The Genetic Modification Of Escherichia Coli For CIS, CIS-Muconic Acid Production

cis,cis-Muconic acid (ccMA) is a dicarboxylic acid with two conjugated double bonds that can be potentially used as a raw material for new functional resins, pharmaceuticals and agrichemicals. At present, this chemical is mainly used as the precursor of adipic acid to manufacture nylon 6,6. With the development of worldwide adipic acid industry, the biosynthetic production of ccMA is becoming a research hotspot. In this study the heterogeneous pathway in Escherichia coli AB2834 was genetically modified to improve the production of ccMA with the combination of metabolic engineering, protein engineering and synthetic biology.It is very important to express and modulate the genes of synthetic pathway in metabolic engineering and synthetic biology study, while promoter is a useful element to control the expression of gene and regulate the synthetic pathway. So, a new 88bp synthetic promoter, which contains degenerate bases in some motifs, was designed to construct promoter library.35 promoters with varying strengths were screened out after double-round screenings via the expression of red fluorescent protein under the control of the synthetic promoter. The result of sequence analysis showed that the promoters with varying strengths are base preference. Then, five characterized promoters with varying strengths were chosen to optimize the expression of the key gene cat A, the highest was 1.29 g/L, nearly 21-fold higher than wild strain. Subsequently, three promoters with varying strengths(1:2:3) were chosen to combinatorialoptimize the expression of the three heterogeneous genes, and the highest was 2.11 g/L, nearly 2.5-fold higher than the lowest one’s.The rational design of key enzyme and synthetic scaffold was also applied to regulate the heterogeneous pathway to increase ccMA production. The key enzyme catechol 1, 2-dioxygenase (CatA) was rationally designed to improve its special activity, and the special activity of mutant CatA and ccMA production were increased 64.45% and 50% respectively, and the highest titer of ccMA was 1.84 g/L. On the other hand, the synthetic scaffolds were introduced into the heterogeneous pathway to relieve flux imbalance in cell, decrease the concentration of intermediates, increase ccMA production. Fermentation performance proved that synthetic scaffold effectively decreased the concentration of catechol and increased the accumulation of ccMA, and the highest titer was 1.82 g/L, about 3-fold higher than wild strain.