The protease present in a host may reduce the yield and biological activity of heterologous proteins. In this study, we used protease overexpression and deletion strategies to examine the effect of the Clp protease system in Corynebacterium glutamicum on the recombinant protein and to produce a highly efficient heterologous protein expression host. In this study, we identified seven genes in the Clp protease family in Corynebacterium glutamicum ATCC 13032 through bioinformatics analysis, and studied their effects on the enhanced green fluorescent protein (EGFP) reporter protein. The fluorescence intensity of the knockout strain was significantly higher, and the effect of the clpS deletion strain was the most obvious. To verify the universal effect of the lack of clpS, the excellent industrial strain C. glutamicum 1.15647 was transformed to form recombinant 15647-ΔclpS. Based on the results, 15647-ΔclpS had a more significant effect on improving protein expression. Furthermore, recombinant human teriparatide (rhPTH) and variable domain of heavy chain of heavy-chain antibody (VHH) were selected to verify the universal applicability of the knockout strain for expressing heterologous proteins. Accordingly, we found that protease deficiency could increase the production of heterologous proteins. Finally, through a large-scale fermentation, the 15647-ΔclpS strain was used to produce VHH. Its yield was approximately 530 mg/L, which was 65% higher than that of WT-15647. In this study, a host that could effectively increase heterologous protein expression was successfully obtained.

宿主中存在的蛋白酶可能会降低异源蛋白质的产量和生物活性。在这项研究中，我们使用蛋白酶过表达和缺失策略来检查谷氨酸棒杆菌中 Clp 蛋白酶系统对重组蛋白的影响，并产生高效的异源蛋白表达宿主。在这项研究中，我们通过生物信息学分析鉴定了谷氨酸棒杆菌 ATCC 13032中 Clp 蛋白酶家族中的 7 个基因，并研究了它们对增强型绿色荧光蛋白 (EGFP) 报告蛋白的影响。敲除菌株的荧光强度显着升高，而clpS的影响缺失菌株最为明显。为了验证缺乏clpS的普遍效果，优良的工业菌株谷氨酸棒杆菌1.15647被转化形成重组15647-ΔclpS。根据结果​​，15647-ΔclpS对改善蛋白质表达的作用更为显着。此外，选择重组人特立帕肽（rhPTH）和重链抗体重链可变域（VHH），验证基因敲除株在表达异源蛋白方面的普遍适用性。因此，我们发现蛋白酶缺乏会增加异源蛋白质的产生。最后，通过大规模发酵，15647-ΔclpS菌株用于生产VHH。其产量约为 530 mg/L，比 WT-15647 高 65%。本研究成功获得了能够有效增加异源蛋白表达的宿主。