Combinatorial pathway analysis for improved L tyrosine production in Escherichia coli Identification of enzymatic bottlenecks by systematic gene overexpression英文电子书.pdf

ARTICLE IN PRESS Metabolic Engineering 10 (2008) 69–77 /locate/ymben Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: Identification of enzymatic bottlenecks by systematic gene overexpression Tina Lutke-Eversloh, Gregory Stephanopoulos¨ Department of Chemical Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-469, Cambridge MA, USA Received 22 September 2007; received in revised form 29 November 2007; accepted 3 December 2007 Available online 1 February 2008 Abstract Combinatorial overexpression of aromatic amino acid biosynthesis (AAAB) genes in the L-tyrosine producing Escherichia coli strains T1 and T2 was employed to search for AAAB reactions limiting L-tyrosine production. All AAAB genes except aroG and tyrA, which were substituted by their feedback resistant derivatives in the host strains, were cloned and overexpressed. A total of 72 different strains overexpressing various AAAB gene combinations were generated and from those strains with improved phenotype, enzymatic bottlenecks of the AAAB pathway could be inferred. The two major gene overexpression targets for increased L-tyrosine production in E. coli were ydiB and aroK, coding for a shikimate dehydrogenase and a shikimate kinase, respectively, and the combination of ydiB and aroK for overexpression resulted in the best L-tyrosine producing strains in this study, yielding 45% for strain T1 and 26% for strain T2, respectively, higher L-tyrosine titers. Interestingly, overexpression studies with combinations of more t