Polypeptides having trehalase activity and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-2020140495-A1
Application number	US-201816624164-A
Country	US
Kind code	A1
Filing date	Jun 26, 2018
Priority date	Jun 28, 2017
Publication date	May 7, 2020
Grant date	—

Abstract

The present invention relates to polypeptides having trehalase activity, particularly derived from Talaromyces . The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides for the production of ethanol.

First claim

1 . A polypeptide having trehalase activity, selected from the group consisting of:
(a) a polypeptide having at least 93% sequence identity to the mature polypeptide of SEQ ID NO: 21 or at least 70% sequence identity to the mature polypeptide of SEQ ID NO: 23; (b) a polypeptide encoded by a polynucleotide having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 20 or the cDNA sequence thereof; or at least 80% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 22 or the cDNA sequence thereof; (c) a variant of the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions; and (d) a fragment of the polypeptide of (a), (b), or (c), that has trehalase activity.

2 . The polypeptides of claim 1 , having a thermal denaturing temperature, Td, determined by TSA of at least 60° C., at least 61° C., at least 62° C., at least 63° C., at least 64° C., at least 65° C., at least 66° C., at least 67° C., such as at least 68° C.

3 . A composition comprising the polypeptide of claim 1 .

4 . A whole broth formulation or cell culture composition comprising the polypeptide of claim 1 .

5 . A polynucleotide encoding the polypeptide of claim 1 .

6 . A nucleic acid construct or expression vector comprising the polynucleotide of claim 5 operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host.

7 . A recombinant host cell comprising the nucleic acid construct claim 6 .

8 . The recombinant host cell of claim 7 , wherein the cell is a yeast cell.

9 . A method of producing a polypeptide having trehalase activity, comprising cultivating the host cell of claim 7 under conditions conducive for production of the polypeptide.

10 . A process of producing a fermentation product, comprising
(a) liquefying a starch-containing material with an alpha-amylase; optionally pre-saccharifying the liquefied material before step (b); (b) saccharifying the liquefied material; (c) fermenting using a fermentation organism; wherein
i) a glucoamylase;
ii) a trehalase of claim 1 ;
iii) optionally a cellulolytic enzyme composition and/or a protease;

are present and/or added during
saccharification step (b);
fermentation step (c);
simultaneous saccharification and fermentation;
optionally presaccharification step before step (b).

11 . The process of claim 10 , wherein an alpha-amylase is present and/or added during saccharification step (b); fermentation step (c); simultaneous saccharification and fermentation; or the optional presaccharification step before step (b).

12 . The process of claim 10 , wherein the alpha-amylase is the one disclosed as SEQ ID NO: 7 having one or more of the following substitutions: G128D, D143N, or G128D+D143N (using SEQ ID NO: 7 for numbering), and wherein the alpha-amylase variant has at least 60% identity, at least 70%, at least 75% identity, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% identity to the mature part of the polypeptide of SEQ ID NO: 7 herein.

13 . The process of claim 10 , wherein the fermenting organism is derived from a strain of Saccharomyces.

14 . The process according to claim 13 , wherein the yeast fermenting organism expresses the trehalse according to claim 1 .

15 . A process of producing fermentation products from starch-containing material comprising:
(i) saccharifying a starch-containing material at a temperature below the initial gelatinization temperature; and (ii) fermenting using a fermentation organism; wherein saccharification and/or fermentation is done in the presence of the following enzymes: glucoamylase, alpha-amylase, trehalase of claim 1 , and optionally a protease and/or a cellulolytic enzyme composition.

16 . The recombinant host cell of claim 8 , wherein the yeast cell is a strain selected from the group consisting of Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces , and Yarrowia.

17 . The recombinant host cell of claim 8 , wherein the yeast cell is a strain selected from the group consisting of Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis , and Yarrowia lipolytica.

18 . The recombinant host cell of claim 8 , wherein the yeast cell is a strain of Saccharomyces cerevisiae.

Assignees

• Novozymes As

Inventors

• Morant Marc Dominique
• Matsui Tomoko
• Schnorr Kirk Matthew
• Fukuyama Shiro
• Tsutsumi Noriko
• Kang Zhengfang

Classifications

• C12N9/2402 Primary

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• C12P19/14

  produced by the action of a carbohydrase {(EC 3.2.x)}, e.g. by alpha-amylase {, e.g. by cellulase, hemicellulase} · CPC title

• C12Y302/01093

  Alpha,alpha-phosphotrehalase (3.2.1.93) · CPC title

• C12P7/06

  Ethanol, i.e. non-beverage · CPC title

• C12Y302/01028

  Alpha,alpha-trehalase (3.2.1.28) · CPC title