Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals

Abstract

Bioprocesses conducted under conditions with restricted O2 supply are increasingly exploited for the synthesis of reduced biochemicals using different biocatalysts. The model facultative aerobe Escherichia coli, the microbial cell factory par excellence, has elaborate sensing and signal transduction mechanisms that respond to the availability of electron acceptors and alternative carbon sources in the surrounding environment. In particular, the ArcBA and CreBC two-component signal transduction systems are largely responsible for the metabolic regulation of redox control in response to O2 availability and carbon source utilization, respectively. Significant advances in the understanding of the biochemical, genetic, and physiological duties of these regulatory systems have been achieved in recent years. This situation allowed to rationally-design novel engineering approaches that ensure optimal carbon and energy flows within central metabolism, as well as to manipulate redox homeostasis, in order to optimize the production of industrially-relevant metabolites. In particular, metabolic flux analysis provided new clues to understand the metabolic regulation mediated by the ArcBA and CreBC systems. Genetic manipulation of these regulators proved useful for designing microbial cells factories tailored for the synthesis of reduced biochemicals with added value, such as poly(3-hydroxybutyrate), under conditions with restricted O2 supply. This network-wide strategy is in contrast with traditional metabolic engineering approaches, that entail direct modification of the pathway(s) at stake, and opens new avenues for the targeted modulation of central catabolic pathways at the transcriptional level.

Keywords: ArcBA; CreBC; Escherichia coli; metabolic flux analysis; polyhydroxyalkanoates; redox homeostasis; reduced biochemicals.

Figure 1

Simplified representation of the oxic and anoxic pathways for D-glucose catabolism in E. coli. Oxic pathways are sketched to the right of the outline, and anoxic pathways are represented to the left along with the main fermentation metabolites formed (in red). The initial catabolic steps of D-glucose through the Embden-Meyerhof-Parnas pathway, which are independent of the presence of O₂, are indicated by a dashed vertical arrow. Central metabolites relevant for the production of the reduced biochemicals discussed in the text are shown in boldface. The double arrow representing the conversion of pyruvate into acetyl-CoA illustrate the activity of either the pyruvate dehydrogenase complex (mostly under oxic conditions, right), or pyruvate-formate lyase (mostly under anoxic conditions, left). Secretion of fermentation metabolites and active H⁺ pumping are indicated by red arrows. Slanted arrowheads identify metabolic steps that are subjected to regulation by the ArcBA system (red) and/or the CreBC system (purple). Abbreviations are as follows: NDHI and NDHII, NADH:ubiquinone oxido-reductases I and II, respectively; Q, (ubi)quinone/(ubi)quinol; Cyt d and Cyt o; cytochromes d and o oxidases, respectively; CoA, coenzyme A; TCA cycle, tricarboxylic acid cycle. Nota bene, although the TCA cycle is depicted as an entirely oxic sequence of reactions in this scheme, the reductive branch, active under conditions with restricted O₂ supply, produces succinate as a fermentation metabolite.

Figure 2

Biosynthetic pathways of two model reduced biochemicals, poly(3-hydroxybutyrate) and 1,3-propanediol. Biochemical steps that consume reducing equivalents are highlighted in blue. Abbreviations are as follows: CoA, coenzyme A; PhaA, 3-ketoacyl-CoA thiolase; PhaB, acetoacetyl-CoA reductase; PhaC, poly(3-hydroxyalkanoate) synthase; DhaB, glycerol dehydratase; DhaT, 1,3-propanediol oxidoreductase. In most of the E. coli recombinants described in the text, the phaBAC genes were obtained from Azotobacter sp. strain FA8 [70].