The physiological effects and metabolic alterations caused by the expression of Rhizobium etli pyruvate carboxylase in Escherichia coli

Abstract

Oxaloacetate (OAA) plays an important role in the tricarboxylic acid cycle and for the biosynthesis of a variety of cellular compounds. Some microorganisms, such as Rhizobium etli and Corynebacterium glutamicum, are able to synthesize OAA during growth on glucose via either of the enzymes pyruvate carboxylase (PYC) or phosphoenolpyruvate carboxylase (PPC). Other microorganisms, including Escherichia coli, synthesize OAA during growth on glucose only via PPC because they lack PYC. In this study we have examined the effect that the R. etli PYC has on the physiology of E. coli. The expressed R. etli PYC was biotinylated by the native biotin holoenzyme synthase of E. coli and displayed kinetic properties similar to those reported for alpha4 PYC enzymes from other sources. R. etli PYC was able to restore the growth of an E. coli ppc null mutant in minimal glucose medium, and PYC expression caused increased carbon flow towards OAA in wild-type E. coli cells without affecting the glucose uptake rate or the growth rate. During aerobic glucose metabolism, expression of PYC resulted in a 56% increase in biomass yield and a 43% decrease in acetate yield. During anaerobic glucose metabolism, expression of PYC caused a 2.7-fold increase in succinate concentration, making it the major product by mass. The increase in succinate came mainly at the expense of lactate formation. However, in a mutant lacking lactate dehydrogenase activity, expression of PYC resulted in only a 1.7-fold increase in succinate concentration. The decreased enhancement of succinate formation in the /dh mutant was hypothesized to be due to accumulation of pyruvate and NADH, metabolites that affect the interconversion of the active and inactive form of the enzyme pyruvate formate-lyase.

Minimizing acetate formation in E. coli fermentations

“…Lowering the pyruvate pool has been many times described in the literature as a way to reduce acetate production [47,48].…”

Ethanol synthesis from glycerol by Escherichia coli redox mutants expressing adhE from Leuconostoc mesenteroides

“…Fermentative lactate dehydrogenase (LdhA) competes for glycolytic intermediates and reducing power at the pyruvate branch point (Bunch et al 1997;Gokarn et al 2001). We decided to delete ldhA rather than pta-ackA (which encode the above-mentioned Pta-AckA pathway) or pfl (encoding pyruvate-formate lyase) as it was suggested that the latter genes are necessary for efficient anoxic glycerol dissimilation by E. coli (Dharmadi et al 2006;Gonzalez et al 2008).…”

Process Design for the Biocatalysis of Value-Added Chemicals from Carbon Dioxide

“…While the plasmids that harbor these gene cassettes are quite stable and produce high levels of pyruvate carboxylase enzyme that are equal to or higher than the pTrc99A-pyc reference plasmid (Gokarn et al, 2001), when the cassettes are introduced into the chromosome, pyruvate carboxylase enzyme levels begin to diminish and disappear after several generations of growth. Interestingly, the drug resistance is still present and the pyc region is intact and can be amplified by the polymerase chain reaction.…”

“…The pyruvate carboxylase protein from Rhizobium etli was initially cloned into the pTrc99A vector to enable the expression of this anaplerotic enzyme by our laboratory (Gokarn et al, 2001). The pTrc99A-pyc construct has been utilized in a variety of metabolic engineering approaches which rely on pyruvate carboxylase, an enzyme that does not naturally occur in wildtype Escherichia coli (March et al, 2002;Vemuri et al, 2002;Vemuri et al, 2005).…”

“…Strain ALS874 was AFP111 transformed with the pTrc99A-pyc plasmid as previously described (Gokarn et al, 2001). Strain ALS1000 was ALS942 transformed with pTrc99A-pyc.…”