amino acid at position 300 is substituted with M, G or A. Preferably, the amino acid at position 433 is substituted with C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid at position 474 is substituted with C, V or H. Preferably, the amino acid at position 476 is substituted with V or C. Preferably, the amino acid at position 479 is substituted with A, R or N. Preferably, the amino acid at position 482 is substituted with S, T or V. Preferably, the amino acid at position 483 is substituted with N, H or G. Preferably, the amino acid at position 513 is substituted with K, Q or I. Preferably, the amino acid at position 515 is substituted with R or S. Preferably, the amino acid at position 518 is substituted with Y, V, I, L, F or T. Preferably, the amino acid at position 529 is substituted with V, T or H. Preferably, the amino acid at position 533 is substituted with F or L. Preferably, the amino acid at position 534 is substituted with F, W or Y. Preferably, the amino acid at position 543 is substituted with L. Preferably, the amino acid at position 544 is substituted with I. Preferably, the amino acid at position 585 is substituted with K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid at position 630 is substituted with N, H or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid at position 641 is substituted with R or K.Preferably, the amino acid at position 644 is substituted with A, V or S. Preferably, the amino acid at position 664 is substituted with Y. Preferably, the amino acid at position 676 is substituted with K. Preferably, the amino acid at position 697 is substituted with V, I or K. Preferably, the amino acid at position 713 is substituted with L or M. Preferably, the amino acid at position 715 is substituted with L or V. Preferably, the amino acid at position 726 is substituted with D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid at position 741 is substituted with C or G. Preferably, the amino acid at position 768 is substituted with P, E or A. Preferably, the amino acid at position 769 is substituted with P or Q. Preferably, the amino acid at position 773 is substituted with L. Preferably, the amino acid at position 788 is substituted with R. Preferably, the amino acid at position 790 is substituted with K or R. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133 and 135, amino acid substitutions at positions 529 and 768, amino acid substitutions at positions 136 and 768, or amino acid substitutions at positions 133 and 529. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally, amino acid substitutions at one or more positions selected from the group consisting of positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741. In some embodiments, the modified SUS polypeptides of the invention comprise amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.

[0241] In some embodiments, the modified SUS of the present invention further comprises one or more conservative substitutions of amino acids compared to SEQ ID NO: 1.

[0242] The term "conservative substitution", also known as substitution by a "homologous" amino acid residue, refers to substitutions in which the amino acid residue is replaced by an amino acid residue having a similar side chain, for example, amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), non-charged polar side chain amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chain amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chain amino acids (e.g., threonine, valine, isoleucine), and aromatic side chain amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0243] Conservative amino acid substitutions generally have minimal effects on the activity of the resulting protein. Such substitutions are described below. Conservative substitutions replace an amino acid with one that is similar in size, hydrophobicity, charge, polarity, steric properties, aromaticity, etc. These substitutions are often made when fine-tuning the properties of a protein is desired.

[0244] As used herein, "homologous" amino acid residues refer to amino acid residues having similar chemical properties, such as hydrophobicity, charge, polarity, steric characteristics, aromatic characteristics, etc. Examples of amino acids that are homologous to each other include positively charged lysine, arginine, and histidine, negatively charged glutamic acid and aspartic acid, hydrophobic glycine, alanine, valine, leucine, isoleucine, proline, and phenylalanine, polar serine, threonine, cysteine, methionine, tryptophan, tyrosine, asparagine, and glutamine, aromatic phenylalanine, tyrosine, and tryptophan, chemically similar side chain groups of serine and threonine, or glutamine and asparagine, or leucine and isoleucine.

[0245] Examples of conservative amino acid substitutions in proteins include: Ser for Ala, Lys for Arg, Gln or His for Asn, Glu for Asp, Ser for Cys, Asn for Gln, Asp for Glu, Pro for Gly, Asn or Gln for His, Leu or Val for Ile, Ile or Val for Leu, Arg or Gln for Lys, Leu or Ile for Met, Met, Leu or Tyr for Phe, Thr for Ser, Ser for Thr, Tyr for Trp, Trp or Phe for Tyr, and Ile or Leu for Val.

