Recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone

一种泛解酸内酯、重组工程菌的技术，应用在基因工程领域，能够解决D-泛解酸内酯分离困难、大废料、增加处理困难等问题，达到缩短生产周期、降低生产成本、避免繁琐步骤的效果

Inactive Publication Date: 2022-05-10
ANHUI HUAHENG BIOTECH +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, glucose dehydrogenase is easy to induce the production of gluconic acid, a by-product of the reaction, which makes it difficult to separate D-pantolactone, and produces a large amount of waste, which increases the difficulty of processing.

Method used

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  • Recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone
  • Recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone
  • Recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0114] Example 1 Construction of Recombinant Engineering Bacteria

[0115] 1. Design and synthesis of target genes 1-3

[0116] Step 1, the nucleotide sequence of the L-pantolactone dehydrogenase coding gene derived from Humibacter sp.BT305 (actinomycetes) is codon-optimized according to the codon preference of Escherichia coli (E.coli) , obtain the modified gene sequence of L-pantolactone dehydrogenase, the nucleotide sequence of which is shown in SEQ ID NO.1;

[0117] Step 2, the nucleotide sequence of the ketopantolide reductase gene encoding gene derived from Candida magnolia was codon-optimized according to the codon preference of Escherichia coli (E.coli) to obtain D-ketopantolide Acid lactone modified gene sequence, the nucleotide sequence of which is shown in SEQ ID NO.2;

[0118] Step 3, the nucleotide sequence of the formate dehydrogenase encoding gene derived from Burkholderia is optimized according to the codon preference of Escherichia coli (E.coli), and the m...

Embodiment 2

[0156] Example 2 Induced Expression of Recombinant Engineering Bacteria

[0157] Inoculate the recombinant engineered bacteria prepared in Example 1 into 5mL LB medium according to the inoculum size of 2%, culture it at 37°C and 200rpm for 10-15h, and obtain the first-grade seed liquid;

[0158] The primary seed liquid was inoculated in 100 mL LB medium according to the inoculum amount of 2%, and it was placed at 37°C and 200 rpm for 8 hours to obtain the secondary seed liquid;

[0159] Secondary seed liquid is inoculated in the fermentor that contains 6L fermentation medium according to 0.2% inoculation amount, and described fermentation medium comprises magnesium sulfate heptahydrate 2g / L, potassium dihydrogen phosphate 7g / L, citric acid monohydrate 2g / L. L, ammonium sulfate 3g / L, yeast powder 1g / L and glucose 6g / L. Add glucose solution to keep the glucose concentration in the fermentation broth less than 5g / L, ferment and cultivate at pH 7, 37°C, and aerobic conditions f...

Embodiment 3

[0160] Example 3 Conversion of L-pantolactone

[0161] Add 130 g of L-pantolactone and 31.5 g of ammonium formate into the reaction vessel, add water to make the total volume 1 L, stir and dissolve, adjust the pH of the solution to 6.2 with 20-25% ammonia water, add the recombined solution collected in Example 2 After the engineered bacteria (OD=2), the mixed solution was stirred and reacted at a constant temperature of 37°C for 32 hours to obtain a reaction solution, in which the final concentration of D-pantolactone was 91g / L, and the final concentration of L-pantoate was 91g / L. The final concentration of the ester was 39 g / L, and the conversion rate of L-pantolactone was 70%.

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Abstract

The invention relates to a recombinant engineering bacterium and application thereof in efficient conversion of L-pantoic acid lactone, the recombinant engineering bacterium is induced to efficiently express L-pantoic acid lactone dehydrogenase, ketone pantoic acid lactone reductase and formate dehydrogenase, and the L-pantoic acid lactone is efficiently converted to generate DL-pantoic acid lactone. According to the recombinant engineering bacterium, the purity and quality of DL-pantoic acid lactone, the reaction efficiency and the resource utilization rate are remarkably improved, the production cost is reduced, and the recombinant engineering bacterium has the advantages of being easy and convenient to operate, environmentally friendly and the like.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a recombinant engineering bacterium and its application for efficient transformation of L-pantolactone. Background technique [0002] D-pantothenolactone is a pharmaceutical intermediate used in the synthesis of vitamin drug D-panthenol and neurotrophic drug D-homopantothenate calcium, and is used as a synthetic precursor for feed additives and daily chemical products. The annual output exceeds tens of thousands Ton. The industrial preparation of D-pantolactone often adopts the method of combining chemical method and resolution method, including the following steps: isobutyraldehyde and formaldehyde undergo aldol condensation to generate hydroxypivalaldehyde, and then react with cyano group to generate cyanaldehyde, Hydrolyze to generate DL-pantolactone racemate, and use chemical resolution and enzyme resolution to obtain D-pantolactone. However, the separation prod...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P17/04
CPCC12P17/04C12N15/70C12N9/0006C12N9/18C12P41/001C12Y101/01168C12Y101/01169C12N2800/22C12N9/0008C12Y102/01002Y02A50/30
Inventor 周芳芳刘树蓬刘磊张大伟刘美霞
Owner ANHUI HUAHENG BIOTECH
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