Table2_Minimizing acetate formation from overflow metabolism in Escherichia coli: comparison of genetic engineering strategies to improve robustness toward sugar gradients in large-scale fermentation processes.docx

Introduction:Escherichia coli, a well characterized workhorse in biotechnology, has been used to produce many recombinant proteins and metabolites, but have a major drawback in its tendency to revert to overflow metabolism. This phenomenon occurs when excess sugar triggers the production of mainly acetate under aerobic conditions, a detrimental by-product that reduces carbon efficiency, increases cell maintenance, and ultimately inhibits growth. Although this can be prevented by controlled feeding of the sugar carbon source to limit its availability, gradients in commercial-scale bioreactors can still induce it in otherwise carbon-limited cells. While the underlying mechanisms have been extensively studied, these have mostly used non-limited cultures. In contrast, industrial production typically employs carbon-limited processes, which results in a substantially different cell physiology. Objective: The objective of this study was to evaluate and compare the efficiency of different metabolic engineering strategies with the aim to reduce overflow metabolism and increase the robustness of an industrial 2’-O-fucosyllactose producing strain under industrially relevant conditions. Methods: Three distinct metabolic engineering strategies were compared: i) alterations to pathways leading to and from acetate, ii) increased flux towards the tricarboxylic acid (TCA) cycle, and iii) reduced glucose uptake rate. The engineered strains were evaluated for growth, acetate formation, and product yield under non-limiting batch conditions, carbon limited fed-batch conditions, and after a glucose pulse in fed-batch mode. Results and Discussion: The findings demonstrated that blockage of the major acetate production pathways by deletion of the pta and poxB genes or increased carbon flux into the TCA cycle by overexpression of the gltA and deletion of the iclR genes, were efficient ways to reduce acetate accumulation. Surprisingly, a reduced glucose uptake rate did not reduce acetate formation despite it having previously been shown ...