Search

US-12624344-B2 - Variant polypeptides having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase and microorganisms for producing pantothenic or pantoic acid

US12624344B2US 12624344 B2US12624344 B2US 12624344B2US-12624344-B2

Abstract

A 3-methyl-2-oxobutanoate hydroxymethyltransferase variant, a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, a composition for producing pantothenic acid and/or pantoic acid comprising the microorganism, and a method for preparing pantothenic acid and/or pantoic acid comprising culturing the microorganism are provided.

Inventors

  • Yee-Seul So
  • Kwang Soo Shin
  • Jihyun Shim
  • Kwang woo Lee
  • Yeon-Jae JANG

Assignees

  • CJ CHEILJEDANG CORPORATION

Dates

Publication Date
20260512
Application Date
20220318
Priority Date
20210510

Claims (6)

  1. 1 . A polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 37, wherein the amino acid at the position corresponding to position 159 of the polypeptide of SEQ ID NO: 37 is substituted with another amino acid, wherein the polypeptide has 3-methyl-2-oxobutanoate hydroxymethyltransferase activity.
  2. 2 . The polypeptide according to claim 1 , wherein the amino acid at the position corresponding to position 159 of the polypeptide of SEQ ID NO: 37 is substituted with arginine (R), serine(S), tyrosine (Y), cysteine (C), proline (P), histidine (H), leucine (L), isoleucine (I), threonine (T), lysine (K), valine (V), methionine (M), aspartic acid (D), glutamic acid (E), asparagine (N), or glutamine (Q).
  3. 3 . The polypeptide according to claim 1 , wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, SEQ ID NO: 123, SEQ ID NO: 124 and SEQ ID NO: 125.
  4. 4 . The polypeptide according to claim 1 , wherein the amino acid at the position corresponding to position 116 of the polypeptide of SEQ ID NO: 37 is substituted with another amino acid.
  5. 5 . The polypeptide according to claim 4 , wherein the amino acid at the position corresponding to position 116 of the polypeptide of SEQ ID NO: 37 is substituted with alanine (A), asparagine (N), threonine (T), glutamic acid (E), serine(S), valine (V), isoleucine (I), leucine (L), aspartic acid (D), cysteine (C), glutamine (Q), or methionine (M).
  6. 6 . The polypeptide according to claim 4 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 128.

Description

TECHNICAL FIELD Cross-Reference to Related Application(S) The present application claims the priority based on Korean Patent Application No. 10-2021-0060009 filed on May 10, 2021, and all the contents disclosed in the document of the corresponding Korean Patent Application are incorporated by reference as a part of the present description. A 3-methyl-2-oxobutanoate hydroxymethyltransferase variant, a microorganism having enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase, a composition for producing pantothenic acid and/or pantoic acid comprising the microorganism, and a method for preparing pantothenic acid and/or pantoic acid comprising culturing the microorganism are provided. BACKGROUND ART Pantothenic acid is a substance belonging to the vitamin B complex, also called vitamin B5, and is one of commercially important substances that are variously applied to cosmetics, medicines, human nutrition, animal nutrition, and the like. Pantothenic acid is a structure in which beta-alanine is linked to pantoic acid by an amide bond. Pantothenic acid or pantoic acid may be prepared by chemical synthesis, or may be prepared biotechnologically by fermenting an appropriate microorganism in a suitable medium. An advantage of the biotechnological preparation method using a microorganism is that a desired stereo-isomeric D-form of pantothenic acid or pantoic acid is formed. Accordingly, it is required to develop a microorganism having an advantageous effect in biotechnologically preparing pantothenic acid and/or pantoic acid and a technology for preparing pantothenic acid and/or pantoic acid with high efficiency using the same. PRIOR ART Patent Document (Prior document 1) U.S. Registered U.S. Pat. No. 7,718,205 DISCLOSURE Technical Problem One example of the present application provides a polypeptide having 3-methyl-2-oxobutanoate hydroxymethyltransferase activity. In one embodiment, (1) the polypeptide may comprise the amino acid sequence in which an amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid. In another embodiment, (2) the polypeptide may comprise the amino acid sequence in which an amino acid corresponding to the 159th residue from the N-terminus is substituted with another amino acid and the 116th residue from the N-terminus is substituted with another amino acid in the amino acid sequence of SEQ ID NO: 37. Counting amino acids from the N-terminus in the amino acid sequence as described above may mean counting with methionine (Met, M) translated from the initiation codon as the first amino acid. Another example, provides a polynucleotide encoding the polypeptide. Other example, provides a recombinant vector comprising the polynucleotide. The recombinant vector, may be an expression vector. Other example provides a microorganism producing pantothenic acid or pantoic acid, which comprises one or more (one, two or all) selected from the group consisting of the polypeptide, polynucleotide encoding the polypeptide, and recombinant vector comprising the polynucleotide, and has enhanced activity of 3-methyl-2-oxobutanoate hydroxymethyltransferase. The microorganism, may comprise (1) one or more (one, two or all) selected from the group consisting of a polypeptide in which an amino acid corresponding to the 159th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide and a recombinant vector comprising the polynucleotide; or(2) (1) above, and one or more (one, two or all) selected from the group consisting of a polypeptide in which an amino acid corresponding to the 116th residue from the N-terminus in the amino acid sequence of SEQ ID NO: 37 is substituted with another amino acid, a polynucleotide encoding the polypeptide and a recombinant vector comprising the polynucleotide. The microorganism may be a genus Corynebacterium microorganism or a genus Escherichia microorganism. The genus Corynebacterium microorganism may be Corynebacterium glutamicum. Other example, provides a composition for producing pantothenic acid or pantoic acid, comprising the microorganism. Other example, provides a use of the microorganism in production of pantothenic acid and/or pantoic acid. Other example, provides a method for preparation of pantothenic acid or pantoic acid, comprising culturing the microorganism in a medium. The method for preparation may further comprise recovering pantothenic acid or pantoic acid from the cultured microorganism, medium or both of them, after the culturing. Technical Solution The present description is to provide a recombinant strain with excellent production ability of pantothenic acid and/or pantoic acid, by searching 3-methyl-2-oxobutanoate hydroxymethyltransferase or its variant which can enhance the production ability of pantothenic acid and/or pantoic acid and introducing this to a mic