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Corynebacterium acetoacidophilum strain and method for producing succinic acid therefrom

A technology of Corynebacterium acetoacetophilus and succinic acid, which is applied in the field of bioengineering, can solve the problems of complex operation process, long fermentation cycle, and reduced activity, and achieve the effects of simple culture conditions, rapid growth, and high conversion rate

Active Publication Date: 2012-08-15
JIANGNAN UNIV
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AI Technical Summary

Problems solved by technology

Many scholars have reported E. coli The method for producing succinic acid by genetic engineering fermentation, such as U.S. Patent US5770435 and Chinese Patent CN1886516A, provides Pts G, pfl B , ldh A mutation (i.e. non-functioning phosphotransferase system, pyruvate formate lyase system, and lactate dehydrogenase system) E. coli Engineering bacteria AEP111, after 6 hours of aeration culture, closed the aeration and applied anaerobic conditions, fermented for 144 hours, produced 59.35 g / L of succinate, and its fermentation period was longer; Chinese patent CN1268972A announced the use of genetic engineering technology to construct a kind of acetic acid and lactate formation pathway deficiencies E. coli Engineering bacteria SS373, the aerobic cultured SS373 was transferred to anaerobic culture, but the acid production was not high (only 2.7 g / L); coli Engineering bacterium NTN111 adopts the method of aerobic culture first and then anaerobic fermentation to produce succinic acid, and the yield is also low. The succinic acid produced by anaerobic fermentation for 41 hours is 25g / L
In addition, in terms of research on the production of succinic acid by Corynebacterium glutamicum, Chinese patents CN1989238A, CN1989239A and CN1389752A respectively announced the use of genetic engineering technology to construct acetyl-CoA hydrolase activity reduction, pyruvate oxidase activity reduction and enhanced 2-oxopentane Corynebacterium mutant strain of diacid dehydrogenase activity, but its operation process is more complicated; C. glutamicum ATCC 13032 was the starting strain, and it was subjected to mutagenesis treatment, and a strain that did not produce lactic acid and high-yield succinic acid was screened C. glutamicum CGMCC No.3991, the anaerobic fermentation of this strain for 36 hours, its succinic acid production can reach 35.8 g / L, but its disadvantage is still that the production is not high

Method used

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  • Corynebacterium acetoacidophilum strain and method for producing succinic acid therefrom
  • Corynebacterium acetoacidophilum strain and method for producing succinic acid therefrom
  • Corynebacterium acetoacidophilum strain and method for producing succinic acid therefrom

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Embodiment 1

[0024] Embodiment 1, gene knockout vector pK19mob sacB -Δ ldh build

[0025] 1) PCR amplification ldh Upstream and downstream fragments of the gene: Extract Corynebacterium acetoacetophilus with a genomic DNA extraction kit (product of Sangon Company) C. acetoacidophilum Genome of ATCC 13870. According to Gene Bank C. glutamicum Primers were designed for the whole genome sequence of ATCC 13032 (Gene Bank No: Nc_006958.1). Using the extracted genome as a template, P1 (SEQ ID No.1) and P2 (SEQ ID No.2) as primers, PCR amplification under the action of Taq enzyme (product of Guangzhou Dongsheng Company) ldh Gene upstream fragment ( ldh 503bp upstream of the start codon of the gene, namely SEQ ID No.5). With P3 (SEQ ID No.3) and P4 (SEQ ID No.4) as primers, PCR amplification ldh The downstream fragment of the gene ( ldh 500bp downstream of the gene termination codon, namely SEQ ID No.6). The sequences of the above primers are respectively:

[0026] P1: AAG...