[0246] In some embodiments, the modifications in the modified SUS polypeptide of the invention consist of amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally at one or more positions selected from the group consisting of amino acid substitutions at positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741, compared to SEQ ID NO: 1. In some embodiments, the modified SUS polypeptide of the invention comprises amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790. Preferably, the amino acid at position 41 is substituted with D, E, or W. Preferably, the amino acid at position 133 is substituted with K or R. Preferably, the amino acid at position 136 is substituted with M, T, or K. Preferably, the amino acid at position 300 is substituted with M, G, or A. Preferably, the amino acid at position 433 is substituted with C or R. Preferably, the amino acid at position 473 is substituted with G. Preferably, the amino acid at position 474 is substituted with C, V, or H. Preferably, the amino acid at position 476 is substituted with V or C. Preferably, the amino acid at position 479 is substituted with A, R, or N. Preferably, the amino acid at position 483 is substituted with N, H, or G. Preferably, the amino acid at position 513 is substituted with K, Q, or I. Preferably, the amino acid at position 515 is substituted with R or S. Preferably, the amino acid at position 518 is substituted with Y, V, I, L, F, or T. Preferably, the amino acid at position 529 is substituted with V, T, or H. Preferably, the amino acid at position 533 is substituted with F or L. Preferably, the amino acid at position 534 is substituted with F, W, or Y. Preferably, the amino acid at position 543 is substituted with L. Preferably, the amino acid at position 544 is substituted with I. Preferably, the amino acid at position 585 is substituted with K or R. Preferably, the amino acid at position 629 is substituted with D. Preferably, the amino acid at position 630 is substituted with N, H, or S. Preferably, the amino acid at position 640 is substituted with P. Preferably, the amino acid at position 641 is substituted with R or K. Preferably, the amino acid at position 644 is substituted with A, V, or S. Preferably, the amino acid at position 664 is substituted with Y. Preferably, the amino acid at position 676 is substituted with K. Preferably, the amino acid at position 697 is substituted with V, I, or K. Preferably, the amino acid at position 713 is substituted with L or M. Preferably, the amino acid at position 715 is substituted with L or V. Preferably, the amino acid at position 726 is substituted with D or E. Preferably, the amino acid at position 729 is substituted with D. Preferably, the amino acid at position 741 is substituted with C or G. Preferably, the amino acid at position 768 is substituted with P, E or A. Preferably, the amino acid at position 790 is substituted with K or R.

[0247] In some embodiments, the modifications in the modified SUS polypeptide of the invention are amino acid substitutions at positions 133, 136, 529, 768, and 790, and optionally at one or more positions selected from the group consisting of amino acid substitutions at positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741, as compared to SEQ ID NO: 1. wherein the amino acid at position 41 is substituted with D, E or W, the amino acid at position 133 is substituted with K or R, the amino acid at position 136 is substituted with M, T or K, the amino acid at position 300 is substituted with M, G or A, the amino acid at position 433 is substituted with C or R, the amino acid at position 473 is substituted with G, the amino acid at position 474 is substituted with C, V or H, the amino acid at position 476 is substituted with V or C, the amino acid at position 479 is substituted with A, R or N, the amino acid at position 483 is substituted with N, H or G, the amino acid at position 513 is substituted with K, Q or I, the amino acid at position 515 is substituted with R or S, the amino acid at position 518 is substituted with Y, V, I, L, F or T, the amino acid at position 529 is substituted with V, T or H, the amino acid at position 533 is substituted with F or L, the amino acid at position 534 is substituted with F, W or Y, the amino acid at position 543 is substituted with L, the amino acid at position 544 is substituted with I, the amino acid at position 585 is substituted with K or R, the amino acid at position 629 is substituted with D, the amino acid at position 630 is substituted with N, H or S, the amino acid at position 640 is substituted with P, the amino acid at position 6 In some embodiments, the modified SUS polypeptide comprises an amino acid substitution at position 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790.

