Brevibacterium flavum recombinant strain producing L-isoleucine and construction method thereof

A technology of Brevibacterium flavum and isoleucine, which is applied in the field of genetic engineering and can solve problems such as large impact

Inactive Publication Date: 2019-04-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, key enzymes have a greater impact on growth and metabolism. How to construct a strain that can increase the production of L-isoleucine without affecting growth has become an urgent research topic.

Method used

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  • Brevibacterium flavum recombinant strain producing L-isoleucine and construction method thereof
  • Brevibacterium flavum recombinant strain producing L-isoleucine and construction method thereof
  • Brevibacterium flavum recombinant strain producing L-isoleucine and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Construction of recombinant expression vector pEC-XK99E-ilvBNC

[0029] According to the ilvBNC gene sequence in the whole genome nucleic acid sequence of C. glutamicum ATCC 13032 in NCBI, the ilvBNC gene sequence is obtained by connecting the ilvBN gene and the ilvC gene, and the restriction endonuclease Sma I is added to the 5' end of the upstream primer, and the ilvBNC gene sequence is Add the Xba I restriction site downstream, synthesize the upstream and downstream primers ilvBNC-F and ilvBNC-R, use the whole genome of C. glutamicum ATCC 13032 as a template to obtain the gene ilvBNC gene fragment by PCR, and purify the fragment with the plasmid After pEC-XK99E was digested with the same restriction endonuclease, it was enzyme-linked overnight and transformed into JM109 competent cells. After colony PCR verification, the correct colony was selected, and the extracted plasmid was verified by electrophoresis. After obtaining the correct target band ( 7 018 a...

Embodiment 2

[0030] Example 2: Construction of recombinant expression vector pEC-XK99E-ilvBNCE

[0031] According to the ilvE gene sequence in the whole genome nucleotide sequence of C. glutamicum ATCC 13032 in NCBI, a restriction endonuclease Xba I and a Corynebacterium glutamicum SD recognition sequence GAAAGGAGATATACC were added to the 5' end of the primer upstream of the gene, and Xba I was added downstream of the gene Restriction site, the ilvE gene fragment was obtained by PCR, after dephosphorylation and gel recovery kit purification, the fragment and the plasmid pEC-XK99E-ilvBNC were digested with the same restriction endonuclease, enzyme ligated overnight and transformed Enter JM109 competent cells, select the possible correct colonies after colony PCR verification, extract the plasmid for verification by digestion and electrophoresis, and obtain the correct target band (2 fragments of 9 340 and 681bp), submit it to General Biosystems (Anhui) Co., Ltd. for sequencing . The constr...

Embodiment 3

[0032] Example 3: Construction of recombinant expression vector pEC-XK99E-ilvBNCE-tdcB

[0033]According to the tdcB gene sequence in the whole genome nucleic acid sequence of E.coli W3110 in NCBI, restriction endonuclease EcoR I and Kpn I enzyme cutting sites were added to the 5' end of the gene upstream and downstream primers, and the gene tdcB gene fragment was obtained by PCR. After the recovery kit is purified, the fragment and the plasmid pEC-XK99E-ilvBNCE are digested with the same restriction enzyme, then enzyme-linked overnight and transformed into JM109 competent cells. After colony PCR verification, the correct colony is selected and the plasmid is extracted. Enzyme digestion and electrophoresis verification, after obtaining the correct target bands (10021 and 990bp), submitted to General Biosystems (Anhui) Co., Ltd. for sequencing. The construction of pEC-XK99E-ilvBNCE-tdcB was completed. The recombinant plasmids of Examples 1-3 were verified by enzyme digestion, ...

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Abstract

The present invention discloses a brevibacterium flavum recombinant strain producing L-isoleucine and a construction method thereof, and belongs to the technical field of genetic engineering. The genetic engineering means is utilized to strengthen acetohydroxyacid synthase, dihydroxy acid reductoisomerase and branched chain amino acid aminotransferase in the brevibacterium flavum, and heterologously expresses catabolic threonine dehydratase encoded by tdcB, and thus constructs a L-isoleucine high-yield strain B. flavum I12/pEC-XK99E-ilvBNCE-tdcB. The yield of the accumulated isoleucine is increased to 22.65 g/L in the recombinant strain and is 25.48% higher than that of an original strain, and the maximum biomass reaches 20.95 g DCW/L. The strain can turn lysine pathway carbon source to the L-isoleucine and a brand-new idea for constructing the L-isoleucine high-yield strain is provided.

Description

technical field [0001] The invention relates to a Brevibacterium flavum recombinant strain producing L-isoleucine and a construction method thereof, belonging to the technical field of genetic engineering. Background technique [0002] L-isoleucine, also known as L-isoleucine, is one of the eight essential amino acids for the human body. Widely used in food, feed, cosmetics and pharmaceutical industries. Therefore, breeding a strain with high L-isoleucine yield and stable metabolism plays an important role in production. Metabolic engineering provides an effective transformation path for L-isoleucine production, but the existing L-isoleucine production strains modified through metabolic pathways still have the problem of low yield. [0003] The first key rate-limiting enzyme in the L-isoleucine synthesis pathway is threonine dehydratase, which is further divided into anabolic threonine dehydratase encoded by ilvA and catabolic threonine dehydratase encoded by tdcB The enz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/13
CPCC12N9/0006C12N9/1022C12N9/1096C12N9/88C12N15/77C12P13/06C12Y101/01086C12Y202/01006C12Y206/01042C12Y403/01019
Inventor 张伟国徐建中王壮壮魏佳
Owner JIANGNAN UNIV
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