当前位置: X-MOL 学术Bioresource Technol. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Enhanced production of recombinant calf chymosin in Kluyveromyces lactis via CRISPR-Cas9 engineering
Bioresource Technology ( IF 9.7 ) Pub Date : 2025-01-23 , DOI: 10.1016/j.biortech.2025.132116
Yanli Zheng, Shiqing Wang, Yuhui Deng, Ping Hu, Qingxin Xue, Jiaxin Li, Lei Lei, Zhuhua Chan, Jiangke Yang, Wenfang Peng

As an important industrial enzyme, chymosin has been widely used in cheese manufacturing. Fermentation with Kluyveromyces lactis has allowed recombinant chymosin production to fit the growing global demand for cheese consumption; yet improvements can be made to allow for stable and larger-scale production. In this work, various chymosin producing (CP) strains were constructed via targeted chromosomal integration of various copies of a prochymosin expression cassette (PEC) using a CRISPR-Cas9 platform optimized for K. lactis. It enabled the demonstration that chymosin yields could be increased along with gradual chromosomal accumulation of PEC inserts within up to 3 copies. Finally, an optimal CP3i strain was constructed, and with which high yields of recombinant chymosin were attained, reaching ca. 1,200 SU/mL in shake-flask fermentation and ca. 28,000 SU/mL in batch-mode bioreaction, respectively. The activity of the product in milk-curding was observed. These findings provide direction to apply K. lactis-based platforms in the subsequent industrial-scale production of recombinant chymosin.

中文翻译:


通过 CRISPR-Cas9 工程增强乳酸克鲁维酵母中重组小牛凝乳酶的生产



凝乳酶作为一种重要的工业酶,已广泛应用于奶酪制造中。使用乳酸克鲁维酵母发酵使重组凝乳酶生产能够满足全球对奶酪消费日益增长的需求;然而,可以进行改进以实现稳定和更大规模的生产。在这项工作中,使用针对乳酸克雷伯菌优化的 CRISPR-Cas9 平台,通过对促凝酶表达盒 (PEC) 的各种拷贝进行靶向染色体整合来构建各种产生凝乳酶 (CP) 菌株。它能够证明凝乳酶产量可以增加,同时 PEC 插入片段的染色体逐渐积累,最多可在 3 个拷贝内。最后,构建了最优的 CP3i 菌株,并获得了高产率的重组凝乳酶,在摇瓶发酵中分别达到约 1,200 SU/mL,在分批生物反应中分别达到约 28,000 SU/mL。观察该产品在牛奶凝结中的活性。这些发现为将基于乳酸克雷伯菌的平台应用于随后的重组凝乳酶的工业规模生产提供了方向。
更新日期:2025-01-23
down
wechat
bug