[0248] As used herein, enzyme activity refers to the amount of substrate reduced or product increased per unit time in a chemical reaction catalyzed by a unit mass of enzyme under certain conditions. For example, the activity of the modified SUS of the present invention is represented by the amount of sucrose reduced or the amount of NDP-glucose, such as UDP-glucose, increased per unit time in the presence of a unit mass of the modified SUS polypeptide and an NDP, such as UDP, under certain conditions.

[0249] As used herein, enzyme activity may also refer to the relative activity of an enzyme, expressed as a ratio of the activity of the enzyme of interest to the activity of a given enzyme catalyzing the same reaction, such as percent relative activity.

[0250] In some embodiments, the activity of the modified SUS of the present invention is expressed as a percentage relative activity compared to SEQ ID NO: 1.

[0251] Improving the stability of SUS is also beneficial for its application in industrial production. In some embodiments, the stability is thermal stability, which refers to the ability of an enzyme to maintain activity after being incubated at a certain temperature (such as 40-70°C or higher) for a certain period of time (such as 10 minutes to 1 hour). In some embodiments, the modified SUS has a thermal stability that is better than that of the polypeptide of SEQ ID NO: 1. For example, after incubation at 50-70°C, for example, after 1 hour, the activity of the modified SUS of the present invention is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or more of the activity of the polypeptide of SEQ ID NO: 1. Alternatively, the modified SUS of the present invention has a higher T50, where T50 refers to the temperature at which the activity of the enzyme decreases by 50% after incubation at that temperature, for example, after one hour. In some embodiments, the T50 of the modified SUS of the invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10° C. or more higher than that of the polypeptide of SEQ ID NO: 1.

[0252] In some embodiments, without prior incubation, the activity of the modified SUS in catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more of the activity of SEQ ID NO: 1 in catalyzing the above reaction.

[0253] In some embodiments, the modified SUS has a better thermal stability than the polypeptide of SEQ ID NO: 1, for example, after incubation at 50-70°C for 1 hour, the activity of the modified SUS is at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300% or more of the activity of the polypeptide of SEQ ID NO: 1, or the T50 of the modified SUS is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10°C or more higher than that of the polypeptide of SEQ ID NO: 1; and without prior incubation, its activity in catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP is higher than that of the polypeptide of SEQ ID NO: 1. NO:1 has an activity of catalyzing the above reaction of at least 100%, 105%, 110%, 120%, 130%, 150%, 170%, 200%, 250%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more.

[0254] 2. Polynucleotide encoding modified SUS.

[0255] As used herein, the term "polynucleotide" or "nucleic acid molecule" includes DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA produced using nucleotide analogs. The nucleic acid molecules can be single-stranded or double-stranded, preferably double-stranded DNA. Nucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides) can be used in the synthesis of the nucleic acids. Such nucleotides can be used, for example, to prepare nucleic acids with altered base pairing ability or increased nuclease resistance.

[0256] The present invention also provides polynucleotides encoding the modified SUS of the present invention. Therefore, in the present invention, the term modification also includes genetic manipulation of the polynucleotide encoding the SUS polypeptide of the present invention. The modification can be a substitution, deletion, insertion and / or addition of nucleotides.

[0257] As used herein, the term "encoding" refers to a polynucleotide that directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or other start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG and TGA. The coding sequence can be a DNA, cDNA or recombinant nucleotide sequence.

[0258] Furthermore, nucleic acid molecules encompassing all or part of the nucleic acid sequences of the present invention can be isolated by polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based on the sequence information contained in the sequences.

[0259] The polynucleotides of the present invention can be amplified using cDNA, mRNA or genomic DNA as templates and suitable oligonucleotide primers according to standard PCR amplification techniques. The nucleic acids amplified in this way can be cloned into suitable vectors and characterized by DNA sequence analysis.

