A single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application

An aminobutyric acid, single-cell technology, applied in the field of microorganisms, can solve the problems of high cost, increased production cost of α-aminobutyric acid, cumbersome process and the like

Active Publication Date: 2019-12-10
JIANGNAN UNIV
View PDF6 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the enzyme conversion process, it was found that the amount of L-threonine deaminase needs to be precisely controlled, otherwise it will cause the accumulation of the intermediate product ketobutyric acid, thereby inhibiting the conversion of ketobutyric acid to α-aminobutyric acid, causing the enzyme conversion Interruption of production of alpha-aminobutyric acid
At the same time, using the enzyme transformation system to produce α-aminobutyric acid requires cell disruption of the three enzyme-producing recombinant bacteria, which is cumbersome and costly. At the same time, the stability of the transformation is affected by the inactivation of the enzyme during the transformation process. In addition, due to the loss of cofactors, it is necessary to continuously add exogenous sources, which further increases the production cost of α-aminobutyric acid

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application
  • A single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: The rbs sequence optimization of L-threonine deaminase and the construction of recombinant L-threonine deaminase Escherichia coli

[0039] [1] Combining the L-threonine deaminase gene ltd sequence from Escherichia coli with the T7 promoter, we designed rbs sequences with different expression intensities according to its expression in Escherichia coli, and then sent them to Shanghai Sangon Biotech for gene expression synthesis. PCR primers include primers rbs1, rbs2, rbs3, rbs4, rbs5, rbs6 and L-threonine containing rbs sequences with different expression intensities (indicated in bold with underline, and the sequences are shown in SEQ ID NO: 1 to SEQ ID NO: 6) The terminal primer ltdR of the acid deaminase gene (sequences such as SEQ ID NO: 7 to SEQ ID NO: 13).

[0040]

[0041] ltdR: 5'-CGGGATCCTTAACCCGCCAAAAAGAACCTG-3' (BamH I)

[0042] [2] Use primers containing rbs sequences with different expression intensities and terminal primer ltdR to form a pri...

Embodiment 2

[0047] Example 2: Construction of recombinant plasmids and recombinant bacteria co-expressing L-threonine deaminase and L-amino acid dehydrogenase

[0048] [1] Genomic DNA of Bacillus cereus, Rhodococcus, Bacillus subtilis and Streptomyces coelicolor were used as templates.

[0049] [2] Design L-amino acid dehydrogenase gene primers according to the restriction site of L-amino acid dehydrogenase gene sequence and pET-28a plasmid, including the L-leucine dehydrogenase gene Bcldh of Bacillus cereus ( Primers are PBcldhF, PBcldhR), Rhodococcus L-phenylalanine dehydrogenase gene Rjpdh (primers are PRjpdhF, PRjpdhR), Bacillus subtilis L-alanine dehydrogenase gene Bsadh (primers are PBsadhF, PBsadhR) , the valine dehydrogenase gene Scvdh of Streptomyces coelicolor (primers are PScvdhF, PScvdhR). The primer sequences are as follows (such as SEQ ID NO: 14 to SEQ ID NO: 21):

[0050] PBcldhF: 5'-CGGGATCCAAGGAGATATACATGACATTAGAAATCTTCG-3'(BamH I)

[0051] PBcldhR: 5'-CGAGCTCTTAGCGACG...

Embodiment 3

[0061] Example 3: Construction of recombinant Escherichia coli providing cofactor NADH circulating dehydrogenase and construction of recombinant Escherichia coli optimized for the promoter and rbs sequence of formate dehydrogenase

[0062][1] According to the gene sequence of the dehydrogenase that provides the cofactor NADH cycle from different sources and the restriction site connected in series to the pET-28a plasmid, primers are designed, including the formate dehydrogenase fdh of Candida boidinii ( The primers are PfdhF, PfdhR), the glucose dehydrogenase Bsglcdh of Bacillus subtilis (the primers are PBsglcdhF, PBsglcdhR), the glucose dehydrogenase Ppglc of Pseudomonas putida (the primers are PPpglcdhF, PPpglcdhR). Carry out PCR with corresponding primers, genome template respectively, obtain the gene fragment of the dehydrogenase that provides the cofactor NADH circulation of corresponding bacterial strain origin, connect it with pET-28a plasmid (nucleotide fragment and pl...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application, belonging to the technical field of microorganisms. In the present invention, L-threonine deaminase, L-amino acid dehydrogenase, and the dehydrogenase gene that provides the cofactor NADH cycle are expressed in series to construct a recombinant E. coli single-cell factory for efficient synthesis of α-aminobutyric acid, through Optimized rbs sequence controls the expression of L-threonine deaminase, solves the problem of rapid accumulation of the intermediate product ketobutyric acid and inhibits transformation, and controls the expression of dehydrogenase that provides the cofactor NADH cycle through promoter and rbs sequence optimization amount, enhancing the rate of NADH regeneration ultimately increases production. The use of single-cell factories for whole-cell transformation can reduce material entry and exit barriers, speed up the transformation rate, and promote the intracellular circulation of cofactors without exogenous additions, and the cost is low. Within 20 hours, the single-cell factory yield of recombinant E. coli in a 5L fermenter was 204g / L, and the space-time yield was 10.2g / L·h, providing a practical and effective strategy for its industrial production.

Description

technical field [0001] The invention relates to a single-cell factory for efficiently synthesizing α-aminobutyric acid and its construction and application, belonging to the technical field of microorganisms. Background technique [0002] Unnatural α-amino acids are a large class of amino acids that are different from the 22 natural α-amino acids that can be synthesized by organisms themselves. They have important biological activities and physiological effects, and are widely used in compounds such as polypeptides, chiral drugs, and alkaloids. Synthesis. α-aminobutyric acid is an unnatural amino acid that inhibits the transmission of human nerve information. It can enhance the activity of glucose phosphatase and promote the metabolism of brain cells. α-aminobutyric acid is also an important chemical raw material and pharmaceutical intermediate, which has been widely used in the synthesis of drugs, such as the synthesis of anti-tuberculosis drug ethambutol hydrochloride and...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12P13/04C12P7/40C12R1/19
CPCC12N9/0016C12N9/88C12P7/40C12P13/04C12Y104/01005C12Y403/01019C12N15/73C12N1/205C12R2001/07C12R2001/19
Inventor 饶志明周俊平杨套伟张显徐美娟张蔡喆戚云龙郑俊贤
Owner JIANGNAN UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap