Recombinant plasmid, recombinant engineering bacterium as well as construction method and application thereof in improving yield of D-psicose

Abstract

The invention discloses a recombinant plasmid, a recombinant engineering bacterium as well as a construction method and application thereof in improving the yield of D-psicose, and belongs to the technical field of enzyme directed evolution. The invention designs a directed evolution recombinant plasmid, and the directed evolution recombinant plasmid can be regarded as a gene circuit, namely, a gene circuit with DTE enzyme efficiency. At the same time, the invention also discloses the recombinant engineering bacterium constructed based on the recombinant plasmid, specifically E. coli DH5alphapSB1C3-psiR-pPsi-EGFP-Cp-KanR-DTE. The gene circuit of the present invention uses a repressor protein PsiR and a corresponding promoter pPsi to realize a biosensor of D-psicose; and the bacterium can sense the production of D-psicose in the body to express different levels of antibiotics resistance genes, so that the efficiency of DTE enzyme is transformed into survival advantages of bacterium.

Description

technical field [0001] The invention belongs to the technical field of enzyme directed evolution, and relates to a recombinant plasmid, a recombinant engineered bacterium, a construction method thereof and an application for increasing the output of D-psicose. Background technique [0002] D-Psicose is a rare monosaccharide naturally occurring in small amounts and is the C-3 epimer of fructose. As a rare sugar, it has a good taste and a non-bitter aftertaste. It is a new type of sweetener. At a 10% dilution, D-psicose is 70% of the sweetness of sucrose but only 0.3% of its calories. It is an ideal substitute for sucrose. D-psicose can inhibit intestinal α-glucosidase and liver lipase, and can inhibit the accumulation of fat. D-psicose has the function of suppressing blood sugar. Dietary supplementation with allulose alleviates postprandial blood glucose rise and enhances insulin sensitivity. Animal experiments have also reported that D-psicose has a protective effect on...

AI Technical Summary

Problems solved by technology

If the production efficiency of a certain enzyme is directed evolution by the traditional method, it is often necessary to use quantitative methods such as HPLC to detect the activity of the enzyme in each mutant strain, which is time-consuming and labor-intensive and the screening throughput is very low

If the phenotype to be evolved can be characterized by fluorescent proteins, mutants can traditionally be screened in 96-well plates, thereby increasing the screening throughput to 10 2 order of magnitude, but still far from meeting the needs of library screening

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