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.

The CreC Regulator of Escherichia coli, a New Target for Metabolic Manipulations

“…As NADH is required for its formation, different strategies have been used to increase cofactor availability and to improve succinate production (66). It has been proposed that the manipulation of the CreBC system (as well as ArcAB) could provide a relevant tool for the modulation of central metabolism and reducing power availability aimed at biotechnological purposes (16), such as succinate production. Although the positive effect of the creC deletion on succinate formation may not seem obvious from the point of view of the redox balance, the increase in succinate production in E. coli DC1060 compared to the parental strain was observed under all aeration conditions, and also when plasmids overexpressing decarboxylating enzymes were added to both strains.…”

“…The expression of over 30 operons (more than 70 genes) is under the control of the Fnr regulator (56), and at least 40 operons are regulated by the ArcAB two-component regulatory system (57,58), including 16 genes that encode proteins playing roles in carbon metabolism (16,59). The ArcAB and Fnr global regulation systems are major controlling factors of gene expression, and in most cases, they operate coordinately to fine-tune catabolism in response to oxygen availability (60).…”

“…On the other hand, concern about the costs of energy used for aeration in bioprocesses has renewed attention to the regulation of aerobic and anaerobic bacterial metabolism as a means to achieve the sustainable synthesis of a variety of bioproducts under these conditions (14). Among the different regulatory systems of E. coli, ArcAB has attracted significant attention in the last few years, as manipulations in this sensor/regulator pair offer the possibility of directing carbon flow toward the synthesis of reduced bioproducts under low-aeration conditions (15)(16)(17). In our laboratory, we analyzed the effects of arcA mutants on the central carbon catabolism of E. coli using glucose and glycerol in microaerobiosis and anaerobiosis and observed that mutations in arcA resulted in significant increases in the synthesis of polyhydroxyalkanoates (18)(19)(20)(21) and ethanol (22,23).…”

Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440

“…in industrial settings, hampers the use of P. putida in such endeavours. This state of affairs makes this simple and abundant polyol an attractive C source for microbial-based processes, such as the production of polyhydroxyalkanoates and other bulk chemicals (Murarka et al, 2008;Ashby et al, 2012;Escapa et al, 2012;Gomez et al, 2012;Ruiz et al, 2012). In contrast, the use of glycerol as a C source for P. putida has been somewhat overlooked.…”

Pseudomonas 2.0: genetic upgrading of P. putida KT2440 as an enhanced host for heterologous gene expression

“…The implicit assumption is that DNA acts as a sort of software which, if entered in a reading machine already in place (the host), will result in the expression of the genes at stake at the user's will [2,3]. This somewhat naïve concept has proven, however, very successful, and the number of genes and pathways that have been functionally expressed in archetypal hosts such as Escherichia coli just by knocking-in the DNA sequences of interest is very large [4][5][6]. This view has been exacerbated in the recent times with the inception of Synthetic Biology, which entertains the performance of a biological chassis (i.e., the basic, complete genetic, and biochemical scaffold needed for the gene expression flow [7]) in which different engineered DNA constructs are plugged-in and out for specific purposes.…”