Enhancement of heterologous protein production in Corynebacterium glutamicum via atmospheric and room temperature plasma mutagenesis and high-throughput screening

• Development of protocols for ARTP mutagenesis and high-throughput screening strategies. • Three strains of Corynebacterium glutamicum with high yield of heterologous protein were obtained. • Compared with the original strain, the yield of MA6 was increased by nearly 91 % to approximately 862 mg/L. • Genome analysis was performed to locate the target gene that caused the increase in protein production. Atmospheric and room temperature plasma (ARTP) is a new and efficient mutation breeding technique. In this study, we discuss a strategy combining ARTP mutagenesis and high-throughput screening to engineer Corynebacterium glutamicum towards high yield production of heterologous proteins. First, three target strains, MC2, MA8, and MA6, were screened from the mutant library with enhanced green fluorescent protein (EGFP) as the reporter protein, and their growth stability and the influence of heterologous protein production were verified. Second, genes encoding three high-value medicinal proteins (glycoprotein D, gD; endoxylanase, XynA; and variable domain of heavy chain of heavy-chain antibody, VHH) were expressed in the mutagenized strain, which confirmed its applicability for an increased biosynthesis of other heterologous proteins. During the large-scale fermentation of C. glutamicum for VHH production, the fermentation characteristics of the best mutant MA6 were verified. Compared to the original strain, the yield of VHH obtained with strain MA6 was increased by nearly 91 % to approximately 862 mg/L. Finally, through systematic genome analysis mutations in five genes were obtained. These genes code for putative proteases or are potentially related to the bacterial restriction repair systems. These findings will help to obtain optimized chassis cells and provide a direction for in-depth research on genetic targets that can increase protein production.