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

Abstract

Different glucose transport systems are examined for their impact on phosphoenolpyruvate availability as reflected by the yields of 3-dehydroshikimic acid and byproducts 3-deoxy-d-arabino-heptulosonic acid, 3-dehydroquinic acid, and gallic acid synthesized by Escherichia coli from glucose. 3-Dehydroshikimic acid is an advanced shikimate pathway intermediate in the syntheses of a spectrum of commodity, pseudocommodity, and fine chemicals. All constructs carried plasmid aroF(FBR) and tktA inserts encoding, respectively, a feedback-insensitive isozyme of 3-deoxy-d-arabino-heptulosonic acid 7-phosphate synthase and transketolase. Reliance on the native E. coli phosphoenolpyruvate:carbohydrate phosphotransferase system for glucose transport led in 48 h to the synthesis of 3-dehydroshikimic acid (49 g/L) and shikimate pathway byproducts in a total yield of 33% (mol/mol). Use of heterologously expressed Zymomonas mobilis glf-encoded glucose facilitator and glk-encoded glucokinase resulted in the synthesis in 48 h of 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 41% (mol/mol). Recruitment of native E. coli galP-encoded galactose permease for glucose transport required 60 h to synthesize 3-dehydroshikimic acid (60 g/L) and shikimate pathway byproducts in a total yield of 43% (mol/mol). Direct comparison of the impact of altered glucose transport on the yields of shikimate pathway products synthesized by E. coli has been previously hampered by different experimental designs and culturing conditions. In this study, the same product and byproduct mixture synthesized by E. coli constructs derived from the same progenitor strain is used to compare strategies for increasing phosphoenolpyruvate availability. Constructs are cultured under the same set of fermentor-controlled conditions.

Phosphotransferase System-Independent Glucose Utilization in Corynebacterium glutamicum by Inositol Permeases and Glucokinases

“…No hints for enhanced carbon flux via PEP carboxylase to oxaloacetate have been shown during PTS-independent growth and L-lysine production, since PEP carboxylase activity was comparable to that of the parental strain. In E. coli, strains engineered for PTSindependent glucose uptake showed the improved production of aromatic compounds such as phenylalanine, shikimate, and anthranilate (3,7,15,17,18,26,35,70) as a consequence of reduced pyruvate and increased PEP levels in the cells. For example, the expression of the genes for galactose permease and glucokinase in PTS-negative E. coli strains bypassed PEP usage for glucose phosphorylation and resulted in a higher yield of 3-deoxy-D-arabinoheptulosonate-7-phosphate, which was assumed to be a consequence of an increased level of PEP, the immediate precursor of 3-deoxy-D-arabinoheptulosonate-7-phosphate (18,21).…”

Experimental design-aided systematic pathway optimization of glucose uptake and deoxyxylulose phosphate pathway for improved amorphadiene production

“…Transcriptome analysis in these fast-growing strains indicated that the expressions of gene galP (coding for galactose permease), glk (coding for glucose kinase), and pgi (coding for phosphoglucose isomerase) were increased as compared to the wild-type strain, suggesting that these genes were important in enhancing glucose uptake Flores et al 2002). Hence, it was reported that the overexpression of galP and glk enhanced the growth rate of microbes in glucose medium Lu et al 2012) and improved the yields of valuable products such as aromatics (Yi et al 2003), recombinant protein (De Anda et al 2006), and ethanol (Hernandez-Montalvo et al 2003). Combinatorial modulation of galP and glk gene expressions has been used to improve cell growth rate (Lu et al 2012), but it is unclear if such an approach will increase the production of isoprenoids.…”

Modular Engineering of l -Tyrosine Production in Escherichia coli

“…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).…”