Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: Identification of enzymatic bottlenecks by systematic gene overexpression

Modular Engineering of l -Tyrosine Production in Escherichia coli

“…ditions, strain A, harboring pS0 and pY0, yielded 746 Ϯ 18 mg/ liter L-tyrosine (Table 3), consistent with the previous studies (25,26). When we reversed the orientation of the genes in the module S0 or Y0, the production dropped significantly to ϳ180 mg/liter in either case (data not shown).…”

“…Previous studies had made advances in engineering the L-tyrosine pathway (24)(25)(26), providing us with useful information from which to build the modular system described here. In their initial constructs, Lütke-Eversloh and Stephanopoulos achieved L-tyrosine production of 621 Ϯ 26 mg/liter (26) by overexpressing aroG* (D146N), tyrA* (M53I; A354V), ppsA, and tktA on a pSC101 plasmid under the control of a constitutive promoter (P LtetO-1 with no tetR) in the tyrR knockout strain; following combinatorial analysis, production was increased by ϳ26% when the isozymes YdiB and AroK were additionally coexpressed on a pBR322 plasmid (25).…”

“…In their initial constructs, Lütke-Eversloh and Stephanopoulos achieved L-tyrosine production of 621 Ϯ 26 mg/liter (26) by overexpressing aroG* (D146N), tyrA* (M53I; A354V), ppsA, and tktA on a pSC101 plasmid under the control of a constitutive promoter (P LtetO-1 with no tetR) in the tyrR knockout strain; following combinatorial analysis, production was increased by ϳ26% when the isozymes YdiB and AroK were additionally coexpressed on a pBR322 plasmid (25). Utilizing this work as a starting point, we selected pZS21 (27), consisting of the pSC101 origin of replication and P LtetO-1 , to express the genes of the first shikimate module (S0), creating plasmid pS0, and pRBS01 (http://registry.jbei.org), a medium-copy-number plasmid with a p15A origin of replication and the IPTG-inducible P lac-UV5 , to express the genes of the first tyrosine module (Y0), creating plasmid pY0.…”

“…We first increased the expression of AroK, since its protein level was relatively low compared to the other four enzymes in the operon (Fig. 1C) and because it catalyzes the conversion of shikimate to S3P, which is known to be a rate-limiting step (25). However, repeated attempts to insert a second promoter 5= of the gene or to modify the RBS of aroK failed consistently, which might have been be due to plasmid instability, as reported previously for an aroK-containing plasmid (37).…”

“…Previous L-tyrosine engineering work has most often focused on the transcriptional deregulation of the tyrR and/or trpR regulons, followed by removing the feedback inhibition on two key enzymes, 3-deoxy-D-arabino-heptulosonate (DAHP) synthase (AroG), which catalyzes the first step committed to the shikimate pathway, and the dual-function chorismate mutase/prephenate dehydrogenase (TyrA), which catalyzes the first two steps in L-tyrosine biosynthesis from chorismate (26,33). Coexpression of the rate-limiting enzymes, shikimate kinase (AroK or AroL) and quinate (QUIN)/shikimate dehydrogenase (YdiB), and deletion of the L-phenylalanine branch of the aromatic amino acid biosynthetic pathway have been shown to increase L-tyrosine production (12,25,33). Furthermore, overexpression of phosphoenolpyruvate synthase (PpsA) and transke-tolase A (TktA), altering glucose transport and the use of other carbon sources, such as xylose and arabinose, have also been shown to increase the pools of precursors to the shikimate pathway (1,9,22,26,34,47,48).…”