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A kind of recombinant gene engineering bacterium and its fermentative application to produce vanillin

A technology of genetically engineered bacteria and vanillin, applied in the direction of genetic engineering, fermentation, application, etc., to achieve good application prospects, enhance tolerance, and improve the effect of ability

Active Publication Date: 2021-04-06
ZHEJIANG UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the relationship between AcrAB-TolC efflux pump and E. coli tolerance to ferulic acid and vanillin; and there is no use of overexpression of AcrAB-TolC efflux pump-related genes to improve the production efficiency of vanillin produced by recombinant E. coli related reports

Method used

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  • A kind of recombinant gene engineering bacterium and its fermentative application to produce vanillin
  • A kind of recombinant gene engineering bacterium and its fermentative application to produce vanillin
  • A kind of recombinant gene engineering bacterium and its fermentative application to produce vanillin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Obtaining of the plasma membrane fusion protein AcrA amino acid sequence and nucleic acid sequence

[0030] The periplasmic membrane fusion protein gene sequence was screened using NCBI database, and a periplasmic membrane fusion protein gene was obtained. The sequence is derived from Escherichia coli (E.coli). According to the characteristics of the cloning vector pACYCDuet, the restriction endonucleases Nco I and Hind III restriction sites were designed. Using the genome of E. coli MG1655 strain as a template, primers acrA- u, acrA-d were amplified by PCR to obtain the periplasmic membrane fusion protein AcrA gene (shown in SEQ ID NO.1 (1-1394)), and the amino acid sequence of the encoded enzyme was shown in SEQ ID NO.2 (1-397) Show. The primer sequences are as follows:

[0031] acrA-u: 5′-taataaggagatataccatggGAATGTATGTACCATAGCACGACGA-3′;

[0032] acrA-d: 5'-gcattatgcggccgcaagcttCGCCAGCCCCCCTGCCAA-3'.

Embodiment 2

[0033] Embodiment 2 Construction of recombinant vector pACYCDuet-acrA and construction of engineering bacteria

[0034] The periplasmic membrane fusion protein AcrA gene fragment cloned in Example 1 was double-digested and recovered using restriction endonucleases Nco I and Hind III, and the fragment was treated with the same restriction endonuclease using T4 DNA ligase The treated cloning vector pACYCDuet was ligated at 22°C for 2 hours to construct the recombinant vector pACYCDuet-acrA.

[0035] The constructed recombinant vector pACYCDuet-acrA was transformed into recipient bacteria E.coli BW25113, spread on LB solid plates (containing 100 μg / mL ampicillin), and cultured at 37°C. Clones were randomly selected from the colonies grown on the plate, and the plasmids were extracted for agarose gel electrophoresis identification, and the engineering bacteria E.coli BW25113 / pACYCDuet-acrA were screened.

Embodiment 3

[0036] Example 3 Obtaining the Amino Acid Sequence and Nucleic Acid Sequence of Inner Membrane Transporter AcrB

[0037] The NCBI database was used to screen the gene sequence of the inner membrane transporter, and an inner membrane transporter gene was obtained. The sequence is derived from Escherichia coli (E.coli). According to the characteristics of the cloning vector pACYCDuet, the restriction endonucleases Nco I and Hind III restriction sites were designed. Using the genome of E. coli MG1655 strain as a template, primers acrB- u, acrB-d were amplified by PCR to obtain the inner membrane transporter AcrB gene (shown in SEQ ID NO.1 (1395-4744)), and the amino acid sequence of the encoded enzyme was shown in SEQ ID NO.2 (398-1446) . The primer sequences are as follows:

[0038] acrB-u: 5′-taataaggagatataccatggGTAAAAGCACAAGAAGTTACCGCTG-3′;

[0039] acrB-d: 5'-gcattatgcggccgcaagcttTTCGTATGAGATCCTGAGTTGGTG-3'.

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Abstract

The invention discloses a recombinant genetically engineered bacterium and its application to produce vanillin by fermentation. The genes of vanillin or ferulic acid include one or more of the periplasmic membrane fusion protein AcrA gene, the inner membrane transport protein AcrB gene or the outer membrane protein TolC gene; the host includes the large intestine containing the ech gene and the fcs gene bacilli. The recombinant genetically engineered bacteria E.coli BW25113::PegYB::pACYCDuet-acrAB-tolC of the present invention can use ferulic acid as a substrate to produce vanillin, and the highest yield of vanillin is 2335 mg / L. The fermented liquid fermented by the method of the invention contains higher vanillin and has good application prospects.

Description

[0001] (1) Technical field [0002] The invention belongs to the technical field of bioengineering, and in particular relates to a recombinant engineering bacterium and a method for producing vanillin, especially a recombinant engineering bacterium resistant to ferulic acid and vanillin and a construction method thereof, and using the A method for the production of vanillin by bacterial strains using ferulic acid as a substrate. [0003] (2) Background technology [0004] Vanillin, also known as vanillin, has a chemical name of 3-methoxy-4-hydroxybenzaldehyde, and is the main component of the popular creamy vanilla flavor. Vanillin is currently the most used spice in the world, and consumers especially welcome the natural vanillin obtained through biotechnology. The yield of natural vanillin extracted from plants is small and expensive, and the use of microbial fermentation is an important method to produce natural vanillin. [0005] In recent years, some researchers have tri...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12P7/24C12R1/19
CPCC07K14/245C12P7/24
Inventor 嘉晓勤刘昌东侯强波郑王建燕化远陈小龙
Owner ZHEJIANG UNIV OF TECH
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