Glucoamylase variants and polynucleotides encoding same

Patent metadata

Field	Value
Publication number	US-2020131491-A1
Application number	US-201816603639-A
Country	US
Kind code	A1
Filing date	Apr 10, 2018
Priority date	Apr 11, 2017
Publication date	Apr 30, 2020
Grant date	—

Abstract

The present invention relates to glucoamylase variants having an increase in raw starch activity compared to the guy-coamylase disclosed as SEQ ID NO: 2, comprising one or more modifications in the catalytic domain and/or one or more modifications in the starch binding domain selected from: a) at least one, preferably at least two, preferably at least three, preferably at least four of: V18M, T43K, N112L, T116R, A117Q, G120S, A271F, Y295W, Q318Y; and/or b) Introducing at least three, preferably at least four substitutions selected from the group: S458C, S458SCGG, S458SGGC, A493V, A518K, E520Q, N527M, S540K, R, S(G)546P, T(V)549W, N503R, N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.

First claim

1 . A glucoamylase variant, having increased raw starch hydrolytic activity at pH=4.0, T=32° C., compared to the glucoamylase disclosed in SEQ ID NO: 2, wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO: 2 or 1-454 of SEQ ID NO: 4, a linker comprising amino acids 455-462 of SEQ ID NO: 2 or amino acids 455-465 of SEQ ID NO: 4 and a starch binding domain comprising amino acids 463-556 of SEQ ID NO: 2 or amino acids 466-559 of SEQ ID NO: 4, and wherein the variant further comprises a substitution at one or more positions selected from the group consisting of:
V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;
V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W;
V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R;
V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
T43K+Q318Y+S95P+A121P+Y295W+S458SCGG+N527M+T(V)549W;
S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W;
S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A;
T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R;
S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
V18M+T43K+S95P+A121P+A518K+N527M+T549W;
T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;
T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;
S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W;
T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;
T43K+S95P+A121P+Q318Y+N527M+T549W;
V18M+T43K+S95P+A121P+Q318Y+N527M+T549W;
S95P+A121P+Y295W+T116R+N527M+T(V)549W;
V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W;
V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;
T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W;
S95P+A121P+Y295W+A117Q+N527M+T(V)549W;
V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W;
T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;
V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W;
T43K+S95P+A121P+Q318Y+A493V+N527M+T549W;
S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W;
V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W;
T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W;
V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W;
S95P+A121P+Y295W+Q318Y+N527M+T(V)549W;
S95P+G120S+A121P+Y295W+N527M+T(V)549W;
S95P+A121P+Y295W+A518K+N527M+T(V)549W;
S95P+A121P+Y295W+N527M+T(V)549W;
S95P+N112L+A121P+Y295W+N527M+T(V)549W;
S95P+A121P+Y295W+N527M+T(V)549W;
V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W;
T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W;
S95P+G120S+A121P+Y295W+A518K+N527M+T(V)549W;
S95P+N112L+A121P+Y295W+A518K+N527M+T(V)549W;
V18M+T43K+S95P+A121P+Q318Y+A518K+T549W;
S95P+A121P+Y295W+A493V+A518K+E520Q+T549W;
S95P+A121P+Y295W+A518K+N527M+T549W;
S95P+A121P+Y295W+A518K+T(V)549W;
S95P+A121P+Y295W+A518K+N527M;
S95P+A121P+Y295W+S458C+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
S95P+A121P+Y295W+N527M+T549W;
S95P+A121P+A271F+Y295W+A518K+N527M+T549W;
S95P+A121P+Y295W+A493V;
S95P+A121P+Y295W+S540K;
S95P+A121P+Y295W+S540R;
S95P+A121P+Y295W+A518K+N527M;
S95P+A121P+Y295W+S458SGGC+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
T43K+S95P+A121P+Y295W+N527M+T(V)549W; and
S95P+A121P+Y295W+S458SCGG+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A;
wherein the position numbering corresponds to amino acid positions in the amino acid sequence set forth in SEQ ID NO: 2; and wherein the variants have at least 85%, but less than 100%, sequence identity to the amino acid sequence of SEQ ID NO: 2.