[0260] The polynucleotides of the present invention can be prepared by standard synthetic techniques, for example, using an automated DNA synthesizer.

[0261] The present invention also relates to complementary strands of the nucleic acid molecules described herein.A nucleic acid molecule that is complementary to another nucleotide sequence is one that is sufficiently complementary to that nucleotide sequence that it can hybridize to the other nucleotide sequence to form a stable duplex.

[0262] As used herein, the term "hybridize" means that nucleotide sequences at least about 90%, preferably at least about 95%, more preferably at least about 96%, and more preferably at least 98% homologous to each other typically remain hybridized to each other under given stringent hybridization and washing conditions.

[0263] Those skilled in the art are aware of various conditions for hybridization, such as stringent hybridization conditions and highly stringent hybridization conditions. See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, NY; and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, John Wiley & Sons, NY.

[0264] Of course, the polynucleotides of the present invention do not include polynucleotides that hybridize only to a poly A sequence (such as the 3' end poly (A) of mRNA) or to a complementary stretch of poly T (or U) residues.

[0265] 3. Expression and Production of Modified SUS

[0266] To express the modified SUS of the present invention, nucleic acid constructs and vectors, such as expression vectors, comprising the polynucleotides of the present invention are also provided.

[0267] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0268] The term "nucleic acid construct" refers to a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not occur in nature. When the nucleic acid construct contains the control sequences required for expressing a coding sequence of the present invention, the term nucleic acid construct is synonymous with the term "expression cassette."

[0269] The term "expression vector" as used herein refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention operably linked to additional nucleotides, e.g., control sequences, that provide for the expression of the polynucleotide. The expression vector includes a viral vector or a plasmid vector.

[0270] The term "control sequence" as used herein is intended to include all elements necessary or advantageous for expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide, or native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter and transcriptional and translational stop signals.

[0271] For example, the control sequence can be a suitable promoter sequence, a nucleotide sequence that is recognized by the host cell to express a polynucleotide encoding the polypeptide of the present invention. The promoter sequence contains a transcriptional control sequence that mediates the expression of the polypeptide. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, the Escherichia coli lac operon. The promoter also includes mutant, truncated and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0272] The term "operably linked" as used herein refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide sequence such that the control sequences direct the expression of the polypeptide coding sequence.

[0273] The polynucleotide encoding the polypeptide of the present invention may be subjected to various manipulations to achieve expression of the polypeptide. Manipulation of the polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotide sequences using recombinant DNA methods are well known in the art.

[0274] In order to identify and select host cells containing the expression vectors of the present invention, the vectors of the present invention preferably contain one or more selectable markers that allow for easy selection of transformed, transfected, transduced, or other cells. A selectable marker is a gene whose product confers biocide or viral resistance, heavy metal resistance, complements an auxotrophic deficiency, or the like. For example, bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance.

[0275] The vector of the present invention can be integrated into the host cell genome or replicate autonomously in the cell independently of the genome. The elements required for integration into the host cell genome or autonomous replication are known in the art (see, for example, Sambrook et al., 1989, supra).

[0276] Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" refer to various art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into host cells, as described in, for example, Sambrook et al., 1989; Davis et al., Basic Methods in Molecular Biology (1986), and other laboratory manuals.

[0277] The present invention also relates to recombinant host cells comprising a polynucleotide of the present invention, which are advantageously used in the recombinant production of a SUS polypeptide. A vector comprising a polynucleotide of the present invention is introduced into a host cell, whereupon the vector is retained as a chromosomal integrant or as a self-replicating extrachromosomal vector. Conventional vectors and host cells for expressing proteins are known to those skilled in the art.

[0278] In some embodiments, the host cell of the present invention is an E. coli cell, such as E. coli BL21 (DE3). In some embodiments, the expression vector is pET-30a (+).

