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Construction method and application of L-isoleucine producing genetically engineered bacteria

A technology of genetically engineered bacteria and isoleucine, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problem of backward production technology and production equipment, low acid-producing ability of strains, and inability to meet market demand. and other problems to achieve the effect of increasing production

Inactive Publication Date: 2015-04-01
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, domestic manufacturers have produced L-isoleucine in batches, but the strains produced by these enterprises have low acid production capacity, and the production technology and production equipment lag behind Japan and other countries, and the output cannot meet the market demand.

Method used

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  • Construction method and application of L-isoleucine producing genetically engineered bacteria
  • Construction method and application of L-isoleucine producing genetically engineered bacteria
  • Construction method and application of L-isoleucine producing genetically engineered bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 alr Construction of knockout vector pJXW-1

[0031] by C. glutamicum The IWJ001 genome was used as a template, and the Alr -S-(+) / (-) and Alr -X-(+) / (-) are the primers to amplify each 1000bp alr Gene Upstream and Downstream Fragments alr -U, alr -D; based on pDTW-202 (Jinyu Hu, Yanzhen Tan, Yanyan Li, Xiaoqing Hu, Daqing Xu, Xiaoyuan Wang, 2013. Construction and application of an efficient multiple-gene-deletion system in Corynebacterium glutamicum. ) as the template, can -lox-F / R is primer amplification can resistance gene fragments. alr -U starts with xho I. Bam H I digestion, alr -D to Xba I and Pst I digestion, can Fragment ends with Bam H I. Xba I digestion, the three fragments were connected together to xho I and Pst The pBluescript II SK (+) of I digestion, the plasmid that constructs complete is namely alr Knockout vector, named pJXW-1 ( figure 2 ).

Embodiment 2

[0032] Example 2 Construction of expression vector

[0033] First, with the plasmid pDXW-8- FusA - frr for the template, FusA -F, frr -R is the primer, amplification FusA - frr gene fragments, in FusA - frr Introduce at both ends not I and Nhe I restriction site; the PCR product FusA - frr The fragment was digested with the corresponding restriction enzymes and connected into pJYW-4 digested with the same restriction enzymes to complete the construction of the expression plasmid pJYW-4- FusA - frr .

[0034] With the plasmid pDXW-8- ilvBN-ilv A- ppnk (Yin L, Zhao J, Chen C, Hu X, Wang X (2014) Enhancing the carbon flux and NADPH supply to increase L-isoleucine production in Corynebacterium glutamicum .) for the template, wxya -F, ppnk -R is the primer, amplification ilvBN-ilvA-ppnk gene fragments, in ilvBN-ilv A- ppnk Introduce at both ends Sfi I and Asc I restriction site; the PCR product ilvBN-ilv A- ppnk The fragment was digested with c...

Embodiment 3

[0036] Embodiment 3 Shake flask fermentation

[0037] From the deposited strain IWJ001Δ alr / pJYW-4- FusA - frr - ilvBN-ilv A- ppnk Streak a ring of bacterial solution in a glycerol tube, streak on the solid-state activation medium, and incubate at 30°C for 36 h. Use an inoculation loop to scrape a ring of bacterial lawn from the activated plate and transfer it to a 250 mL Erlenmeyer flask containing 25 mL of seed medium, and cultivate at 30 °C for 18 h at 200 r / min. Transfer 2.4 mL of bacterial liquid from the cultured seed liquid to a 250 mL Erlenmeyer flask containing 23 mL of fermentation medium, incubate at 30°C for 6 h, and add a final concentration of 1 mM isopropylthiogalactoside (IPTG) Protein expression was induced until the end of fermentation (t=72h).

[0038] step time %A %B 1 0 92 8 2 27 40 60 3 31.5 0 100 4 32 0 100 5 34 0 100 6 35.5 92 8

[0039] Medium used in this experiment:

[0040] Activ...

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Abstract

The invention discloses L-isoleucine producing genetically engineered bacteria and a construction method and application thereof, belonging to the technical field of genetic engineering and microbial fermentation. According to the method, a knockout vector of the gene alr is constructed and electroporated into the host bacteria to obtain a deficient strain IWJ001 delta alr of the alr. The gene segments are connected to an overexpression vector by use of the PCR amplification genes fusA, frr, ilvBN, ilvA and ippnk and then electroporated into corynebacterium glutamicum IWJ001 delta alr, and the target genetically engineered bacteria, namely L-isoleucine producing genetically engineered bacteria IWJ001 delta alr / pJYW-4-fusA-frr-ilvBN-ilvA-ppnk, are obtained through antibiotic resistance screening. The L-isoleucine producing genetically engineered bacteria constructed by the method disclosed by the invention can be used for increasing the yield of L-isoleucine through fermentation in a shake flask or fermentation tank, and the method is conductive to reducing the difficulty in separation and purification and increasing the yield so as to remarkably reduce the production cost of L-isoleucine.

Description

technical field [0001] The invention relates to a strain of L-isoleucine-producing genetically engineered bacteria and its construction method, and its application for improving amino acid production, belonging to the technical fields of genetic engineering and microbial fermentation. Background technique [0002] L-isoleucine, also known as L-isoleucine, chemically named L-α-amino-β-methylvaleric acid, belongs to the aspartic acid family of non-polar, water-transporting amino acids. Since there are two chiral carbon atoms at the α and β positions, there are 4 stereoisomers, namely D, L, D, and L. The only isoleucine that exists in nature and has physiological effects is L-shaped. L-isoleucine widely exists in a variety of animal and plant proteins. It is one of the eighteen common amino acids and one of the eight amino acids in the human body. In addition, due to its interaction with L-leucine and L-valine The molecular structure contains a methyl side chain and is called...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/77C12P13/06C12R1/15
Inventor 王小元赵建勋胡晓清胡瑾瑜
Owner JIANGNAN UNIV
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