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Recombinant engineering bacterium and method for preparing D-pantoic acid by using recombinant engineering bacterium

A technology of recombinant engineering bacteria and pantothenic acid, applied in the direction of microorganism-based methods, recombinant DNA technology, botany equipment and methods, etc., can solve the problems of limiting industrial application prospects, cumbersome reaction steps, long reaction time, etc., and achieve The raw material is cheap, the reaction cost is low, and the effect of the extraction process is reduced

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 method for preparing D-pantoic acid by using recombinant engineering bacterium
  • Recombinant engineering bacterium and method for preparing D-pantoic acid by using recombinant engineering bacterium
  • Recombinant engineering bacterium and method for preparing D-pantoic acid by using recombinant engineering bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Construction of the first recombinant engineering bacteria expressing L-amino acid deaminase

[0108]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: 5) 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 amino acid sequence is shown in SEQ ID NO: 1;

[0109] 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.

[0110] 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.

[0111] The primer sequences are a...

Embodiment 2

[0120] Example 2 Construction of the second recombinant engineering bacteria expressing aldolase

[0121] Step 1, the nucleotide sequence of the aldolase-encoding gene sequence derived from Escherichia coli (the encoded amino acid sequence is shown in SEQ ID NO: 6) is based on the codon of Escherichia coli (E.coli). Codon optimization is carried out preferentially to obtain an aldolase-optimized gene sequence, whose nucleotide sequence is shown in SEQ ID NO: 2;

[0122] The nucleotide sequence shown in SEQ ID NO: 2 was sent to Hangzhou Aoqian Biotechnology Co., Ltd. for artificial synthesis, and XhoI and NdeI restriction sites were added to obtain target gene 2.

[0123] Step 2, using the DNA molecule of the target gene 2 as a template, using primer pairs ald-for and ald-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 2. gene fragments.

[0124] The primer sequences ...

Embodiment 3

[0132] Example 3 Construction of the third recombinant engineering bacteria co-expressing formate dehydrogenase and ketopantoate reductase

[0133] 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: 7) is based on Escherichia coli (E. .coli) codon preference carries out codon optimization, obtains formate dehydrogenase optimized gene sequence, and its amino acid sequence is as shown in SEQ ID NO:3;

[0134] The nucleotide sequence of the ketopantoate reductase-encoding gene sequence (the encoded amino acid sequence thereof is shown in SEQ ID NO: 8) derived from Stenotrophomonas maltophilia was based on Escherichia coli (E. coli), the codons are optimized to obtain a ketopantoate reductase-optimized gene sequence, and its nucleotide sequence is shown in SEQ ID NO: 4;

[0135] The amino acid sequence shown in described SEQ ID NO:3 and SEQ ID NO:4 was sent to ...

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Abstract

The invention relates to a preparation method of D-pantoic acid, which is characterized in that valine and formaldehyde are used as substrates, and recombinant engineering bacteria are added to obtain the D-pantoic acid. The genetically engineered bacterium for biological preparation 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 field of biosynthesis, in particular to a recombinant engineering bacterium and a method for preparing D-pantoate. 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. CN108456701A discloses a preparation method of D-pantolactone. The method uses valine as a substrate and is catalyzed by a combination of multiple enzymes to prepare D-pantolactone. However, there are disadvantages such as cumbersome reaction steps, long reaction time, and high cost caused by the consumption of coenzyme NADPH in the process of converting ketopantoate into pantoate, which limits its...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/53C12N15/60C12N1/21C12P7/42C12P17/04C12P13/02C12R1/19
CPCC12N15/70C12N15/52C12N9/0022C12N9/88C12N9/0008C12N9/0006C12P7/42C12P17/04C12P13/02C12Y104/03002C12Y401/02C12Y102/01002C12Y101/01169C12N2800/22Y02A50/30
Inventor 周芳芳刘树蓬刘磊
Owner ANHUI HUAHENG BIOTECH
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