[0279] The modified SUS of the present invention can be operably linked to a non-SUS polypeptide (e.g., a heterologous amino acid sequence) to form a fusion protein. For example, in one embodiment, the fusion protein is a GST-SUS fusion protein, in which the SUS sequence is fused to the C-terminus of the GST sequence. This fusion protein can facilitate the purification of recombinant SUS. In another embodiment, the fusion protein is a SUS protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and yeast host cells), the expression and / or secretion of SUS can be increased by using a heterologous signal sequence.

[0280] In some embodiments, the host cells of the present invention also include host cells expressing the SUS polypeptide of SEQ ID NO: 1.

[0281] 4. Producing NDP-glucose

[0282] The present invention provides a method for producing NDP-glucose, comprising contacting the SUS polypeptide of SEQ ID NO: 1, the modified SUS of the present invention, or a host cell with D-glufosinate.

[0283] In some embodiments, the method of producing NDP-glucose of the present invention comprises the steps of:

[0284] (a) providing SUS activity, sucrose and NDP such as ADP or UDP to the reaction medium, and

[0285] (b) incubating the reaction medium to decompose sucrose and produce NDP-glucose, such as ADP-glucose or UDP-glucose,

[0286] The SUS activity is provided by the SUS polypeptide of SEQ ID NO: 1 or the modified SUS or host cell of the present invention.

[0287] In some embodiments, L-phosphinothricin is produced using a cell-free catalytic method, and in step (a), a SUS polypeptide of SEQ ID NO: 1 or a modified SUS of the present invention is provided. In some embodiments, free or immobilized SUS polypeptide of SEQ ID NO: 1 or a modified SUS of the present invention can be used.

[0288] In some embodiments, the incubating is performed at a temperature of 50-70° C. In some embodiments, the incubating is performed at a temperature of 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70° C. or more.

[0289] In some embodiments, the medium is a buffer, such as PBS, Tris-HCl buffer. In one embodiment, the medium is a Tris-HCl buffer, such as 50 mM Tris-HCl buffer, pH 8.0.

[0290] In some embodiments, the reaction medium is a medium composed partially or entirely of cell culture medium, and the SUS activity is provided by the host cells of the present invention, which are cultured in the reaction medium.

[0291] In some embodiments, the host cell of the present invention and / or the second host cell is cultured and expanded in a cell culture medium, and the expanded host cells are then separated from the cell culture medium and the biomass is resuspended in a buffer or water. Sucrose and NDP are added to the buffer or water before, during, or after the addition of the expanded host cells.

[0292] In some embodiments, bacterial cells, such as E. coli cells, may be used.

[0293] By the method of the present invention, NDP-glucose, such as ADP-glucose or UDP-glucose, can be produced at a higher temperature than that in the prior art. Example

[0294] The present invention will be more clearly understood by those skilled in the art through the following examples. It should be understood that the examples are only for illustration and are not intended to limit the scope of the present invention.

[0295] Example 1. Materials and methods

[0296] Unless otherwise specified, the experimental methods used in the present invention are conventional methods. For details of gene cloning procedures, please refer to Sambrook et al., 1989.

[0297] i) Reagents: DNA polymerase (PrimeSTAR Max DNA Polymerase) and DpnI endonuclease were purchased from TaKaRa, a plasmid extraction kit was purchased from Axygen, sucrose and MgCl2 were purchased from MacLean, and UDP (uridine-5′-diphosphate sodium salt) and UDP-G (uridine diphosphate glucose disodium salt) were purchased from Aladdin.

[0298] ii) Vectors and strains: The expression vector used was pET-30a(+), the plasmid was purchased from Novagen, and the host cell used was Escherichia coli BL21(DE3), purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0299] iii) Sequencing and primer synthesis were completed by Suzhou Hongxun Biotechnology Co., Ltd., and synthesis of the wild-type gene (encoding the nucleotide sequence of SEQ ID NO: 1) was completed by Universal Biotechnology.

