Combinatorial analysis of enzymatic bottlenecks of l-tyrosine pathway by p-coumaric acid production in Saccharomyces cerevisiae
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.
KeywordsARO2 p-Coumaric acid Gene overexpression Saccharomyces cerevisiae TYR1 l-Tyrosine
The work was financially supported by the National Natural Science Foundation of China (31470004), the Sino-Swiss scientific and technological cooperation project supported by the Ministry of Science and Technology of China (2015DFG32140) and the National Basic Research Program of China (2011CBA00802).
Supplementary Table 1—Strains and plasmids used.
Supplementary Table 2—Primers used.
Supplementary Fig. 1—Schematic representation of the engineered strains.
Supplementary Fig. 2—Schematic representation of markless promoter replacement in S. cerevisaie.
Supplementary Fig. 3—The schematic maps of dual-promoter vectors pLC41 and pLC42 construction by marker substitution.
Supplementary Fig. 4—The qPCR analysis of gene expression levels of the engineered strains compared with NK-L70 strain.