Manipulation of the Anoxic Metabolism in Escherichia coli by ArcB Deletion Variants in the ArcBA Two-Component System

Abstract

Bioprocesses conducted under conditions with restricted O 2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative anaerobe Escherichia coli has elaborate sensing and signal transduction mechanisms for redox control in response to the availability of O 2 and other electron acceptors. The ArcBA two-component system consists of ArcB, a membrane-associated sensor kinase, and ArcA, the cognate response regulator. The tripartite hybrid kinase ArcB possesses a transmembrane, a PAS, a primary transmitter (H1), a receiver (D1), and a phosphotransfer (H2) domain. Metabolic fluxes were compared under anoxic conditions in a wild-type E. coli strain, its ⌬arcB derivative, and two partial arcB deletion mutants in which ArcB lacked either the H1 domain or the PAS-H1-D1 domains. These analyses revealed that elimination of different segments in ArcB determines a distinctive distribution of D-glucose catabolic fluxes, different from that observed in the ⌬arcB background. Metabolite profiles, enzyme activity levels, and gene expression patterns were also investigated in these strains. Relevant alterations were observed at the P-enol-pyruvate/pyruvate and acetyl coenzyme A metabolic nodes, and the formation of reduced fermentation metabolites, such as succinate, D-lactate, and ethanol, was favored in the mutant strains to different extents compared to the wild-type strain. These phenotypic traits were associated with altered levels of the enzymatic activities operating at these nodes, as well as with elevated NADH/NAD ؉ ratios. Thus, targeted modification of global regulators to obtain different metabolic flux distributions under anoxic conditions is emerging as an attractive tool for metabolic engineering purposes.A noxic fermentation of different carbon sources by Escherichia coli is increasingly gaining momentum in biotechnological setups designed to obtain reduced biochemicals. Relevant examples in this sense include (but are certainly not limited to) the production of ethanol (31, 58, 69), succinate (64), D-lactate (45), and polyhydroxyalkanoates (24, 40), often by using redox and/or regulatory E. coli mutants as the biocatalyst. These metabolic engineering approaches underscore the need for a complete understanding of the cell physiology and metabolic network operativity under anoxic growth conditions. In fact, the relative lack of knowledge on the cellular wiring of these regulatory networks under conditions relevant to both laboratory and industrial applications represents a hurdle that has to be overcome for the efficient design of industrial processes. Metabolic fluxes through the central carbon pathways constitute the backbone of cell metabolism and represent the in vivo reaction rates of cognate enzymatic steps (62). The observed fluxome is the phenotypic consequence of both gene transcription and translation, as well as of the enzymatic activity and the regulation exerted at the metabolite level (48). Fluxome analysis is thus a useful approach to...

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