[0300] iv) Site-directed mutagenesis:

[0301] Design specific primer pairs to introduce the desired substitution at the base corresponding to the amino acid position of the desired mutation. Using the extracted pre-mutation plasmid (containing the wild-type GluDH coding sequence, pET-30a(+) backbone) as a template, the mutation was introduced by PCR using Quickchange technology (Nucleic Acids Research, 2004, 32(14):e115). After PCR amplification, the amplified product was digested with Dpn I for 4 hours to remove the template plasmid. The digested product was transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB agar medium (containing 50 mg / L kanamycin), and a single colony was picked and cultured in LB liquid medium (containing 50 mg / L kanamycin). The correctness of the mutation was verified by sequencing. The verified clone was stored at -80°C for future use.

[0302] v) Protein expression and preparation of crude enzyme solution:

[0303] The stored clones were activated on LB agar. A single colony was then inoculated into LB liquid medium (containing 50 mg / L kanamycin) and incubated at 37°C with shaking for 12 h. One mL of the culture was transferred to 50 mL of fresh LB liquid medium (containing 50 mg / L kanamycin) and incubated at 37°C with shaking until the OD600 reached approximately 0.6. IPTG (final concentration 0.4 mM) was added and incubated at 25°C for 16 h to induce protein expression.

[0304] After incubation, the culture was centrifuged at 4,000 g for 10 minutes at 4°C, and the supernatant was discarded to collect the E. coli cells. The collected E. coli cells were resuspended in 15 mL of pre-chilled 50 mM PBS, pH 6.5, and disrupted by sonication at 4°C. The cell lysate was centrifuged at 6,000 g for 15 minutes at 4°C to remove the precipitate. The resulting supernatant was the crude enzyme solution containing the recombinant enzyme.

[0305] vi) Enzyme activity assay

[0306] Prepare a mixture with the following final concentrations: 50 mM PBS, 50 g / L sucrose, 2 mM UDP, 10 mM MgCl2, pH 6.5. Add crude sucrose synthase prepared as described in section v) to this solution (the amount of enzyme used was adjusted based on the UDP conversion percentage, ensuring that the UDP conversion percentage was below 15%). Incubate on a shaker (400 rpm) at the specified temperature for 1 hour, then heat at 95°C for 10 minutes to inactivate the protein. UDP-G concentration was then measured by high-performance liquid chromatography to determine the initial catalytic reaction velocity.

[0307] vii) T50 determination

[0308] The enzyme was incubated for one hour at a range of temperatures, with an enzyme incubated at 4°C for one hour serving as a control. The enzyme activity after incubation was measured as described in (vi) and plotted. The temperature at which the enzyme activity after incubation reached 50% of the control activity was defined as T50. For the wild type, the incubation temperature was 50-60°C, with 1°C intervals. For mutants with improved stability, the temperature was increased as needed.

[0309] Example 2: Characterization of sucrose synthase EsSUS from Ectothiorhodospira sp. BSL-9

[0310] The nucleic acid encoding sucrose synthase EsSUS (SEQ ID NO: 1) from Ectothiorhodospira sp. BSL-9 was cloned into pET-30a(+) plasmid and expressed as described in Example 1, and the enzyme activities at 50°C and 56°C and T50 were measured.

[0311] The results showed that the enzyme activity of the wild-type EsSUS was about 1.27 U / ml; the T50 was about 54°C; and the enzyme activity of the wild-type EsSUS decreased by about 60% at 56°C compared to that at 50°C.

[0312] Example 3: Introducing a single mutation into EsSUS to improve thermal stability

[0313] Using the encoding nucleic acid of SEQ ID NO: 1 as a template, mutants containing single mutations were prepared according to the method of Example 1. The resulting mutants are shown in Table 1. The enzyme activities of the resulting mutants at 56°C were measured according to the method described in Example 1. The results are shown in Table 1, where the relative enzyme activity is the ratio of the activity of the mutant to the activity of the wild type.

[0314] Table 1. EsSUS mutants containing single mutations

[0315] It can be seen that the introduction of mutations into EsSUS resulted in the generation of active mutants and improved the thermal stability of the enzyme.

