[NiFe] Hydrogenase Accessory Proteins HypB–HypC Accelerate Proton Conversion to Enhance the Acid Resistance and d-Lactic Acid Production of Escherichia coli

Escherichia coli is a major industrial producer of d-lactic acid due to its well-known advantages, such as short cycle times and low demand. However, acid sensitivity limits production capacity and increases costs. Enhancing the resistance of E. coli to acid stress is essential for improving the cell performance and production value. Here, we propose a feasible strategy to increase the acid tolerance of cells by strengthening intracellular proton conversion. The transcriptome test of the acid-tolerant adaptive evolution strain identified the hydrogenase accessory proteins HypB and HypC as a class of acid-tolerant factors that can assist the hydrogenase in catalyzing the reduction of protons to produce hydrogen. Strengthening the expression of HypB and HypC can increase the cell survival rate by 336.3 times during the lethal stress of d-lactate. In addition, HypB and HypC will assist d-lactate-producing strains to show higher sustainable productivity in an acidic fermentation environment, and d-lactate titer will increase by 113.6%. In order to further improve the expression system of the hydrogenase accessory protein, the introduction of a strong acid stress-driven promoter tdcAp can reduce the demand for neutralizer delivery in the fermentation process by about 26.7% while maintaining the maximum intensity of d-lactic acid production. Therefore, this research developed a method to improve the acid resistance of E. coli cells and reduce the cost of organic acid production by transforming protons.