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.

常压室温等离子体（ARTP）是一种新型高效的突变育种技术。在这项研究中，我们讨论了一种结合 ARTP 诱变和高通量筛选来改造谷氨酸棒状杆菌的策略实现异源蛋白质的高产生产。首先，从突变体库中筛选出以增强型绿色荧光蛋白（EGFP）为报告蛋白的三个目标菌株，MC2、MA8和MA6，并验证了它们的生长稳定性和异源蛋白产生的影响。其次，在诱变菌株中表达了编码三种高价值药用蛋白质（糖蛋白 D、gD；木聚糖内切酶、XynA 和重链抗体重链可变域 VHH）的基因，这证实了其适用于增加其他异源蛋白质。谷氨酸棒杆菌大规模发酵过程中对于 VHH 生产，验证了最佳突变体 MA6 的发酵特性。与原始菌株相比，用菌株 MA6 获得的 VHH 产量增加了近 91%，达到约 862 mg/L。最后，通过系统的基因组分析，获得了五个基因的突变。这些基因编码假定的蛋白酶或可能与细菌限制修复系统有关。这些发现将有助于获得优化的底盘细胞，并为深入研究可以增加蛋白质产量的遗传靶点提供方向。