[0316] Example 4: Introducing multiple mutations into EsSUS to improve thermal stability

[0317] Using the nucleic acid encoding SEQ ID NO: 1 as a template, mutants containing 2-5 mutations were prepared according to the method of Example 1. The resulting mutants are shown in Table 2. The relative enzyme activity of the resulting enzyme at 56°C was measured according to the method described in Example 1. The results are shown in Table 2, where the relative enzyme activity is the ratio of the activity of the mutant to the activity of the wild type.

[0318] Table 2. EsSUS mutants containing 2-5 mutations

[0319] As shown in Table 2, the introduction of multiple mutations can increase the enzyme activity after incubation at 56°C for 1 h by more than 2-fold compared to the wild type.

[0320] The T50 of the mutants was determined as described in Example 1. The results showed that the T50 of the mutants was increased by at least 3°C ​​compared to the wild type, with the T50 of the mutants of SEQ ID NOs: 63-70 reaching 63-64°C. In other words, the introduction of mutations at five positions (133, 136, 529, 768, and 790) significantly increased the T50 of the enzyme.

[0321] It can be seen that the effects of introducing mutations in EsSUS are additive, significantly improving the thermal stability of the enzyme.

[0322] Example 5: Introducing additional mutations into EsSUS mutants to improve enzyme activity

[0323] Amino acid substitutions were further introduced based on SEQ ID NO: 64, and the enzyme activity of the mutant was measured at 56° C. as described in Example 1. The results are shown in Table 3, where the relative enzyme activity refers to the ratio of the activity of the mutant to the activity of SEQ ID NO: 64.

[0324] Table 3

[0325] Furthermore, amino acid substitutions were further introduced based on SEQ ID NO: 118, and the enzyme activity of the mutant was measured at 56° C. as described in Example 1. The results are shown in Table 4, where the relative enzyme activity refers to the ratio of the activity of the mutant to the activity of SEQ ID NO: 118.

[0326] Table 4

[0327] Furthermore, the T50 of the mutants obtained was measured as described in Example 2. The T50 of the mutant of SEQ ID NO: 131 was 63.5°C. The T50 of the other mutants in Tables 3 and 4 were also comparable to that of SEQ ID NO: 64.

[0328] These results indicate that the introduction of additional mutations greatly improved activity without compromising thermostability, and that the effects of the mutations were at least additive.

[0329] Example 6: Introducing further mutations to improve enzyme thermostability

[0330] Furthermore, amino acid substitutions were further introduced based on SEQ ID NO: 131, and the enzyme activity of the mutant was measured at 63.5°C as described in Example 1. The results are shown in Table 5, where the relative enzyme activity refers to the ratio of the activity of the mutant to the activity of SEQ ID NO: 131 after incubation at 63.5°C for 1 h.

[0331] Table 5

[0332] The T50 of the obtained mutants was determined as described in Example 1. The results showed that the T50 of the mutants was significantly higher than that of SEQ ID NO: 131, wherein the T50 of SEQ ID NOs: 159-189 and 191-203 was 66-66.5°C.

[0333] It can be seen that the effects of introducing mutations in EsSUS are additive, significantly improving the thermal stability of the enzyme.

