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Recombinant engineering bacterium and application thereof in preparation of generic compounds

A technology for recombining engineering bacteria and recombinant plasmids, applied in the field of genetic engineering, can solve the problems of limiting industrial application prospects, cumbersome reaction steps, and long reaction time, and achieve the effects of shortening the production cycle, low reaction cost, and avoiding cumbersome steps

Active Publication Date: 2022-06-24
ANHUI HUAHENG BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are defects such as cumbersome reaction steps, long reaction time, and high cost caused by the consumption of the coenzyme NADPH in the process of converting ketopantoate into pantoate, which limits its industrial application prospects.

Method used

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  • Recombinant engineering bacterium and application thereof in preparation of generic compounds
  • Recombinant engineering bacterium and application thereof in preparation of generic compounds
  • Recombinant engineering bacterium and application thereof in preparation of generic compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] Example 1 Construction of the first recombinant engineering bacteria expressing L-amino acid deaminase

[0122] Step 1, according to the nucleotide sequence of the L-amino acid deaminase encoding gene of Proteus mirabilis (the encoded amino acid sequence is shown in SEQ ID NO: 6) according to Escherichia coli (E.coli) The codon preference of L-amino acid deaminase is optimized, and the L-amino acid deaminase optimized gene sequence is obtained, and its nucleotide sequence is shown in SEQ ID NO: 1;

[0123] The nucleotide sequence shown in SEQ ID NO: 1 is artificially synthesized, and XhoI and NdeI restriction sites are added to obtain the target gene 1.

[0124] Step 2, using the DNA molecule of the target gene 1 as a template, using primer pairs LA-for and LA-rev to carry out PCR amplification, 1% agarose gel electrophoresis to separate the PCR products, and then use a gel recovery kit to recover the target gene 1. gene fragments.

[0125] The primer sequences are ...

Embodiment 2

[0134] Example 2 Construction of the second recombinant engineering bacteria co-expressing methanol dehydrogenase and aldolase

[0135] Step 1, according to the nucleotide sequence of the methanol dehydrogenase encoding gene sequence of Bacillus methanolicus (the encoded amino acid sequence is shown in SEQ ID NO: 7) according to the code of Escherichia coli (E.coli) Codon optimization was carried out according to the sub-preference to obtain the methanol dehydrogenase optimized gene sequence, and its nucleotide sequence is shown in SEQ ID NO: 2;

[0136] The nucleotide sequence of the aldolase-encoding gene sequence derived from Escherichia coli (the encoded amino acid sequence is shown in SEQ ID NO: 8) was determined according to the codon bias of Escherichia coli. Codon optimization, the aldolase optimization gene sequence is obtained, and its nucleotide sequence is as shown in SEQ ID NO:3;

[0137] The nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 were arti...

Embodiment 4

[0156] Example 4Construction of the third recombinant engineering bacteria co-expressing formate dehydrogenase and ketopantoate reductase

[0157] In step 1, the nucleotide sequence of the gene sequence encoding formate dehydrogenase from Burkholderia stabilis (the encoded amino acid sequence is shown in SEQ ID NO: 9) is based on Escherichia coli (E. .coli) codon preference carries out codon optimization, and adds BamHI and NotI restriction enzyme cleavage site, obtains formate dehydrogenase optimization gene sequence, and its nucleotide sequence is as shown in SEQ ID NO:4;

[0158] The nucleotide sequence of the ketopantoate reductase-encoding gene sequence (the encoded amino acid sequence thereof is shown in SEQ ID NO: 10) derived from Stenotrophomonas maltophilia was based on Escherichia coli (E. coli), the codon preference is optimized, and XhoI and NdeI restriction sites are added to obtain a ketopantoate reductase-optimized gene sequence, whose nucleotide sequence is s...

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Abstract

The invention relates to a recombinant engineering bacterium and application thereof in preparation of a generic compound, in particular to a recombinant engineering bacterium and application thereof in preparation of D-pantoic acid, valine and methanol are taken as substrates, thalli generated by induction of the recombinant engineering bacterium are added, and a D-pantoic acid solution is obtained. The genetically engineered bacterium for biosynthesis of D-pantoic acid is successfully constructed for the first time, valine is used as a substrate for fermentation and conversion to generate D-pantoic acid, raw materials are cheap and easy to obtain, and the reaction cost is low.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacteria and its application in the preparation of pan-compounds. Background technique [0002] Pantothenic acid also known as vitamin B 5 , is an essential nutrient element for mammals including humans and livestock, and is used in the biosynthesis of coenzyme A (CoA) and acyl carrier protein (ACP) in body cells, and then participates in more than one hundred cellular metabolic reactions. [0003] D-pantolactone is an important precursor for the synthesis of D-pantothenic acid. CN110423717A discloses a preparation method of D-pantoic acid lactone, wherein DL-pantoic acid lactone is selectively hydrolyzed and separated by enzymatic method or synthesized by enzymatic method to prepare D-pantoic acid lactone. However, there are the following defects. The synthesis of DL-pantolactone requires the use of a large amount of isobutyraldehyde and f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/53C12N9/02C12N9/04C12N9/06C12N9/88C12P7/42C12P17/04C12N1/21C12R1/19
CPCC12N15/70C12N9/0006C12N9/0008C12N9/88C12N9/0022C12P7/42C12P17/04C12Y101/01244C12Y102/01002C12Y101/01169C12Y104/03002C12N2800/22Y02A50/30
Inventor 周芳芳刘树蓬刘磊
Owner ANHUI HUAHENG BIOTECH
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