Combinatorial Optimization Of CO_-2 Transportation And Fixation In Escherichia Coli By Promoter Engineering

Escherichia coli can fix CO₂ with native PPC and PCK to generate OAA from PEP, and it is the limiting step in the process of succinic acid biosynthesis. In previous work, CO₂ transporter gene and CO₂ fixation gene were overexpressed for collaborative metabolic regulation of transportation and fixation. Precise control of foreign gene expression might be a good strategy to further enhance succinate production. In the study, a synthetic promoter library was rapidly built using energy matrix. Then, two reporter genes were applied to library characterization. At last, combinatorial optimization of CO₂ transportation and fixation by promoter library was implemented, which made the optimization of collaborative expression system come true.Firstly, a synthetic promoter library was generated. Based on the consensus-35 and-10 sequences of trc promoter, a library containing 20 promoters with different energy was created by energy matrix.Secondly, characterization of promoter library was completed. RFU detection showed that using energy matrix, a synthetic promoter library containing 20 promoters with strength ranging from 0.8% to 100% compared with trc promoter was built. High correlation between the readings of rfp transcripts and the red fluorescence assays indicated that expression was mainly transcriptionally controlled. And the fact that the activities of designed promoters were highly similar using rfp and cat as reporter genes provided evidence that the synthetic promoters were stably and had certain applicability. So the promoter library could be applied to the synthesis of succinic acid.At last, optimization of collaborative expression system was implemented. In order to achieve different levels of CO₂ transport gene and CO₂ fixed gene expression, four designed promoters with different strengths were used for the construction of pT-P_x-bicA, pT-P_x-sbtA, pACYC-P_x-pck, pACYC-P_x-ppc.With dual-plasmid system, 64 combinations of single transport gene and single fixed gene were obtained. Among them, 3 types were obtained by preliminary screening in shake flasks and secondary screening in bioreactor, with a more than 10 % increase in succinate production. Next, combinations of double transport genes and double fixed genes based on the screened 3 types were investigated. The best combination was Tang1527, and the succinate production was 37.5% higher than that of control. So optimization of transporter gene and fixed gene expression benefits the succinic acid production.In this study, combinatorial optimization of CO₂ transportation and fixation was investigated and implemented, and ultimately the production of succinic acid was improved. The results provide a new idea on promoter library construction and engineered bacteria of succinic acid, and it also has a certain significance on combinatorial optimization of metabolic pathways.