Li Qinghua, Zhang Chen, Li Jianghua, Du Guocheng, Li Zhaofeng, Zhou Jingwen, Zhang Guoqiang
Science Center for Future Foods, Jiangnan University, Wuxi, 214122, China.
School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
Synth Syst Biotechnol. 2024 Dec 20;10(2):365-372. doi: 10.1016/j.synbio.2024.12.003. eCollection 2025 Jun.
The expression system has been developed into a chassis for the production of heterologous lipases, attributed to its strong capabilities in protein production and secretion, robust post-translational modifications, and favourable safety profile. However, the system's relatively low expression levels remain a challenge, hindering its ability to meet the increasing demands of large-scale production. Strain C19, screened by high-throughput methods combining droplet microfluidics and flow cytometry, was demonstrated to be a potential chassis cell based on fermentation kinetic analysis and transcriptome sequencing. By leveraging the endogenous α-amylase's expression elements and integration sites, a combination of random and site-directed integration strategies was employed to enhance the expression of heterologous lipases in strain C19. As a result, lipase production in shake-flask fermentation reached a titer of 113.6 U/L. The study further demonstrated that the different α-amylase gene loci could serve as effective integration sites for the multi-copy expression of heterologous proteins because the lipase activity of the 3-amylase site integrated strain C19#1-ABC was 3.3 times higher than that of C19#1. Furthermore, fermentation results in a 5-L bioreactor indicated that optimization of fermentation processes and facilities had the potential to further increase heterologous protein expression levels. These findings offered valuable insights into the advancement of expression systems and the potential for scaling engineered strains for industrial applications.
该表达系统已发展成为用于生产异源脂肪酶的底盘，这归因于其在蛋白质生产和分泌方面的强大能力、强大的翻译后修饰以及良好的安全性。然而，该系统相对较低的表达水平仍然是一个挑战，阻碍了其满足大规模生产日益增长的需求的能力。通过结合微滴微流控和流式细胞术的高通量方法筛选出的C19菌株，基于发酵动力学分析和转录组测序被证明是一种潜在的底盘细胞。通过利用内源性α-淀粉酶的表达元件和整合位点，采用随机和定点整合策略相结合的方法来提高C19菌株中异源脂肪酶的表达。结果，摇瓶发酵中的脂肪酶产量达到了113.6 U/L。该研究进一步证明，不同的α-淀粉酶基因位点可以作为异源蛋白多拷贝表达的有效整合位点，因为3-淀粉酶位点整合菌株C19#1-ABC的脂肪酶活性比C19#1高3.3倍。此外，在5-L生物反应器中的发酵结果表明，发酵工艺和设施的优化有潜力进一步提高异源蛋白的表达水平。这些发现为表达系统的改进以及工程菌株扩大到工业应用的潜力提供了有价值的见解。
