Engineering of Escherichia coli central metabolism for aromatic metabolite production with near theoretical yield

Abstract

Escherichia coli and many other microorganisms synthesize aromatic amino acids through the condensation reaction between phosphoenolpyruvate (PEP) and erythrose 4-phosphate to form 3-deoxy-D-arabinoheptulosonate 7-phosphate (DAHP). It has been shown that overexpression of transketolase increases the production of DAHP in an aroB mutant strain (unable to further metabolize DAHP) with elevated DAHP synthase. However, the yield (percent conversion) of DAHP from glucose is still low. Stoichiometric analysis shows that many enzymes compete for intracellular PEP. In particular, the phosphotransferase system, responsible for glucose transport in E. coli, uses PEP as a phosphate donor and converts it to pyruvate, which is less likely to recycle back to PEP. This stoichiometric limitation greatly reduces the yield of aromatic metabolites. To relieve this limitation, we overexpressed PEP synthase in the presence of glucose and showed that it increased the final concentration and the yield of DAHP by almost twofold, to a near theoretical maximum. The PEP synthase effect is not observed without overproduced transketolase, suggesting that erythrose 4-phosphate is the first limiting metabolite. This result demonstrates the utility of pathway analysis and the limitation of central metabolites in the high-level overproduction of desired metabolites.

L‐Tyrosine production by recombinant Escherichia coli: Fermentation optimization and recovery

“…Their calculated molar maximum yields from glucose were 0.529 mol L-phe/mol glucose, 0.548-mol L-tyr/mol glucose, and 0.414 mol trp/mol glucose. Patnaik and Liao (1994) reported that the yield of glucose to DAHP is fairly low since many enzymes compete for PEP in the cell. One of the sinks on PEP is conversion to pyruvate (PYR).…”

“…Historically, metabolic engineering approaches geared towards increasing carbon flux to the aromatic amino acid pathway concentrated on overexpressing mutant aroG, F, H enzymes that are feedback resistant (fbr) to end products accompanied by overexpression of rate controlling pathway enzymes and deletion of by-product generating pathways. A second generation of quantitative metabolic engineering approaches resulted in balancing fluxes in a manner that enhanced the availability of intracellular precursors required for biosynthesis of aromatic amino acids (Ikeda, 2006;Patnaik and Liao, 1994;Takors et al, 2007). Applications of these approaches of metabolic engineering have resulted in E. coli and Corynebacterium glutamicum that produce L-phe and L-trp in titers as high as 50 and 60 g/L respectively (Ikeda, 2006;Ikeda and Katsumata, 1992;Ikeda et al, 1994;Konstantinov et al, 1991;Konstantinov and Yoshida, 1992).…”

Stimulation of glucose catabolism through the pentose pathway by the absence of the two pyruvate kinase isoenzymes inEscherichia coli

“…It is evident that if we are interested in optimizing carbon flow for the production of certain metabolites with maximal yields, this basic scheme needs to be modified. Accordingly, for the production of several aromatic compounds (Frost and Draths, 1995), the modification of the phosphoenolpyruvate node (PEP) has been the focus of several groups (Gubler et al, 1994;Miller et al, 1987;Mori and Shiio, 1987;Patnaik and Liao, 1994;Sano and Ito, 1987). Flux distribution at the PEP node is tightly regulated by allosteric regulation of the enzymes involved in this node.…”

Altered Glucose Transport and Shikimate Pathway Product Yields in E.coli

“…The Bailey study (47) also did not amplify expression of either transketolase or transaldolase. Increased availability of phosphoenolpyruvate in E. coli is not reflected in increased shikimate pathway product yields until the availability of D-erythrose 4-phosphate is increased with amplified expression of transketolase (50).…”