Embodiment 2

[0033] Embodiment 2, ldh gene deletion C. acetoacidophilum Δ ldh preparation of

[0034] 1) C. acetoacidophilum Competent preparation: pick a fresh plate C. acetoacidophilum ATCC 13870, inoculated into liquid LB medium containing 1% glucose, cultured overnight at 30°C, 200r / min, and then transferred to 30 mL containing 1% (mass volume ratio) glycine and 0.5% (volume ratio) Tween 80 In LB medium, make the starting OD 600 reach 0.5, continue culturing at 37°C, 100r / min for 20 hours to OD 600 reached 0.9. After the bacterial solution was ice-bathed, the bacterial cells were collected by centrifugation, washed 4 times, and the cells were suspended with pre-cooled 15% (volume percentage) glycerol, and distributed in 1.5 mL centrifuge tubes, 100 μL per tube. Store the competent cells in a -70°C refrigerator or directly use them for electroshock transformation.

[0035] 2) Electric shock transformation plasmid pK19mob sacB -Δ ldh : Take the competent cells out of th...

Embodiment 3-4

[0037] Embodiment 3-4, the method for producing succinic acid by Corynebacterium acetoacetophilus

[0038] (1) Aerobic medium (g / L): glucose 20, urea 1.2, yeast extract 4, casein 4, (NH 4 ) 2 SO 4 10, KH 2 PO 4 1.5,K 2 HPO 4 1.5, MgSO 4 ·7H 2 O 1.5, MnSO 4 ·H 2 O 0.005, FeSO 4 ·7H 2 O 0.06, thiamine 0.0004, biotin 0.0004, pH 7.0-7.2, sterilized at 115°C for 20 min.

[0039] (2) Culture medium in anoxic environment (g / L): glucose 80, carbonate 60, KH 2 PO 4 1.5,K 2 HPO 4 1.5, MgSO 4 ·7H 2 O 1.5, MnSO 4 ·H 2 O 0.005, FeSO 4 ·7H 2 O 0.06, Thiamine 0.0004, Biotin 0.0004. pH 7.0-7.2, sterilized at 115°C for 20 minutes.

[0040] Corynebacterium acetoacetophilus ATCC 13870 and YF / Δ ldh Pick a ring of fresh slant and inoculate them in the Erlenmeyer flasks with aerobic medium, culture at 35°C and 300 r / min for 36 hours, expand the culture according to the inoculum size of 1%, and collect by centrifugation after 48 hours bacteria. The wet cells were tran...

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Abstract

A corynebacterium acetoacidophilum strain and a method for producing succinic acid therefrom belong to the technical field of bioengineering. The invention discloses a corynebacterium acetoacidophilum strain YF / delta ldh and a method for producing succinic acid therefrom. The corynebacterium acetoacidophilum strain is an ldh (L-lactate dehydrogenase) missing bacterium built by gene knockout technology, is preserved in china center for type culture collection on February 29th, 2012, and encoded as CCTCC NO.M2012041. The strain is high in acid yield, highly resistant to sodions and high in foodsafety, can accumulate succinic acid more than 95g / L by being culture for 48 hours in the anaerobic environment using glucose as substrate and sodium bicarbonate as carbon dioxide donor, and can accumulate succinic acid 136g / L by being cultured for 94 hours.

Description

technical field [0001] The invention relates to constructing an engineering bacterium of Corynebacterium acetoacetophilus by using genetic engineering technology and producing succinic acid by using the genetic engineering bacterium, which belongs to the technical field of bioengineering. Background technique [0002] Succinic acid (butanedioic acid), also known as succinic acid (succinic acid), widely exists in animals, plants and microorganisms, and is one of the intermediate products of the TCA cycle. Named for its earliest isolation from amber. Succinic acid is an important four-carbon compound in industry. It is widely used in food, medicine, pesticides, dyes, spices, paints, plastics and material industries as organic synthesis raw materials, intermediate products or special chemicals. [0003] At present, the production methods of succinic acid mainly include chemical synthesis and biological methods. Obtaining succinic acid by chemical methods not only consumes a l...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/77C12P7/46C12R1/15
Inventor 郑璞于芳杨倩
Owner JIANGNAN UNIV
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