Method for improving riboflavin production capacity of escherichia coli engineering bacteria by DNA shuffling

Abstract

The invention provides a method for improving riboflavin production capacity of escherichia coli engineering bacteria by using DNA shuffling, and relates to the field of genetic engineering, and the method comprises the following steps: constructing riboflavin-producing escherichia coli engineering bacteria containing 26 genes; carrying out DNA rearrangement and directional screening on the T7RNA polymerase gene expression unit, and extracting to obtain a first batch of positive plasmids; carrying out DNA rearrangement and directional screening on a riboflavin biosynthesis and transport system, and extracting to obtain a second batch of positive plasmids; carrying out DNA rearrangement and directional screening on the Escherichia coli sigma factor gene, and extracting to obtain a third batch of positive plasmids; introducing the third batch of plasmids into different chassis cells to obtain recombinant strains; and selecting the positive strain with the highest riboflavin yield. The toxicity problem of T7RNAP to an escherichia coli host in the construction process of the riboflavin-producing escherichia coli engineering bacteria, the feedback inhibition problem of a product and the moderate and coordinated expression problem of a constructed riboflavin biosynthetic gene are effectively eliminated. The efficiency and the yield of biosynthesis of riboflavin by the escherichia coli engineering strain are further improved.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for improving the riboflavin-producing ability of Escherichia coli engineering bacteria by using DNA shuffling. Background technique [0002] On the basis of the construction of E. coli engineering bacteria that synthesize riboflavin, two problems were encountered: [0003] The first aspect: the toxicity of T7 RNAP to the E. coli host. The T7 promoter is one of the strongest prokaryotic promoters. The highly active T7 RNA polymerase can synthesize mRNA 5 times faster than E. coli RNA polymerase; at the same time, because the T7 promoter cannot be recognized by E. coli RNA polymerase, only It can be specifically recognized and regulated by the T7 RNAP encoded by the phage itself; in addition, the length of the T7 promoter and terminator is very short. In view of the above characteristics and advantages, the T7 expression system has become the first choice for the high...

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Problems solved by technology

However, T7 RNAP has strong toxicity to Escherichia coli, which makes the application of this system face many difficulties

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