2 . The glucoamylase variant of claim 1 , wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO: 2, a linker comprising amino acids 455-465 of SEQ ID NO: 4 and a starch binding domain comprising amino acids 466-559 of SEQ ID NO: 4, and wherein the variant comprises a combination of specific substitutions selected from the group consisting of:
V18M+T43K+S95P+T116R+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; S95P+A121P+Y295W+T116R+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; T43K+S95P+G120S+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A; V18M+T43K+S95P+T116R+A121P+Q318Y+S458SCGG+N527M+T(V)549W; V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M+T(V)549W+N503R; V18M+T43K+S95P+A121P+Q318Y+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; T43K+S95P+A121P+Y295W+Q318Y+S458SCGG+N527M+T(V)549W; S95P+A121P+Y295W+G120S+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; V18M+T43K+S95P+A121P+Q318Y+S458SCGG+N527M T(V)549W; S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546PCGV+G547S+S548T+T549A; T43K+S95P+A121P+Y295W+Q318Y+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A; S95P+T116R+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A; T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W+N503R; and S95P+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A; and wherein the increase in raw starch hydrolytic activity compared to SEQ ID NO: 2 is at least 30%.

3 . The glucoamylase variant of claim 1 , wherein the variant is derived from the glucoamylase of SEQ ID NO: 2, and wherein the variant comprises a combination of specific substitutions selected from the group consisting of:
V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W; V18M+T43K+S95P+A121P+A518K+N527M+T549W; T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W; V18M+T43K+S95P+A121P+Y295W+A518K+N527M+T549W; T43K+S95P+A121P+Q318Y+N527M+T549W; V18M+T43K+S95P+A121P+Q318Y+N527M+T549W; V18M+T43K+S95P+T116R+A121P+Q318Y+A493V+N527M+T549W; V18M+T43K+S95P+A121P+Q318Y+A493V+N527M+T549W; V18M+T43K+S95P+A121P+Q318Y+A493V+A518K+N527M+T549W; T43K+S95P+A121P+Q318Y+A518K+N527M+T549W; V18M+T43K+S95P+T116R+A121P+Q318Y+N527M+T549W; T43K+S95P+A121P+Q318Y+A493V+N527M+T549W; T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T549W; V18M+T43K+S95P+T116R+A121P+Q318Y+A518K+T549W; V18M+T43K+S95P+A121P+Q318Y+A518K+N527M+T549W; T43K+S95P+A121P+Y295W+Q318Y+N527M+T549W; V18M+T43K+S95P+A121P+Q318Y+A518K+T549W; S95P+A121P+Y295W+A493V+A518K+E520Q+T549W; S95P+A121P+Y295W+A518K+N527M+T549W; S95P+A121P+Y295W+N527M+T549W; S95P+A121P+A271F+Y295W+A518K+N527M+T549W; S95P+A121P+Y295W+A493V; S95P+A121P+Y295W+S540K; S95P+A121P+Y295W+S540R; and S95P+A121P+Y295W+A518K+N527M; and wherein the increase in raw starch hydrolytic activity compared to SEQ ID NO: 2 is at least 5%.

4 . The glucoamylase variant of claim 1 , wherein the variant is derived from a glucoamylase having a catalytic domain comprising amino acids 1-454 of SEQ ID NO: 2, a linker comprising amino acids 455-465 of SEQ ID NO: 4 and a starch binding domain comprising amino acids 466-559 of SEQ ID NO: 4, and wherein the variant comprises a substitution at one or more positions selected from the group consisting of:
T43K+S95P+G120S+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W; S95P+G120S+A121P+Y295W+N539R+I541Y+T543V+A545S+S546GCGV+G547S+S548T+T549A; T43K+S95P+A121P+Y295W+Q318Y+N527M+T(V)549W; S95P+A121P+Y295W+T116R+N527M+T(V)549W; T43K+S95P+A121P+Y295W+Q318Y+A518K+N527M+T(V)549W; S95P+A121P+Y295W+A117Q+N527M+T(V)549W; S95P+T116R+A121P+Y295W+A518K+N527M+T(V)549W; V18M+T43K+S95P+A121P+Q318Y+A518K+T(V)549W; S95P+A121P+Y295W+Q318Y+N527M+T(V)549W; S

Assignees

• Novozymes As

Inventors

• Namoto Tomoko
• Tsutsumi Noriko
• Ayabe Keiichi
• Kang Zhengfang

Classifications

• 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

• C12N9/2428 Primary

  Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase · CPC title

• C12P19/02

  Monosaccharides (2-ketogulonic acid C12P7/60) · CPC title

• Y02E50/10

  Biofuels, e.g. bio-diesel · CPC title