S.cerevisiae has been extensively engineered as a cell factory platform for flavonoids production.The first step of flavonoids biosynthesis pathway is catalyzed the conversion of tyrosine to p-coumaric acid by tyrosine ammonia lyase,which is also a rate-limiting step for many secondary metabolites in plants.Here we designed a combinatorial metabolic engineering approach to improve p-coumaric acid production via L-tyrosine biosynthesis pathway.First,the ENO2 and the TAL1 genes were over-expressed in the wild stain to enhance the supply of precursor substrates for the further analysis of key genes in aromatic amino acid biosynthesis pathway.Then DAHP synthase and chorismate mutase were eliminated feedback inhibition by site-directed mutagenesis,resulting in more than 4.6-fold in the yield of p-coumaric acid.Second,all the L-tyrosine branch pathway genes were cloned and over-expressed in the precursor and feedback inhibition-resistant over-expressing strain background.The titer of p-coumaric acid was significantly increased by 9.6-fold and 9.4-fold compared to the wild type,when over-expressing ARO2 and TYR1,respectively.Subsequently,we improved the strain by over-expressing multiplecombination of pathway genes increasing the yield of p-coumaric acid by 12.7-fold.Interestingly,over-expression studies revealed that p-coumaric acid production was tightly correlated with cell growth,exhibiting the maximum productivity at the end of the exponential growth phase.It is also noteworthy that the highest strain not only has a lower cell density,but also has a higher by-product acetate formation in batch culture.This study provides a novel insights to identify multiple bottlenecks of p-coumaric production and can be used to engineer subsequent yeast for biosynthesis of flavonoids or valuable L-tyrosine derived secondary metabolites.