In this study, we applied a series of genetic modifications to wild-type S. cerevisiae strain BY4741 to address the bottlenecks in the l-tyrosine pathway. A tyrosine ammonia-lyase (TAL) gene from Rhodobacter capsulatus, which can catalyze conversion of l-tyrosine into p-coumaric acid, was overexpressed to facilitate the analysis of l-tyrosine and test the strain's capability to synthesize heterologous derivatives. First, we enhanced the supply of precursors by overexpressing transaldolase gene TAL1, enolase II gene ENO2, and pentafunctional enzyme gene ARO1 resulting in a 1.55-fold increase in p-coumaric acid production. Second, feedback inhibition of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase and chorismate mutase was relieved by overexpressing the mutated feedback-resistant ARO4 K229L and ARO7 G141S , and a 3.61-fold improvement of p-coumaric acid production was obtained. Finally, formation of byproducts was decreased by deleting pyruvate decarboxylase gene PDC5 and phenylpyruvate decarboxylase gene ARO10, and p-coumaric acid production was increased 2.52-fold. The best producer-when TAL1, ENO2, ARO1, ARO4 K229L , ARO7 G141S , and TAL were overexpressed, and PDC5 and ARO10 were deleted-increased p-coumaric acid production by 14.08-fold (from 1.4 to 19.71 mg L-1). Our study provided a valuable insight into the optimization of l-tyrosine metabolic pathway.
Keywords: Metabolic engineering; Saccharomyces cerevisiae; l-Tyrosine; p-Coumaric acid.
© King Abdulaziz City for Science and Technology 2020.