Study on Global Transcription Machine Engineering Breeding of Escherichia coli Producing Eketodoxin

Abstract

Ectoin, whose chemical name is tetrahydromethylpyrimidine carboxylic acid, is a specific amino acid derivative, derived from the metabolic process of some halophilic bacteria, and ectoin has been widely used in many fields such as enzyme preparations, cosmetics, health care products and medical treatment because of its diverse physiological activities. In view of the fact that the accumulation of heterologous natural products in synthetic biology technology is often related to complex problems such as the adjustment of metabolic networks, the construction of heterologous pathways, and the matching of genes and hosts, it was decided to adopt global transcription machine engineering (gTME) technology, which is different from classical mutagenesis and pathway engineering. We successfully cloned the endogenous global transcription factor rpoD by PCR, and constructed a mutant expression library based on pET28a using error-prone PCR. We used fluorescence-based biosensors as a preliminary high-throughput screening system, and further combined with 96 deep-well microculture technology for quantitative screening, and the yield of ectoin was used as the re-screening criterion. After this series of studies, we have successfully increased the yield of ectoin in E. coli engineered bacteria using gTME technology. The successful implementation of this project will not only deepen our understanding of the biosynthetic mechanism of ectoin, but also lay a solid foundation for its future industrial application.