Consequences of phosphoenolpyruvate:sugar phosphotranferase system and pyruvate kinase isozymes inactivation in central carbon metabolism flux distribution in Escherichia coli

Eugenio Meza,Judith Becker,Guillermo Gosset,Francisco Bolivar,Christoph Wittmann

doi:10.1186/1475-2859-11-127

Abstract

BackgroundIn Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. In this work we generated W3110 derivative strains that lack the main PEP consumers PEP:sugar phosphotransferase system (PTS-) and pyruvate kinase isozymes PykA and PykF (PTS-pykA- and PTS-pykF-). To characterize the effects of these modifications on cell physiology, carbon flux distribution and aromatics production capacity were determined.ResultsWhen compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose. These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Carbon flux analysis revealed that PTS inactivation causes a redirection of metabolic flux towards biomass formation. A cycle involving PEP carboxylase (Ppc) and PEP carboxykinase (Pck) was detected in all strains. In strains W3110, VH33 (PTS-) and VH35 (PTS-, pykF-), the net flux in this cycle was inversely correlated with the specific rate of glucose consumption and inactivation of Pck in these strains caused a reduction in growth rate. In the PTS- background, inactivation of PykA caused a reduction in Ppc and Pck cycling as well as a reduction in flux to TCA, whereas inactivation of PykF caused an increase in anaplerotic flux from PEP to OAA and an increased flux to TCA. The wild-type and mutant strains were modified to overproduce L-phenylalanine. In resting cells experiments, compared to reference strain, a 10, 4 and 7-fold higher aromatics yields from glucose were observed as consequence of PTS, PTS PykA and PTS PykF inactivation.ConclusionsMetabolic flux analysis performed on strains lacking the main activities generating pyruvate from PEP revealed the high degree of flexibility to perturbations of the central metabolic network in E. coli. The observed responses to reduced glucose uptake and PEP to pyruvate rate of conversion caused by PTS, PykA and PykF inactivation included flux rerouting in several central metabolism nodes towards anabolic biosynthetic reactions, thus compensating for carbon limitation in these mutant strains. The detected cycle involving Ppc and Pck was found to be required for maintaining the specific growth and glucose consumption rates in all studied strains. Strains VH33 (PTS-), VH34 (PTS-pykA-) and VH35 (PTS-pykF-) have useful properties for biotechnological processes, such as increased PEP availability and high biomass yields from glucose, making them useful for the production of aromatic compounds or recombinant proteins.

Highlights

In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes
This bacterium can grow in defined media using glucose as sole carbon source, and under this condition, central metabolism is mainly constituted by the EmbdenMeyerhof-Parnas (EMP) pathway, the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle
When E. coli grows on glucose as carbon source, this sugar is imported by the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) that couples the transfer of the phosphate group from PEP to this sugar, generating as products glucose 6-phosphate (G6P) and pyruvate (PYR) [2,3,4] (Figure 1)

Summary

Introduction

In Escherichia coli phosphoenolpyruvate (PEP) is a key central metabolism intermediate that participates in glucose transport, as precursor in several biosynthetic pathways and it is involved in allosteric regulation of glycolytic enzymes. Results: When compared to reference strain W3110, strain VH33 (PTS-) displayed lower specific rates for growth, glucose consumption and acetate production as well as a higher biomass yield from glucose These phenotypic effects were even more pronounced by the additional inactivation of PykA or PykF. Escherichia coli is a fast growing microorganism that can be modified by a wide variety of molecular tools, enabling the generation of industrial strains for the production of metabolites and recombinant proteins This bacterium can grow in defined media using glucose as sole carbon source, and under this condition, central metabolism is mainly constituted by the EmbdenMeyerhof-Parnas (EMP) pathway, the pentose phosphate pathway (PPP) and the tricarboxylic acid (TCA) cycle. An inhibitory effect of PEP has been described on the EMP pathway enzymes glucokinase (Glk), phosphoglucoisomerase (Pgi), phosphofructokinase (Pfk) and aldolase (FbaA) [9] whereas an activation effect has been described in the enzymes phosphate acetyltransferase (Pta) and acetate kinase (Aks) [10] (Figure 1)

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