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Single-cell plant for efficiently synthesizing alpha-aminobutyric acid, as well as construction and application thereof

An aminobutyric acid and single-cell technology, applied in the field of microorganisms, can solve the problems of cumbersome process, accumulation of intermediate product ketobutyric acid, high cost, etc.

Active Publication Date: 2017-02-15
JIANGNAN UNIV
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  • 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

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  • Single-cell plant for efficiently synthesizing alpha-aminobutyric acid, as well as construction and application thereof
  • Single-cell plant for efficiently synthesizing alpha-aminobutyric acid, as well as construction and application thereof

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...

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Abstract

The invention discloses a single-cell plant for efficiently synthesizing alpha-aminobutyric acid, as well as construction and application thereof, and belongs to the technical field of microorganisms. An escherichia coli single-cell plant which is constructed and recombined by tandem expression of L-threonine deaminase, L-amino acid dehydrogenase and a dehydrogenase gene for providing cofactor NADH circulation is used for efficiently synthesizing the alpha-aminobutyric acid, and the expression quantity of the L-threonine deaminase is optimized and controlled by rbs sequence, so that the problem of conversion inhibition caused by quick accumulation of an intermediate product ketobutyric acid is solved; meanwhile, the expression quantity of the dehydrogenase for providing cofactor NADH circulation is optimized and controlled by a promoter and the rbs sequence, so that the regeneration rate of the NADH is increased and the yield is finally increased. The single-cell plant is used for performing full-cell conversion, so that substances in and out obstacles can be reduced, the conversion rate is increased, cofactor intracellular circulation is promoted, an external source does not need to be added, and the cost is low. Within 20 hours, the yield of the recombinant escherichia coli single-cell plant in a 5-liter fermentation tank is 204 g / L, the space time yield is 10.2 g / L*h, and an actual and effect strategy is provided for 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

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Application Information

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Patent Type & Authority Applications(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
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