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A kind of fermentation production process of α-ketobutyric acid

A technology of ketobutyric acid and production method, which is applied in the directions of microorganism-based methods, microorganisms, biochemical equipment and methods, etc., can solve the problems of high substrate production cost, decreased bacterial biomass and high product toxicity, and achieves improved The effect of bacterial biomass, increasing fermentation level, and easy operation

Inactive Publication Date: 2020-08-18
TIANJIN UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] At present, the industrial production of α-ketobutyric acid mainly adopts chemical synthesis method, but this method has problems such as complex reaction conditions, large energy consumption, and heavy pollution.
However, the precursor conversion method of the biological method has problems such as high substrate production cost and difficult cultivation of microbial preparations; the biggest problem of using recombinant Escherichia coli to directly ferment α-ketobutyric acid in the biological method is that the product is highly toxic, such as figure 1 As shown, when the concentration of α-ketobutyric acid reaches more than 8g / L, α-ketobutyric acid has a serious inhibitory effect on the growth of Escherichia coli and product synthesis
At the same time, it was found through experiments that knocking out the acetohydroxyacid synthase I gene ilvBN would affect the synthesis of valine, and the biomass of the bacteria decreased significantly, and had no obvious effect on increasing the accumulation of α-ketobutyric acid

Method used

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  • A kind of fermentation production process of α-ketobutyric acid
  • A kind of fermentation production process of α-ketobutyric acid
  • A kind of fermentation production process of α-ketobutyric acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Construction of a genetically engineered strain E.coli THRD△ilvIH+PBV220-ilvA producing ɑ-ketobutyric acid

[0054] 1. Knockout of gene ilvI encoding acetohydroxyacid synthase III large subunit

[0055] (1) Design upstream and downstream homology arm amplification primers ilvI-1, ilvI-2, ilvI-5 and ilvI-6 according to the 5' and 3' end 500bp sequences of the ilvI gene in E.col iMG1655 in the NCBI database, and use E The genome DNA of the coliTHRD strain (Accession No. CGMCC No. 11074) is used as a template to amplify homology arm fragments. The PCR amplification conditions were 1 cycle at 95°C for 5 min, 25 cycles at 94°C for 30 s, 56°C for 30 s, 1 min at 72°C, and 1 cycle at 72°C for 10 min, and the reaction system was 50 μL.

[0056] (2) Design amplification primers CM-3 and CM-4 according to the chloramphenicol resistance gene sequence in the plasmid pKD3, and amplify the chloramphenicol resistance gene fragment using the plasmid pKD3 as a template, and t...

Embodiment 2

[0070] Embodiment 2: Shake flask fermentation of genetically engineered bacteria producing α-ketobutyric acid

[0071] Strain: E.coli THRD△ilvIH+PBV220-ilvA Resistance: Amp R

[0072] Seed culture: strains passed through Amp R After two generations of slant activation, scrape 1 ring of wet bacteria with an inoculation needle under sterile conditions and inoculate into 30mL containing 100μg / mL Amp R in the seed medium. Cultivate at 37°C and 200rpm for 8h, during which the pH is adjusted to ≈7.0 with ammonia water according to the color change of the indicator.

[0073] Fermentation conditions: After culturing the seed liquid for 8 hours, the seed liquid OD 600=8, use a sterilized 5mL pipette to draw 3mL seed solution under aseptic conditions and connect to 27mL containing 100μg / mL Amp R In the fermentation medium, the inoculum size was 10%. 35°C, 200rpm to start fermentation culture, when the fermentation broth OD 600 =15, directly increase the temperature to 42°C to in...

Embodiment 3

[0077] Embodiment 3: 7.5L fermenter fermentation of genetically engineered bacteria producing α-ketobutyric acid

[0078] Strain: E.coli THRD△ilvIH+PBV220-ilvA Resistance: Amp R

[0079] Seed culture: strains passed through Amp R After two generations of slant activation, under aseptic conditions, all the wet thallines on 4 slants were inoculated into a 5L seed tank with 2L seed culture medium with an inoculation needle, and 25% (W / V) ammonia water was added to regulate the seeds. The pH of the solution is 7.0, the dissolved oxygen is maintained at about 30-50% by increasing the rotating speed and the ventilation rate, and the culture is carried out at a constant temperature of 37°C.

[0080] Fermentation conditions: waiting for seed liquid OD 600 When=15, 500mL seed liquid is transferred in the 7.5L fermentor that contains aseptic fermentation medium, and total liquid volume is 4L, and inoculum size is 12%. Cultivate at 35°C, add 25% (W / V) ammonia water during the period...

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Abstract

The invention relates to a fermentation production technology of alpha-ketobutyric acid. A threonine dehydrase encoding gene ilvA subjected to site-directed mutagenesis is excessively expressed through a heat-induced expression vector, and a large-subunit encoding gene ilvI of acetohydroxyacid synthase III is knocked out; a threonine production strain E.coliTHRD is adopted as a starting strain. According to the production technology, the inhibition problem of alpha-ketobutyric acid to cell growth and acid production in the fermentation process can be effectively solved, and the highest yield of alpha-ketobutyric acid can reach 22.8 g / L after fermentation is conducted for 28 h.

Description

technical field [0001] The invention relates to the field of compound biotechnology production, in particular to a fermentation production process of α-ketobutyric acid. Background technique [0002] The production methods of α-ketobutyric acid include chemical synthesis and biosynthesis. The chemical method is to obtain α-ketobutyric acid by mixing and hydrolyzing diethyl oxalate and ethyl propionate. Biological synthesis of α-ketobutyric acid mainly includes precursor transformation and direct fermentation. Nakahara et al. (1994) used 1,2-butanediol as carbon source and substrate, and used Rhodococcus equi IF03730 to convert it into α-ketobutyric acid, and the conversion rate was 68.2% after 32 hours of cultivation. Subsequent studies have found that Pseudomonas sputita and Pseudomonas stutzeri SDM can convert crotonic acid and DL-2-hydroxybutyric acid into α-ketobutyric acid, respectively, with a yield of 4.8 g / L. Ma Cuiqing et al. (ZL201110376233.8) reported the prep...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12R1/19
Inventor 谢希贤陈宁齐俊生范晓光徐庆阳张成林
Owner TIANJIN UNIV OF SCI & TECH
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