Claims

1. A modified sucrose synthase (SUS) polypeptide, Compared to its wild-type SUS, comprising an amino acid substitution at one or more positions selected from positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788, and 790, wherein the positions are numbered with reference to SEQ ID NO: 1, wherein the amino acid at position 4 is substituted with V, the amino acid at position 24 is substituted with L, the amino acid at position 41 is substituted with D, E or W, the amino acid at position 108 is substituted with C or M, the amino acid at position 114 is substituted with E, the amino acid at position 133 is substituted with K or R, the amino acid at position 136 is substituted with M, T or K, the amino acid at position 161 is substituted with D, the amino acid at position 300 is substituted with M, G or A, the amino acid at position 433 is substituted with C or R, the amino acid at position 473 is substituted with G, and the amino acid at position 4 the amino acid at position 474 is substituted with C, V or H, the amino acid at position 476 is substituted with V or C, the amino acid at position 479 is substituted with A, R or N, the amino acid at position 482 is substituted with S, T or V, the amino acid at position 483 is substituted with N, H or G, the amino acid at position 513 is substituted with K, Q or I, the amino acid at position 515 is substituted with R or S, the amino acid at position 518 is substituted with Y, V, I, L, F or T, the amino acid at position 529 is substituted with V, T or H, and the amino acid at position 533 is substituted with F or L, the amino acid at position 534 is substituted with F, W or Y, the amino acid at position 543 is substituted with L, the amino acid at position 544 is substituted with I, the amino acid at position 585 is substituted with K or R, the amino acid at position 629 is substituted with D, the amino acid at position 630 is substituted with N, H or S, the amino acid at position 640 is substituted with P, the amino acid at position 641 is substituted with R or K, the amino acid at position 644 is substituted with A, V or S, the amino acid at position 664 is substituted with Y, and the amino acid at position 676 is substituted with K, the amino acid at position 697 is substituted with V, I or K, the amino acid at position 713 is substituted with L or M, the amino acid at position 715 is substituted with L or V, the amino acid at position 726 is substituted with D or E, the amino acid at position 729 is substituted with D, the amino acid at position 741 is substituted with C or G, the amino acid at position 768 is substituted with P, E or A, the amino acid at position 769 is substituted with P or Q, the amino acid at position 773 is substituted with L, the amino acid at position 788 is substituted with R, the amino acid at position 790 is substituted with K or R, The modified SUS has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of nucleoside diphosphate (NDP).

2. The modified SUS polypeptide of claim 1, comprising an amino acid substitution at positions 133 and 135, an amino acid substitution at positions 529 and 768, an amino acid substitution at positions 136 and 768, or an amino acid substitution at positions 133 and 529.

3. The modified SUS polypeptide of claim 1 or 2, comprising amino acid substitutions at positions 133, 136, 529, 768, and 790.

4. The modified SUS polypeptide of claim 3, further comprising an amino acid substitution at one or more positions selected from the group consisting of positions 41, 300, 433, 473, 474, 476, 479, 483, 513, 515, 518, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, and 741.

5. The modified SUS polypeptide of claim 3 or 4, comprising amino acid substitutions at positions 133, 136, 513, 515, 518, 529, 640, 641, 644, 768, and 790. 6 . The modified SUS polypeptide of claim 1 , wherein the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO: 1 or a natural variant thereof.

7. A modified SUS polypeptide comprising SEQ ID NO: 2-208, or the modified SUS comprises 1-10 amino acid substitutions at positions other than positions 4, 24, 41, 108, 114, 133, 136, 161, 300, 433, 473, 474, 476, 479, 482, 483, 513, 515, 518, 529, 533, 534, 543, 544, 585, 629, 630, 640, 641, 644, 664, 676, 697, 713, 715, 726, 729, 741, 768, 769, 773, 788 and 790 compared to one of 2-208, wherein the modified SUS polypeptide has the activity of catalyzing the decomposition of sucrose to produce fructose and NDP-glucose in the presence of NDP.

8. A polynucleotide encoding the modified SUS polypeptide of any one of claims 1-7.

9. An expression vector comprising the polynucleotide of claim 8.

10. A host cell comprising the modified SUS polypeptide of any one of claims 1 to 7, the polynucleotide of claim 8, or the vector of claim 14.

11. A method for producing NDP glucose, comprising contacting a SUS polypeptide derived from a microorganism or a host cell comprising the SUS polypeptide with sucrose in the presence of NDP, wherein the SUS polypeptide is a wild-type SUS polypeptide or the modified SUS polypeptide according to any one of claims 1 to 7.

12. The method of claim 11, wherein the wild-type SUS polypeptide comprises the amino acid sequence of SEQ ID NO:

1.

13. The process of claim 11 or 12, which is carried out at a temperature of 50-70°C.