Method for improving alpha-ketoisocaproate yield through RBS optimization

An amino acid and deaminase technology, applied in the field of genetic engineering, can solve the problems of insufficient industrial production, low yield and conversion rate of α-ketoisocaproic acid, etc., and achieve the effects of easy control, fast reaction rate, and serious pollution problems.

Active Publication Date: 2015-11-25
JIANGNAN UNIV
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Problems solved by technology

[0004] However, the output and conversion rate of the current α-ketoisocaproic acid in the conversion process are still low, which is n

Method used

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  • Method for improving alpha-ketoisocaproate yield through RBS optimization
  • Method for improving alpha-ketoisocaproate yield through RBS optimization
  • Method for improving alpha-ketoisocaproate yield through RBS optimization

Examples

Experimental program
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Example Embodiment

[0022] Example 1 Construction of RBS mutant library

[0023] Using pET28a-lad (see the patent application with application number 201410050399.4 and publication number CN103789247A) as the template for the construction method, and the forward primer as the 5'- NNNNNN ATATACCATGGCGATATCTAGAAGAA-3', the reverse primer is 5'-CTTAAAGTTAAACAAAATTATTTCTAGAGG-3' as the primer for PCR reaction, the reaction product is digested with DpnI and its template is recovered using a DNA column recovery kit, phosphorylase is added for end phosphorylation, and added The SolutionI ligase was ligated overnight at 16°C. The ligated plasmid was transformed into the competent E.coli BL21 (DE3), and spread on the kanapenicillin resistant plate.

Example Embodiment

[0024] Example 2 Screening of mutants with increased catalytic activity of L-amino acid deaminase

[0025] The single colony in Example 1 was picked with a sterilized toothpick into a 96-well deep well plate containing 700 μLLB medium, cultured overnight at 37°C with shaking, and inoculated into 96 wells containing 700 μL of fermentation medium at a 2% inoculum In a deep well plate, shake culture at 37℃ to OD 600 It was 0.6. After induction with 0.4mM IPTG, the cells were collected 3h after induction. After centrifugation at 3400rpm for 10min, 1mL of 100mM leucine aqueous solution was added to the deep well plate. After reaction for 10 minutes, 100μL of 1% FeCl was added. 3 The solution develops color.

Example Embodiment

[0026] Example 3 Determination of the catalytic activity of whole-cell conversion of L-leucine and mutation sequence changes

[0027] The recombinant E. coli BL21 (DE3) that developed dark black in Example 2 was transferred from LB medium to a 250 mL Erlenmeyer flask containing 25 mL of seed medium, cultivated overnight, and inoculated with 2% Inoculate 50mL fermentation medium, cultivate to OD at 37℃, 200r / min 600 It was 0.6, induced by adding 0.4mMIPTG, cultured at 37°C for 5h, and centrifuged to collect the bacteria for whole cell transformation. Take 0.8g / L cells, 100mM substrate L-leucine, react at 37°C for 0.5h, centrifuge at 12000rpm for 2min, and take the supernatant to determine the content of α-ketoisocaproic acid.

[0028] According to the formula Calculate the enzyme activity. (Where C (KIC) It is the measured content of α-ketoisocaproic acid, DCW is the dry weight of the bacterial cell 0.8g / L, and T is the reaction time 0.5h). Its mutant catalytic activity is as f...

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Abstract

The invention discloses a method for improving alpha-ketoisocaproate yield through RBS optimization, and belongs to the field of genetic engineering. By virtue of a genetic engineering bacterium which is subject to RBS optimization, alpha-ketoisocaproate is produced through full-cell catalysis by taking leucine as a substrate. The method disclosed by the invention has the advantages of low production cost, mild production condition, less impurities in a transformation system, simple process step, safe production operation and the like. The alpha-ketoisocaproate produced by the method disclosed by the invention is high in yield, and each liter of transformation liquid contains 81.41g of alpha-ketoisocaproate; and the transformation rate of L-leucine reaches 88.66%.

Description

technical field [0001] The invention relates to a method for optimizing the production of α-ketoisocaproic acid by optimizing RBS, and belongs to the field of genetic engineering. Background technique [0002] α-Ketoisocaproic acid (KIC), also known as α-oxoisovaleric acid, is an intermediate metabolite in the synthesis and decomposition of leucine. As a nitrogen-free substitute for leucine, it can be used as a nutritional supplement for patients with kidney disease . In addition, KIC can be used in the fields of feed, health products, and chemical synthesis. [0003] At present, the production methods of KIC are mainly chemical synthesis methods, mainly including the hydrolysis reaction of the addition product of diethyl oxamide and Grignard reagent, double carbonylation method, Heine method and so on. These methods reaction conditions are harsh, all need special structure as starting material, or expensive catalyst (as the complex compound of octahydroxy dicobalt and zer...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12P7/40C12R1/19
Inventor 刘龙陈坚堵国成李江华宋阳
Owner JIANGNAN UNIV
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