Improving succinate production by engineering oxygen-dependent dynamic pathway regulation in Escherichia coli

Abstract

Succinate is an important building block for chemical synthesis. However, during the fermentation process, excessive osmotic stress and byproduct accumulation substantively impair the performance of the microbial cell factory. To this end, two strategies were proposed. First, an osmo-tolerant mutant, Escherichia coli FMME-N-2, was screened by combined mutagenesis (ARTP and ⁶⁰Co-γ irradiation) to produce 51.8 g L⁻¹ succinate with a productivity of 0.81 g L⁻¹ h⁻¹. Second, an oxygen-dependent bifunctional switch (OBS) was developed with promoter P_fnrF8-based activation and tobacco etch virus protease-based inhibition functions. With ribosomal binding site (RBS) and degron optimization of OBS, the optimal strain E. coli FMME-N-30 achieved a succinate titer and productivity of 119 g L⁻¹ and 1.65 g L⁻¹ h⁻¹, respectively, in a 30-L fermentor, while only 7.1 g L⁻¹ acetate and no formate or lactate were detected. Compared to the wild-type strain E. coli FMME-N, the succinate titer was increased by 3.3-fold. These results highlight the applicability of OBS for the large-scale production of value-added chemicals.

Funding

Innovative Research Group Project of the National Natural Science Foundation of China,(32021005)

Key Programme,(22038005)