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Method for enhancing utilization of acetic acid and improving L-arginine produced by escherichia coli fermentation

A technology of Escherichia coli and recombinant Escherichia coli, applied in the biological field, can solve the problems of reduced yield of target products, loss of carbon atoms, etc., and achieve the effects of reducing growth toxicity, reducing toxicity, and high application value

Pending Publication Date: 2022-08-05
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the accumulation of acetic acid will inhibit the growth and metabolism of E. coli on the one hand, and lead to the loss of carbon atoms and the lower yield of target products on the other hand.

Method used

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  • Method for enhancing utilization of acetic acid and improving L-arginine produced by escherichia coli fermentation
  • Method for enhancing utilization of acetic acid and improving L-arginine produced by escherichia coli fermentation
  • Method for enhancing utilization of acetic acid and improving L-arginine produced by escherichia coli fermentation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Screening of acetyl-CoA synthase

[0045] Specific steps are as follows:

[0046] (1) Take the genomic DNA of Escherichia coli, Bacillus subtilis, and Acetobacter pasteurianus as templates respectively, and use F and R as primers to carry out PCR amplification to obtain nucleotide sequences as shown in SEQ ID No.1 to SEQ ID No.4 respectively. The encoding acetyl-CoA synthase genes acs1~acs4; wherein, PCR amplification primers are shown in Table 1:

[0047] Table 1: PCR primers

[0048]

[0049]

[0050] PCR amplification conditions were: 95°C pre-denaturation, 3 min; 95°C denaturation, 30s, 58°C annealing, 30s, 72°C extension, 90s, 30 cycles; 72°C final extension for 10 min.

[0051] The PCR amplification system was: template 0.5 μL, upstream and downstream primers 0.5 μL each, sterilized double-distilled water 23.5 μL, PrimerStar DNA polymerase 25 μL.

[0052] (2) The genes acs 1 to acs 4 (amplification results as shown in figure 1 shown) and the p...

Embodiment 2

[0053] Example 2: Construction of Acetyl-CoA synthase expression vector

[0054] Specific steps are as follows:

[0055] The recombinant plasmids pET28a-acs 1 to pET28a-acs 4 obtained in Example 1 were chemically transformed into Escherichia coli E.coli BL21 (DE3) to obtain transformed products; the transformed products were spread on LB solid medium (containing 10 μg·mL -1 Kanamycin), incubate at 37°C upside down for 12 hours to obtain transformants; colony PCR verification, pick and verify correct transformants and inoculate into LB liquid medium (containing 10 μg·mL -1 kanamycin), in a constant temperature incubator at 37°C, under the conditions of 120-180rpm shake flask culture for 12h, extract the plasmid for PCR verification and sequencing verification, if the verification is correct, the recombinant Escherichia coli E.coli BL21(DE3) / pET28a-acs 1~E.coli BL21(DE3) / pET28a-acs 4; The recombinant plasmids pET28a-acs 1~pET28a-acs 4 of four acetyl-CoA synthases from differen...

Embodiment 3

[0059] Example 3: Construction of host cells

[0060] Specific steps are as follows:

[0061] (1) Using the genome of Escherichia coli BL21 (DE3) as the DNA template, using the primers in Table 3 to amplify the homology arm fusion fragments (adiA, speA, speC, and speF genes of adiA, speA, speC, and speF gene amplification results are as follows: Figure 4 shown).

[0062] Table 3 Primers

[0063]

[0064] PCR amplification conditions were: 95°C pre-denaturation, 3 min; 95°C denaturation, 30s, 58°C annealing, 30s, 72°C extension, 90s, 30 cycles; 72°C final extension for 10 min.

[0065] The PCR amplification system was: template 0.5 μL, upstream and downstream primers 0.5 μL each, sterilized double-distilled water 23.5 μL, PrimerStar DNA polymerase 25 μL.

[0066] (2) Inoculate Escherichia coli BL21 (DE3) in LB liquid medium, cultivate overnight at 37°C, introduce pCas9 plasmid into BL21 by electroporation, spread the plate, pick positive transformants and inoculate in LB...

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Abstract

The invention discloses a method for enhancing utilization of acetic acid and improving L-arginine produced by escherichia coli fermentation, and belongs to the technical field of bioengineering. Acetobacter pasteurianus sourced acetyl coenzyme A synthetase ACS is successfully expressed on a pET28a carrier, and then a recombinant plasmid is transferred into modified escherichia coli ARG-4. The recombinant escherichia coli is inoculated into a fermentation culture medium containing glucose and is subjected to shake flask fermentation for 48 hours, so that the yield of the L-arginine in the fermentation liquid can reach 7.51 g / L, and meanwhile, the content of a byproduct acetic acid is 2.53 g / L. The recombinant Escherichia coli is inoculated into a fermentation medium containing glucose and is fermented in a fermentation tank for 48 hours, so that the yield of L-arginine in fermentation liquor can reach 15.07 g / L, meanwhile, the content of byproduct acetic acid is only 0.41 g / L. In the whole fermentation process, acetic acid can be effectively utilized to generate acetyl coenzyme A, and the recombinant Escherichia coli has the advantages of low raw material cost, few byproducts and easiness in operation.

Description

technical field [0001] The invention relates to a method for enhancing the utilization of acetic acid and improving the fermentation production of L-arginine by Escherichia coli, and belongs to the field of biotechnology. Background technique [0002] L-arginine is an important food additive, commonly used in the fields of food, medicine and chemical industry, and has been known to be of great value in the treatment of liver diseases and wound healing. L-arginine has a low risk factor. It is very safe to use L-arginine in cosmetics. It not only softens the skin stratum corneum, maintains skin moisture, but also relieves skin inflammation and improves rough and dry skin. Since L-arginine is alkaline and does not harm hair, it can also be added to hair dyes in place of ammonia water to improve hair quality. L-arginine preparations can also be used to prepare biological reagents such as spermone mixture, arginine succinate, arginine glucuronide, arginine aspartic acid and phos...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/52C12P13/10C12R1/19
CPCC12N15/70C12N9/88C12N9/93C12Y401/01019C12Y401/01017C12Y602/01C12P13/10C12N1/20Y02E50/10
Inventor 徐美娟饶志明吴洁琴汤佳冰张显杨套伟
Owner JIANGNAN UNIV
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