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Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

A technology of engineering bacteria, pet-30a, applied in the direction of microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of increasing costs, unrealizable, increasing process flow, etc., to reduce production costs and avoid additives Effect

Active Publication Date: 2014-10-08
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although (3R, 5S) tert-butyl 6-chloro-3,5-dihydroxyhexanoate can be successfully prepared by this preparation method, the required substrate (S)-6-chloro-5-carbonyl-3 -Hydroxyhexanoic acid tert-butyl ester still needs to be prepared by chemical methods, and the conversion from 6-chloro-3,5-dicarbonylhexanoic acid tert-butyl ester to (3R,5S)6-chloro-3,5-dihydroxyhexanoic acid cannot be achieved The one-step production of tert-butyl acid increases the process flow and also increases the cost of production

Method used

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  • Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate
  • Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate
  • Engineering bacteria and method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Construction of plasmid pET30-AdhR

[0064] The adhR gene of the Lactobacillus kefir DSM20587 bacterium was cloned using primers F_AdhR / R_AdhR to obtain an adhR gene with a length of 759bp, and the gene size was verified by nucleic acid electrophoresis, such as figure 1 .

[0065] The sequence of primer F_AdhR is:

[0066] 5'-CATGCCATGGCTATGACTGATCGTTTAAAAG-3';

[0067] The sequence of primer R_AdhR is:

[0068] 5'-CCCGAGCTCTTATTGAGCAGTGTATCCAC-3'.

[0069] The NCBI accession number of the adhR gene sequence is AY267012.

[0070] The adhR gene was digested with NcoI and SacI, and the gene band after digestion was recovered. At the same time, the pET-30a(+) plasmid was digested with NcoI and SacI, the plasmid band after digestion was recovered, and the digested adhR The gene and digested pET-30a(+) plasmid were ligated with ligase and transformed into the cloning host Escherichia coli DH5α. Colony PCR was performed with primers F_AdhR / R_AdhR to verify the...

Embodiment 2

[0071] Example 2 Construction of plasmid pACYCDuet-CB

[0072] The gdhBS gene of Bacillus subtilis168 bacteria was cloned using primers F_GdhBS / R_GdhBS to obtain a gdhBS gene with a length of 786bp, and the gene size was verified by nucleic acid electrophoresis, such as figure 2 .

[0073] The sequence of primer F_GdhBS is:

[0074] 5'-GAAGATCTCATGTATCCGGATTTAAAAGGAAAAGTC-3';

[0075] The sequence of primer R_GdhBS is:

[0076] 5'-CCGCTCGAGTTAACCGCGGCCTG-3'.

[0077] NCBI accession number of gdhBS gene sequence: AL009126.

[0078] The gdhBS gene was digested with BglII and XhoI, and the gene band after digestion was recovered. At the same time, the pACYCDuet-1 plasmid was digested with BglII and XhoI, and the plasmid band after digestion was recovered. The digested gdhBS gene and enzyme The cut pACYCDuet-1 plasmid was ligated with ligase and transformed into the cloning host Escherichia coli DH5α. Colony PCR was performed with primers F_GdhBS / R_GdhBS to verify the trans...

Embodiment 3

[0088] Example 3: Construction and induced expression of genetically engineered bacteria EcoR

[0089] The plasmid pET30-AdhR constructed in Example 1 was transformed into the expression host Escherichia coliBL21 (DE3), and colony PCR was performed with primers F_AdhR / R_AdhR to verify the transformed recombinant. The verified genetically engineered bacteria is EcoR. The EcoR was inoculated into the fermentation medium and cultured with constant temperature shaking for 12-16 hours. The culture conditions were 37° C. and 200 rpm. When the cell concentration grows to OD 600 When =1.0, add 0.6mM IPTG (final concentration) and induce at 16°C for 20h.

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Abstract

The invention discloses an engineering bacteria and a method for preparing tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. The engineered bacteria comprises an alcohol dehydrogenase gene, a glucose dehydrogenase gene and a carbonyl reductase gene; the method comprises the following steps of culturing engineering bacteria and inducing the expression of enzymes, collecting cells by virtue of centrifuging, resuspending with a buffer to obtain a resting cell suspension; adding 6-tert-butyl-chloro-3,5-carbonylhexanoate, glucose and isopropyl alcohol into the resting cell suspension, reacting, after the reaction is completed, separating and purifying the reaction liquid to obtain the tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate. By virtue of the method, one-step production of the tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate from 6-tert-butyl-chloro-3,5-carbonylhexanoate is realized, the process of the tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate is simplified, and the production cost is reduced since the addition of the additional coenzyme is avoided.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an engineering bacterium and a method for preparing (3R, 5S) tert-butyl 6-chloro-3,5-dihydroxyhexanoate by using the engineering bacterium. Background technique [0002] Statins are a class of competitive enzyme inhibitors of hydroxymethylglutaryl coenzyme A (HMG-CoA) reductase, and are also the main blood lipid-lowering drugs. HMG-CoA reductase catalyzes the reduction of HMG-CoA to 3-methyl-3,5-dihydroxyvaleric acid, which is the biosynthesis pathway of cholesterol. Statins can inhibit cholesterol in vivo by inhibiting the synthesis of HMG-CoA reductase Synthesis of free cholesterol, thereby reducing the level of free cholesterol in cells, feedback up-regulation of the expression of low-density lipoprotein receptors on the cell surface, promoting the removal of very low-density lipoprotein cholesterol residues and low-density lipoprotein in circulating blood, and fi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/62C12R1/19
Inventor 吴坚平陈少云何秀娟杨立荣徐刚
Owner ZHEJIANG UNIV
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