Method for improving yield and production intensity of gluconobacter oxydans sorbose

A technology for oxidizing glucose and increasing production intensity, which is applied in the field of fermentation engineering, can solve the problem of D-sorbitol forming by-products, etc., and achieve the effect of increasing production intensity and increasing yield

Active Publication Date: 2019-04-16
ZHEJIANG NHU CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, overexpression of sorbitol dehydrogenase through metabolic engineering can increase the production intensity of sorbose and shorten the fermentation cycle, but only overexpression of sorbitol dehydrogenase cannot solve the problem of other dehydrogenases catalyzing D-sorbitol in the fermentation process. Alcohol by-product problem

Method used

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  • Method for improving yield and production intensity of gluconobacter oxydans sorbose

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1: Construction of the knockout frame of gene

[0032] Use the genome of G.oxydans CGMCC 1.110 as a template to amplify the sequence of 1000bp upstream and downstream of the gene to be knocked out, and use primers to amplify the kana gene using pBBR1MCS-2 as a template, and amplify upp using the G.oxydans 1.110 genome as a template Gene, use fusion PCR to connect the above four fragments to construct a gene knockout box: left homology arm (HAL)-kana-upp-right homology arm (HAR), and connect the knockout box to pMD -19-T vector, transformed into Escherichia coli competent cells JM109, and the transformants were screened on LB plates containing ampicillin (100mg / L), sequenced, and the strains with correct sequencing were preserved.

Embodiment 2

[0033] Embodiment 2: Construction of recombinant bacteria G.oxydans-1

[0034] According to the method of Example 1, the knockout box for knocking out the GDH gene is constructed: GDHL-kana-upp-GDHR, because the dehydrogenase knockout box has kana (Genbank accession number: MH539767.1)-upp gene, will be sequenced The correct dehydrogenase knockout frame fragment was transformed into the receptor strain of Gluconobacter oxidans, CGMCC 1.110, and an upp gene-deficient strain G that grew normally in D-sorbitol medium containing kana resistance and cefoxitin was obtained .oxydans (knockout gene::kana-upp), after the first round of kana antibiotic selection, cultured in D-sorbitol containing 5-fluorouracil (300mg / L) and cefoxitin (50mg / L) The second round of screening was carried out, and the recombinant strain G.oxydans-1 with the GDH gene knocked out was obtained.

Embodiment 3

[0035] Embodiment 3: Construction of recombinant bacteria G.oxydans-2

[0036] According to the method of Example 1, the knockout box for knocking out the GA-5-DH gene was constructed: GA-5-DHL-kana-upp-GA-5-DHR, and the sequenced correct dehydrogenase knockout box fragment was converted to oxidized Gluconobacter CGMCC CGMCC 1.110 was screened by the same method as above, and the recombinant strain G.oxydans-02 with the GA-5-DH gene knocked out was obtained.

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Abstract

The invention discloses a method for improving the yield and production intensity of gluconobacter oxydans sorbose and belongs to the technical field of fermentation engineering. A related gene is formed by knocking out D-sorbitol or an L-sorbose metabolism by-product in gluconobacter oxydans, the formation of the by-products is reduced, the efficiency of converting the D-sorbitol into the L-sorbose is improved, and accordingly the yield and production intensity of the L-sorbose are improved. The L-sorbose conversion rate of a constructed recombinant strain G.oxydan-11 reaches 96.12% comparedwith control bacteria and is improved by 4.47% compared with that of wild bacteria, tthe production intensity is 14g / L h, and the fructose by-product content is only 5.6g / L is decreased by 45.6% compared with that of the wild bacteria.

Description

technical field [0001] The invention relates to a method for increasing the sorbose yield and production intensity of Gluconobacter oxydans, belonging to the technical field of fermentation engineering. Background technique [0002] L-sorbose (L-sorbose) is a ketohexose, which is a kind of direct precursor of vitamin C, 2-keto-L-gulonic acid (2-keto-L-gulonic acid, referred to as 2-KLG). important raw material. The method for industrial production of sorbose is mainly to use Gluconobacter oxidans to ferment D-sorbitol to form L-sorbose. Sorbose is further formed into 2-keto-L-gulonic acid (2-KLG for short), the direct precursor of vitamin C, through the "microbial fermentation method" or "Rayleigh method", 2-KLG Vitamin C is obtained after internal fatification and enolization. Therefore, the conversion rate of D-sorbitol to L-sorbose determines the conversion rate of industrial production of vitamin C. [0003] Gluconobacter oxidans is the main strain for industrial pro...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/53C12P19/02C12R1/01
CPCC12N9/0006C12P19/02C12Y101/9901C12Y101/99012
Inventor 周景文陈坚刘立曾伟主堵国成
Owner ZHEJIANG NHU CO LTD
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