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Escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase as well as construction method and application of escherichia coli

A lactate dehydrogenase and formate dehydrogenase technology, applied in the field of Escherichia coli and its construction, can solve the problems of difficulty in obtaining high optical purity L-phenyllactic acid, the enzyme is easily affected by the external environment, and is not suitable for industrial applications, etc., to achieve High yield, simple reaction system components and low cost

Active Publication Date: 2014-11-05
深圳市华利康纤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the fermentation and synthesis of phenyllactic acid by lactic acid bacteria, not only L-phenyllactic acid and D-phenyllactic acid are produced at the same time, but also organic acids such as lactic acid are often produced. It is difficult to obtain L-phenyllactic acid with high optical purity.
In the process of in vitro enzyme-catalyzed reaction, the enzyme is easily affected by the external environment, the substrate tolerance is poor, and the activity is lost quickly. At the same time, the expensive coenzyme NADH needs to be added from an exogenous source, which is not suitable for industrial application.
At present, there is no report on the simple and efficient synthesis technology of L-phenyllactic acid

Method used

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  • Escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase as well as construction method and application of escherichia coli
  • Escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase as well as construction method and application of escherichia coli
  • Escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase as well as construction method and application of escherichia coli

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Construction of Escherichia coli genetic engineering strain E.coli BL21 (pETDuet-ldhL-fdh).

[0033] (1) Acquisition of formate dehydrogenase gene fdh gene and construction of recombinant plasmid pETDuet-fdh.

[0034] Escherichia coli expression plasmid pETDuet-1 (Novagen) has two multiple cloning sites, which can express two target genes simultaneously. First, synthesize the formate dehydrogenase gene fdh (GenBank accession number is AJ011046) encoding Candida boidinii in the present invention by chemical synthesis, and introduce Nde that can be inserted into the multiple cloning site 2 of the pETDuet-1 plasmid at both ends of the sequence I and Xho I enzyme cutting sites, after double enzyme cutting, it was connected to the pETDuet-1 plasmid, transformed into E. coli competent cells, and after ampicillin resistance screening, the positive clone plasmid was extracted and analyzed by restriction map, and verified. Recombinant plasmid pETDuet-fdh carrying fdh...

Embodiment 2

[0042] Example 2: Coupled expression of L-lactate dehydrogenase and formate dehydrogenase in E. coli BL21 (pETDuet-ldhL-fdh).

[0043] The correct recombinant strain E.coli BL21 (pETDuet-ldhL-fdh) screened in Example 1 was inoculated in 50 mL of LB liquid medium containing 100 μg / mL ampicillin, cultured on a shaker at 37°C for 12 hours, and transferred to 1 L containing 100 μg / mL ampicillin LB liquid medium, cultured on a shaker at 37°C, when the culture medium OD 600nm After reaching 0.4-0.8, IPTG with a final concentration of 1 mmol / L was added to induce expression, and culture was continued for 5 hours at 25°C. After the induction culture was over, centrifuge at 12,000 rpm for 5 minutes to obtain cell pellets, wash twice with saline, and resuspend in 100 mL of phosphate buffer with pH 7.4 and 50 mmol / L. Ultrasound was used to break the cell wall in the ice-water mixture. The breaking time was 30 minutes. The solution after breaking the wall was centrifuged at 16,000 rpm f...

Embodiment 3

[0044] Example 3: E.coli BL21 (pETDuet-ldhL-fdh) produces L-phenyllactic acid under different pH conditions.

[0045] In the 10mL reaction system, add 50mmol / L substrate phenylpyruvate, 100mmol / L auxiliary substrate sodium formate, and recombinant Escherichia coli wet cells with a dry weight of 7g / L, mix well and shake the reaction on a constant temperature shaker at 37°C , the pH of the reaction system was respectively 5.0, 5.5, 6.0, 6.5, 7.0, 7.5. After 1 hour, the reaction mixture was centrifuged to remove the thallus, and the supernatant liquid was taken to detect the output and optical purity of L-phenyllactic acid generated. The results showed (Table 1), when the pH of the reaction system is 5.5-6.0, the yield of L-phenyllactic acid is the highest, which is 40.1 mmol / L, and the optical purity of L-phenyllactic acid is greater than 99.9%.

[0046] The output of L-phenyllactic acid under different pH conditions in table 1

[0047] pH

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Abstract

The invention discloses escherichia coli with coexpression of L-lactate dehydrogenase and formate dehydrogenase. The escherichia coli is characterized in that L-lactate dehydrogenase genes ldhL from bacillus coagulans and formate dehydrogenase genes fdh from candida boidinii are introduced into the escherichia coli, wherein the register number of GenBank of nucleotide sequences of the L-lactate dehydrogenase genes ldhL is KF386111; the register number of GenBank of nucleotide sequences of the formate dehydrogenase genes fdh is AJ011046. The invention further discloses a construction method and an application of the reconstructed escherichia coli. The method has the characteristics of being simple to operate, low in cost, high in product synthesis efficiency and high in optical purity. Thus, an effective way for biosynthesis of L-phenyllactic acid is provided.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and specifically relates to an Escherichia coli co-expressing L-lactate dehydrogenase and formate dehydrogenase, a construction method and application thereof. Background technique [0002] Phenyllactic acid (PLA), namely 2-hydroxy-3-phenylpropionic acid, also known as 3-phenyllactic acid or β-phenyllactic acid, is a new type of natural preservative discovered in recent years. Antibacterial spectrum, not only can inhibit Gram-positive bacteria, Gram-negative bacteria, but also has obvious inhibitory effect on various molds and other fungi that cause food spoilage, and has high safety performance, and has a good application in the food industry prospect. In addition, phenyllactic acid is also the precursor of many drugs. Danshensu (3,4-dihydroxyphenyllactic acid), a derivative of phenyllactic acid, can inhibit platelet aggregation and expand coronary arteries, so it has important medical a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12P7/40C12R1/19
Inventor 郑兆娟赵明月周影徐艳冰姜婷欧阳嘉
Owner 深圳市华利康纤生物科技有限公司
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