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L-threonine aldolase and application of L-threonine aldolase to synthesis of methylsulfonylphenylserine

A technology of thiamphenylphenylserine and threonine aldolase, which is applied in the field of biotechnology and chemical engineering, can solve the problems of high production cost, severe reaction conditions, and large environmental pollution, and achieve the effect of short reaction time

Active Publication Date: 2019-12-17
福建昌生生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to provide an asymmetric biocatalytic synthesis method for chiral intermediates of β-aminoalcohol antibiotics, which solves the problems of cumbersome production steps, severe reaction conditions, high production costs and environmental pollution in organic synthesis methods in the prior art. Major technical bottlenecks

Method used

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  • L-threonine aldolase and application of L-threonine aldolase to synthesis of methylsulfonylphenylserine
  • L-threonine aldolase and application of L-threonine aldolase to synthesis of methylsulfonylphenylserine
  • L-threonine aldolase and application of L-threonine aldolase to synthesis of methylsulfonylphenylserine

Examples

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Embodiment 1

[0038] Example 1 Recombinant expression of L-threonine aldolase

[0039] A novel L-threonine aldolase gene (GenBank No.WP_123664127-1) was screened through gene mining, the amino acid sequence encoded by the gene and the reported L-threonine aldolase (see Table 1) Amino acid sequence similarity is less than 70%, see attached figure 1 The comparison results in .

[0040] Table 1 Information table of L-threonine aldolases reported in the literature

[0041]

[0042]

[0043] The L-threonine aldolase in this example is encoded by the LTA gene (GenBank No. WP_123664127-1) derived from Actinocorallia herbida, and its amino acid sequence is SEQ ID NO:1. The applicant optimized the codon according to the amino acid sequence of L-threonine aldolase, and obtained the optimized base sequence of LTA as SEQ ID NO:2. The optimized LTA gene DNA sequence was artificially synthesized, and the LTA gene was further connected to the plasmid pET28a to construct the recombinant plasmid p...

Embodiment 2

[0046] Embodiment 2 (2S, 3R)-catalyzed synthesis of thiamphenicol phenylserine

[0047] The whole cells of the recombinant Escherichia coli obtained in Example 1 were used, and a 300mL p-thymphenylphenylserine synthesis reaction system was configured: 27.6g p-thymphenylbenzaldehyde, 37.5g glycine, and 6.7mg pyridoxal phosphate were weighed respectively , into a 1L reaction bottle, and 300 mL of phosphate buffer (200 mM, pH 7) containing 30% acetonitrile (v / v) was pre-added in the bottle. After thorough mixing, 3.75 g (wet weight) of recombinant Escherichia coli cells harvested in Example 1 were weighed, and shaken and reacted at 30° C. for 6 h. After the reaction, the supernatant containing the product was collected by centrifugation at 8000 rpm for 5 min. In other embodiments, the recombinant Escherichia coli harvested in Example 1 can also be resuspended with an appropriate amount of phosphate buffer (200 mM, pH 7.0), and then added to the reaction system.

[0048] The L-t...

experiment example 1

[0057] Experimental example 1 Substrate specificity of aldolase catalyzed by L-threonine aldolase

[0058] In order to explore the catalytic activity of L-TA on different benzene ring substituted substrates, the following experiments were designed. Whole-cell catalytic synthesis of (2S,3R)-phenylserine derivatives using benzaldehyde and glycine with different phenyl ring substituents as substrates. The reaction conditions were: 100 mM benzaldehyde with different benzene ring substituents, 50 mg / mL cell concentration, 30% acetonitrile, 37° C. for 4 h. The experimental results are shown in Table 2. React with benzaldehyde (entry1) as substrate, the conversion rate of substrate is 67%, and the de value of product is 17%; With nitrobenzaldehyde (entries2-4), bromobenzaldehyde (entries5-7), Chlorobenzaldehyde (entries8-10) and fluorobenzaldehyde (entries11-13) are reacted as substrates. When the substitution position of the group on the benzene ring of the substrate is in the orth...

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Abstract

The invention provides L-threonine aldolase. The L-threonine aldolase has the activity of asymmetric catalytic synthesis of (2S,3R)-methylsulfonylphenylserine from methylsulfonyl benzaldehyde and glycine. The L-threonine aldolase is selected from any one of the following groups: (1) polypeptide with the amino acid sequence shown in SEQ ID NO: 1; (2) polypeptide with the amino acid sequence shown in SEQ ID NO: 1, wherein the homology of the amino acid sequence is greater than or equal to 80%; and (3) derived polypeptide formed by substituting, missing or adding of 1-5 amino acid residues of theamino acid sequence shown in SEQ ID NO: 1 and retaining catalytic activity. The invention further provides application of the L-threonine aldolase to synthesis of the methylsulfonylphenylserine, an L-threonine aldolase gene derived from Actinocorallia herbida is mined and screened, the L-threonine aldolase derived from recombinant escherichia coli is expressed through a gene engineering technology, the (2S,3R)-methylsulfonylphenylserine is subjected to asymmetric catalytic synthesis by taking the methylsulfonyl benzaldehyde and the glycine as raw materials and adopting a whole-cell catalysismethod, and the reaction conditions are mild and environmentally friendly.

Description

technical field [0001] The invention belongs to the fields of biotechnology and chemical engineering, and in particular relates to an L-threonine aldolase and its application in the synthesis of p-thymphenylphenylserine. Background technique [0002] β-Hydroxy-α-amino acids are important constituents of β-aminoalcohol antibiotics thiamphenicol and florfenicol, glycopeptide antibiotic vancomycin, anticancer drug paclitaxel, and immunosuppressants. p-thiamphenylphenylserine is the key chiral building block for the synthesis of β-aminoalcohol broad-spectrum antibiotics thiamphenicol and florfenicol. It has two chiral centers and can form four chiral isomers. The reported active intermediates are almost all composed of (2S,3R)-configuration. How to efficiently construct two adjacent chiral centers in its structure through asymmetric catalysis, and develop new efficient and green synthetic routes for thiamphenicol and florfenicol have always been the focus and difficulty of orga...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/88C12N15/70C12P13/04C12Y401/02005
Inventor 林娟王力超许炼陈承滔赖凌燕
Owner 福建昌生生物科技发展有限公司
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