Construction and expression of D-LDH-FDH fusion gene capable of improving D-phenyllactic acid yield

A technology of D-LDH-FDH and phenyl lactic acid, which is applied in genetic engineering, plant gene improvement, DNA preparation, etc., can solve the problems of increased reaction costs, etc., and achieve the effects of convenient function assistance, easy operation, and strong catalytic effect

Active Publication Date: 2015-08-19
CHANGSHU INSTITUTE OF TECHNOLOGY
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  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0003] Lactate dehydrogenase is the key enzyme for the conversion of phenylpyruvate to phenyllactate in natural microbial cells, but because the enzyme needs to consume the reduced coenzyme in the system during the catalytic process, the additional addition of the reduced coenzyme will significantly increase the reaction cost

Method used

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  • Construction and expression of D-LDH-FDH fusion gene capable of improving D-phenyllactic acid yield
  • Construction and expression of D-LDH-FDH fusion gene capable of improving D-phenyllactic acid yield
  • Construction and expression of D-LDH-FDH fusion gene capable of improving D-phenyllactic acid yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This example illustrates the construction of a dual-enzyme co-expression vector.

[0046] 1PCR primer design

[0047] On the basis of retaining the functional regions of D-LDH and FDH genes, design PCR primers, design an overlapping complementary fragment between the downstream primer of D-LDH and the upstream primer of FDH, and add a flexible connecting peptide sequence (linker).

[0048] 1.1D-LDH primer: the upstream primer (PL1) is as follows:

[0049] 5'-CGC GGA TCC G AA AAT TAT TGC ATA TGC-3' (the underlined part is the BamHI restriction site)

[0050] The downstream primer (PL2) is as follows:

[0051] 5’-GTC AAA CTT AAC TTG TGTG-3’

[0052] 1.2 FDH primer: a section (underlined part) of the FDH upstream primer overlaps and is complementary to the D-LDH downstream primer, and is used to connect the two genes when fusion PCR is performed. Wherein Gly4-Ser-Gly4-Ser-Gly4-Ser is a specially designed connecting peptide sequence. The FDH upstream primer (PF1) i...

Embodiment 2

[0073] This example illustrates the construction of recombinant bacteria and the induced expression of fusion proteins.

[0074] 1. Construction of recombinant bacteria

[0075] The above recombinant vector pET-Duet-D-LDH-Linker-FDH was transformed into Escherichia coli BL21(DE3), and positive clones were screened to obtain BL21(DE3) / pET-Duet-D-LDH-Linker-FDH recombinant bacteria.

[0076] 2. Induced expression of fusion protein by recombinant bacteria

[0077] Inoculate the transformed BL21(DE3) / pET-Duet-D-LDH-Linker-FDH single colony in LB liquid medium containing 50mg / L ampicillin, and culture overnight at 37°C and 200r / min (about 9h) Inoculate 1% in fresh LB medium containing the same concentration of ampicillin, culture at 37°C and 200r / min until the OD600 is about 0.6-0.8, add IPTG concentration to 0.6mmol / L, and lower the induction temperature to 25°C, After induction at 200r / min for 5h, the cells were collected by centrifugation, washed twice with PBS, resuspended in...

Embodiment 3

[0079] This example illustrates the transformation of whole cells of recombinant Escherichia coli to synthesize phenyllactic acid.

[0080] Pick a single colony of recombinant bacteria and inoculate it in LB liquid medium containing 50mg / L ampicillin, culture overnight at 37°C and 200r / min (about 9h), and inoculate it at 1% in fresh LB medium containing the same concentration of ampicillin At 37°C and 200r / min, when the OD600 was about 0.6-0.8, IPTG concentration was added to 0.6mmol / L, and the induction temperature was lowered to 25°C. After induction at 200r / min for 5h, the bacteria were collected by centrifugation and washed with PBS for two times. Second, the prepared sludge was used for whole cell transformation. Resuspend the collected bacteria in transformation medium containing 20g / L glucose and 9g / L sodium phenylpyruvate at pH 7.0, make the dry weight of recombinant Escherichia coli 20g / L, transform at 37°C and 200r / min constant temperature shaking Cultivate and samp...

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Abstract

The invention relates to a cDNA sequence of a D-LDH-FDH fusion gene capable of improving D-phenyllactic acid yield, wherein the cDNA sequence is formed by a D-LDH gene, a DNA sequence of connecting peptide and an FDH gene through serial connection. The invention further provides a construction method and an expression method for the fusion gene. By serially connecting lactic dehydrogenase and formic dehydrogenase through the connecting peptide to obtain fusion protein, activities of two enzymes are kept after the protein is expressed in colon bacillus, the enzyme activity of lactic dehydrogenase in the fusion protein is higher than the activity during single expression and is higher than the activity during separate expression of lactic dehydrogenase and formic dehydrogenase in the same cell, and a stronger catalytic effect is reflected when phenylpyruvic acid is catalytically converted into phenyllactic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the construction and expression of a D-LDH-FDH fusion gene for enhancing the production of D-phenyl lactic acid. Background technique [0002] Phenyllactic acid (PLA), namely 2-hydroxy-3-phenylpropionic acid, is a new type of biological preservative, which has the function of resisting Gram-positive bacteria, negative bacteria and fungi. In addition, phenyl lactic acid is also an analogue of the traditional Chinese medicine extract "danshensu", which has certain medicinal value. Therefore, the development and utilization of phenyllactic acid has broad application prospects. At present, phenyllactic acid is mainly obtained by chemical synthesis or microbial transformation. The chemical synthesis method has defects such as polluting the environment, complex technology, harsh conditions, and many by-products to varying degrees. Therefore, the researchers turned their resea...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/10C12N9/04
Inventor 朱益波王立梅齐斌蒋卓越季伟
Owner CHANGSHU INSTITUTE OF TECHNOLOGY
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