Method for increasing yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum

A technology of Corynebacterium glutamicum and aminolevulinic acid, which is applied in the field of bioengineering, can solve the problems of low 5-ALA, complex medium components, and expensive 5-aminolevulinic acid, and achieve high yield and optimized fermentation conditional effect

Pending Publication Date: 2021-06-18
JIANGNAN UNIV
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Problems solved by technology

Yang et al. used the strategy of fed-batch fermentation and two-stage fermentation to finally produce 14.7 g / L 5-ALA through the recombinant C. glutamicum C4 pathway, with a productivity of 0.92 g / L / h. The highest yield of 5-ALA synthesized by glutamicum using glucose as raw material (see Yang P, Liu W, Cheng X, et al. A new strategy for production of 5-aminolevulinic Acid in recombinant Corynebacterium glutamicum with highield. Appl Environ Microbiol, 2016 , 82(9):2709-2717.), but the shortcoming of this method is that two-stage fermentation is required, and the medium composition is more complicated, the initial concentration of glycine is higher, and succinic acid also needs to be added
[0004] Because the level of 5-ALA produced by fermentation is generally low at present, the price of 5-aminolevulinic acid is relatively expensive, and the current production capacity of 5-ALA cannot meet domestic demand; therefore, it is necessary to increase the level of fermentation production of 5-ALA Very important

Method used

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  • Method for increasing yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum
  • Method for increasing yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum
  • Method for increasing yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Construction of Corynebacterium glutamicum C1 knocking out α-ketoglutarate inhibitory protein odhI

[0056] Specific steps are as follows:

[0057] (1) With the genomic DNA of Corynebacterium glutamicum ATCC13032 as a template, with odhI F1 (sequence shown in SEQ ID NO.7) and R1 (sequence shown in SEQ ID NO.8), odhI F2 (sequence shown in SEQ ID NO.8) NO.9) and R2 (sequence shown in SEQ ID NO10) were used as primers to amplify the upstream and downstream two homologous arms of about 500 bp each, perform fusion extension PCR, purify and recover the gene fragment and digest it with the pK18 plasmid The obtained linearized product was ligated by DNA ligase, and the ligated product was introduced into E.coli BL21 competent cells by chemical transformation method, cultured on Kan-resistant BHI plate, picked a single colony and transferred to LB medium, and the bacterial liquid was taken Extract the recombinant plasmid pK18-△odhI, sequence pk18-△odhI, and compare t...

Embodiment 2

[0059] Example 2: Construction of recombinant Corynebacterium glutamicum knocking out succinate dehydrogenase sdhA

[0060] Specific steps are as follows:

[0061] (1) Using the genomic DNA of Corynebacterium glutamicum ATCC13032 / pK18-△odhI constructed in Example 1 as a template, with sdhA F1 (sequence shown in SEQ ID NO.11) and R1 (sequence shown in SEQ ID NO.11) NO.12), sdhA F2 (sequence shown in SEQ ID NO.13) and R2 (sequence shown in SEQ ID NO.14) were used as primers to amplify the upstream and downstream two homologous arms of about 500bp each, Fusion extension PCR was performed to obtain a 2200bp gene fragment, which was connected to the pK18mobsacB vector to obtain pk18-△sdhA. The pk18-△sdhA was sequenced and compared by DNAMAN. The results showed that the gene sequence was correct.

[0062] (2) Transfer the recombinant plasmid pk18-△sdhA to Corynebacterium glutamicum ATCC13032 / pK18-△odhI by electroporation to obtain transformants, then expand the prepared transforman...

Embodiment 3

[0063] Example 3: Cloning and expression of 5-aminolevulinic acid synthase hemA in Corynebacterium glutamicum strain C. glutamicum 13032-△odhI△sdhA

[0064] Specific steps are as follows:

[0065] (1) Construction of recombinant plasmids

[0066] The gene hemA (Genebank: AY489557.1) sequence derived from Rhodopseudomonas palustris was synthesized in Suzhou Jinweizhi Biological Co., Ltd.

[0067] Using the synthesized recombinant plasmid containing the target gene as a template, using primers hemA-F and hemA-R respectively, the target gene fragment hemA fragment was obtained by PCR amplification, and the obtained hemA fragment and pXMJ19 plasmid were double-digested with HindIII and EcoRI and After ligation, the recombinant plasmid was obtained, and the recombinant plasmid was transformed into Escherichia coli BL21, the positive transformant was picked to extract the plasmid, and the plasmid was used as a template for PCR verification. The results showed that the plasmid pXMJ1...

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Abstract

The invention discloses a method for increasing the yield of 5-aminolevulinic acid synthesized by corynebacterium glutamicum, and belongs to the technical field of bioengineering. According to the method, the construction of the recombinant corynebacterium glutamicum for knocking out alpha-ketoglutaric acid inhibitory protein and succinate dehydrogenase and overexpressing 5-aminolevulinic acid synthetase is successfully realized. A 5-L fermentation tank batch fermentation strategy is adopted, fermentation conditions are optimized, finally, recombinant corynebacterium glutamicum (C. glutamicum) 13032/delta odhI delta sdhA/pXMJ19-hemA is fermented for 84 h, 25.05 g/L of 5-ALA is synthesized within 48 h under the condition that glycine with the final concentration of 4 g/L is added, the productivity is 0.52 g/L/h, and at the moment, the sugar-acid conversion rate reaches 16.79%. The high yield of the 5-aminolevulinic acid is realized, and the highest yield of one-step fermentation starting from glucose as a carbon source to the current is obtained.

Description

technical field [0001] The invention relates to a method for improving the output of 5-aminolevulinic acid synthesized by Corynebacterium glutamicum, belonging to the technical field of bioengineering. Background technique [0002] 5-Aminolevulinic acid (5-ALA) is a non-proteinogenic amino acid of many tetrapyrrole compounds such as heme, chlorophyll, vitamin B12 precursor. It plays a very important role in many fields, especially medicine and agriculture. In the pharmaceutical industry, 5-ALA is currently mainly used in photodynamic diagnosis and treatment of various cancers and tumor localization of various diseases. In agriculture, it is used as a biodegradable herbicide, insecticide or growth regulator that is non-toxic to crops, animals and humans. [0003] The production methods of 5-ALA mainly include chemical synthesis and microbial fermentation. Due to the low output of the chemical synthesis method and serious environmental pollution problems, it is not suitable...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N9/02C12N9/10C12N15/77C07K14/34C12P13/00C12R1/15
CPCC12N9/001C12N9/1029C12N15/77C07K14/34C12P13/005C12Y103/99001C12Y203/01037
Inventor 饶志明王丽君杨套伟徐美娟张显邵明龙
Owner JIANGNAN UNIV
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