ptsG gene knocked out recombination bacterial efficiently expressing human-like collagen protein, construction method thereof, and protein expression

Abstract

The invention relates to ptsG gene knocked out recombination bacterial efficiently expressing human-like collagen protein, a construction method thereof, and protein expression. In the prior art, when escherichia coli is adopted to carry out fermentation production of human-like collagen protein, acetic acid accumulation can affect bacterial growth and protein expression, and a collagen protein yield can be reduced with the existing acetic acid byproduct reduction method. According to the present invention, escherichia coli BL21 with a preservation number of CGMCC No.0743 is adopted as starting bacterial, Red homologous recombination is adopted, and apramycin resistance gene is adopted to replace ptsG gene on the escherichia coli genome, and FLP incision enzyme expressed by plasmid pCP20 is adopted to eliminate the apramycin resistance gene to obtain the ptsG gene knocked out escherichia coli engineering bacterial CGMCC No.7331. According to the present invention, a gene engineering tool is adopted to transform an escherichia coli glucose absorption way so as to reduce acetic acid accumulation, improve human-like collagen accumulation, reduce glucose consumption by 9-28%, and improve human-like collagen protein yield by 20-30%.

Description

technical field [0001] The invention relates to a recombinant bacterium, in particular to a ptsG gene knockout recombinant bacterium highly expressing human-like collagen and its construction method and protein expression. Background technique [0002] Collagen has the characteristics of bioabsorption, cell adhesion, promotion of new cell formation, promotion of epithelial cell formation and biocompatibility, which makes it have a very wide range of potential uses, and has always been a hot spot in the field of biomedical materials. The development and production of type water-soluble collagen has always been a research hotspot of scientists in the world. Human-like collagen is obtained by reverse-transcribing a section of mRNA of collagen known in the human body to generate cDNA, then repeating and modifying specific sequences, transforming it into Escherichia coli, and undergoing high-density fermentation, separation, extraction and purification. It solves the problem...

AI Technical Summary

Problems solved by technology

In the batch-fed culture mode, the accumulation of acetic acid is reduced by controlling the rate of nutrient addition and cell growth rate, but this leads to low production of human-like collagen. In addition, complex feeding strategies also require Continuous supervision of the fermentation process, which makes the fermentation process very inconvenient and susceptible to human error; another way to reduce the accumulation of acetic acid is to flow out part of the medium through a separation device, such as a hollow fiber, and add fresh medium to maintain a constant reaction volume

But this approach did not significantly increase the yield of human-like collagen per unit volume

These methods only use engineering means to reduce the accumulation of acetic acid to increase the production of human-like collagen, but the effect is often poor and cannot meet the purpose of high production

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