Production Of Shikimic Acid And Resveratrol In Escherichia Coli Based On Synthetic Biology

Shikimic acid(SA) has aroused widespread concern as the key chiral compound for the synthesis of Oseltamivir, which is the front-line treatment for serious cases of influenza. SA and its derivatives have the activities of antitumor, antithrombotic and other pharmacological effects. Resveratrol(Res) and its derivatives have several beneficial effects on human health, such as protective effects against carcinogenesis, aging and cerebrovascular diseases, so they are widely used in food and nutrition supplement. At present, majority of commercial synthesis of SA and Res are extracted from plants, which is dependence on plant resources seriously. With the advantages like short cultivate time and easy genetic operation, microorganisms are often preferred in numbers of studies using synthetic biology for the production of many botanical metabolites. However, the overexpression of genes relies on engineered plasmids mostly, which has its own defects. In recent years, the technology of site-specific integration of key genes into chromosome has raised considerable concern, for which the genes were stably maintained and excellent results were obtained. This study was based on the principle of synthetic biology, and the strain E. coli BW25113 was used for the production of high-yield SA and Res by a de novo biosynthesis pathway through the technology of site-specific genome integration.As the recombinant strains for the production of SA,(1)The downstream shikimate kinase isoenzyme genes aro K and aro L were deleted, the key enzyme genes aro G(encoding 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase), aro B(encoding 3-dehydroquinic acid synthase), tkt A(encoding transketolase I) and aro E(encoding shikimate dehydrogenase) of SA pathway were studied by constructing a quadruple-gene co-expression plasmid p ETDuet-GBAE, which could significantly improve SA production for the effect of synergy, SA production reached to 1.08 g/L.(2)The technology of site-specific genome integration was used first to improve the SA production further. The genes pts HIcrr of phosphoenolpyruvate: carbohydrate phosphotransferase system were deleted to reduce the consumption of phosphoenol pyruvate(PEP), then the genes from plasmid p ETDuet-GBAE were integrated into the locus of pts HIcrr, and the SA production was improved to 2.08 g/L.(3)The genes glk(encoding glucokinase) and gal P(encoding galactose permease) were integrated to increase the rate of glucose transportation, and the fermentation period was shortened.(4)Another key gene pps A(encoding PEP synthase) was integrated into tyr R(encoding aromatic amino acid biosynthesis and transport regulon transcriptional regulator), the supply of PEP was strengthened and SA production reached to 2.83 g/L, which was 94 times than the starting strain BW25113(?aro L/aro K, DE3).(5)Plasmid p ETDuet-GBAE was added for the genes over-expression further, and the SA production got 4.14 g/L, the yield from glucose was 23.56%.(6)For the amplification cultivation in a 5-L bioreactor, glucose-glycerol mixture was used first as carbon source. The glucoseglycerol mixture could circumvent the drawbacks of sole carbon source, a win-win situation of the production rate and continuous production was achieved. As a result, SA production reached to 27.41 g/L, with a productivity of 0.57 g/L·h, which was 2-fold than the glucose as sole carbon source.As the recombinant strains for the production of Res,(1) The gene sts(encoding stilbene synthase) from Vitis vinifera was integrated into tyr R, and the genes tal(encoding tyrosine ammonia-lyase) from Rhodotorula glutinis, 4cl(encoding 4-coumarate coenzyme A ligase) from Petroselinum crispum were integrated into the locus of trp ED(synthetic genes for L-tryptophan). The recombinant strain Res1 was capable of producing 4.61 mg/L of Res from glucose by a genuine de novo biosynthesis pathway.(2)The genes mat C(encoding malonate carrier protein) and mat B(encoding malonyl-Co A synthetase) from Rhizobium trifolii for the synthesis of malonyl-Co A were integrated into the locus of phe LA(synthetic genes for L-phenylalanine), then the Res production was increased to 9.35 mg/L.(3)The genes aro G, aro B, tkt A, aro E, glk, and gal P were integrated into pts HIcrr, Res production was improved to 11.41 mg/L.(4)When the exogenous genes for the production of Res were over-expressed further by the plasmids, the production of Res got 13.70 mg/L, which was nearly 3-fold than the strain Res1.(5)The substrate tyrosine and p-coumaric acid(p-CA) were added in the follow-up study, two by-products Res-der1 and Res-der2 were detected in the fermentation broth, they were derivatives of p-CA. Res-der2 was proved to be 4-vinylphenol, which is decarboxylated from p-CA. In addition, the contents of Res-der2 were increased with the increase of the amount of p-CA. Thus, it can be inferred that the intermediate p-CA was decarboxylated or catalyzed to generate other by-products, the production of Res was affected for by the synthesis of 4-coumaroyl-Co A was blocked.In conclusion, a SA-producing strain with highly yield and a Res-producing strain by a genuine de novo biosynthesis were constructed successfully through the technology of site-specific genome integration based on synthetic biology, which laid the foundation for later study. The genes in the final recombinant could express constitutively, isopropyl β-D-thiogalactoside(IPTG) and antibiotics were avoided in the process of fermentation, which are environmentally friendly, simple to handle and suitable for industrial production.