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Ketolytic acid hydroxymethyltransferase mutant, coding gene and application of mutant

An acid hydroxymethyl, transferase technology, applied in transferase, application, genetic engineering and other directions, can solve the problems of low yield, increased pan-operon transcription, high production cost, and achieve the effect of clear structure

Pending Publication Date: 2021-10-01
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In 2002, Aileen Rubio et al. found that adding a CG base pair upstream of the panB gene derived from Salmonella enterica resulted in a consistent spacing of 17 bp between the 10 and 35 hexamers of the promoter , resulting in increased transcription of the pan-operon
[0007] Generally speaking, the problems reported about the production of D-pantothenic acid by fermentation are low output and high production cost, which cannot meet the requirements of large-scale industrial production.

Method used

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  • Ketolytic acid hydroxymethyltransferase mutant, coding gene and application of mutant
  • Ketolytic acid hydroxymethyltransferase mutant, coding gene and application of mutant
  • Ketolytic acid hydroxymethyltransferase mutant, coding gene and application of mutant

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1: Construction of wild-type ketopantoate hydroxymethyltransferase genetically engineered bacteria panB-CG

[0039] The gene sequence of Corynebacterium glutamicum ATCC 13032 ketopantoate hydroxymethyltransferase KPHMT (GenBank accession number: BX927148.1) in the gene bank was obtained by total gene synthesis to obtain the panB-CG plasmid. Design expression primer 1 (tttgtttaactttaagaaggagatataccATGCCCATGTCAGGCATTGATGCAAAG), primer 2 (tctcagtggtggtggtggtggtgctcgagAAAGGACTCCGCTTCGCCTGGGAAGGT), using Max high-fidelity DNA polymerase was used for PCR amplification to obtain the 813bp ketopantoate hydroxymethyltransferase gene sequence (the amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence is shown in SEQ ID NO.2). Using the pET28a vector as a template, the linearized vector was obtained by PCR amplification technique. Primers were designed as follows, primer 3 (ACCTTCCCAGGCGAAGCGGAGTCCTTTctcgagcaccaccaccaccaccactgaga), primer 4 (CTTTGCATCA...

Embodiment 2

[0040] Example 2: Construction of wild-type ketopantoate reductase gene engineering bacteria panE-EC

[0041] The gene sequence of Escherichia coli str. K-12substr, W3110 ketopantoate reductase KPR (GenBank accession number: BAE76205.1) in the gene bank was obtained by total gene synthesis to obtain the panE-EC plasmid. Design expression primer 5 (tttgtttaactttaagaaggagatataccATGAAAATTACCGTATTGGGATGCGGTGCC), primer 6 (tctcagtggtggtggtggtggtgctcgagCTACCAGGGGCGAGGCAAACCAGTGCCGAT), using Max high-fidelity DNA polymerase performs PCR amplification to obtain a 912bp ketopantoate reductase gene sequence (the nucleotide sequence is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4). Using the pET28a vector as a template, the linearized vector was obtained by PCR amplification technique. Primers were designed as follows, primer 7 (ATCGGCACTGGTTTGCCTCGCCCCTGGTAGctcgagcaccaccaccaccaccactgaga), primer 8 (GGCACCGCATCCCAATACGGTAATTTTCATggtatatctccttcttaaagttaaacaa...

Embodiment 3

[0042] Example 3: Construction of wild-type pantothenate synthetase genetically engineered bacteria panC-EC

[0043] The gene sequence of pantothenate synthase Ps derived from Escherichia coli str. K-12substr, W3110 in the gene bank (GenBank accession number: BAE76042.1) was obtained by total gene synthesis to obtain the panC-EC plasmid. Design expression primer 9 (tttgtttaactttaagaaggagatataccATGTTAATTATCGAAACCCTGCCGCTGC), primer 10 (tctcagtggtggtggtggtggtgctcgagTTACGCCAGCTCGACCATTTTGTTGTCGAT), using Max high-fidelity DNA polymerase performs PCR amplification to obtain a 855bp pantothenate synthetase gene sequence (the nucleotide sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6). Using the pET28a vector as a template, the linearized vector was obtained by PCR amplification technique. Primers were designed as follows, primer 11 (ATCGACAACAAAATGGTCGAGCTGGCGTAActcgagcaccaccaccaccaccactgaga), primer 12 (GCAGCGGCAGGGTTTCGATAATTAACATggtatatctc...

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Abstract

The invention relates to the field of gene engineering, in particular to a corynebacterium glutamicum sourced ketolytic acid hydroxymethyltransferase mutant, a coding gene thereof and application of the mutant in preparation of ketopantoic acid and D-pantothenic acid. By means of a computer simulation technology, key amino acid sites around a substrate channel and an active pocket are predicted and mutated, and a series of mutants are obtained. The ketolytic acid hydroxymethyltransferase mutant is obtained by carrying out site-specific mutagenesis on the 18th site, the 20th site, the 21st site, the 24th site, the 25th site, the 26th site, the 27th site and the 49th site of an amino acid sequence as shown in SEQ ID NO: 1. In the finally obtained ketolytic acid hydroxymethyltransferase mutant, the enzyme activity is obviously changed, and the catalytic activity and substrate tolerance of the mutant are greatly improved when alpha-ketoisovaleric acid is catalyzed to generate ketopantoic acid compared with a wild enzyme.

Description

(1) Technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a ketopantoate hydroxymethyltransferase mutant derived from Corynebacterium glutamicum and its coding gene, a recombinant vector containing the mutant gene, and a ketopantoate Application of acid hydroxymethyltransferase mutant in generating ketopantoic acid and preparing D-pantothenic acid. (2) Background technology [0002] Ketolytic acid hydroxymethyltransferase (KPHMT, EC2.1.2.11), which is the expression product of panB gene, is also a key step in Escherichia coli W3110 catalyzing the biosynthetic pathway of pantothenic acid: the substrate α-ketoisoamyl The acid reacts with 5,10-methylenetetrahydrofolate under the catalysis of ketopantoate hydroxymethyltransferase to generate ketopantoate, and the reaction process is reversible. [0003] D-pantothenic acid, also known as pantothenic acid or vitamin B 5 , is a water-soluble vitamin, the chemical index number (CA...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P13/02C12R1/19
CPCC12N9/1014C12N15/70C12P13/02C12Y201/02011
Inventor 柳志强蔡雪强煜刘思琦张博郑裕国
Owner ZHEJIANG UNIV OF TECH
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