To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives. When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l(-1), respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l(-1)) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain. Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.