基于CRISPR/Cas9和紫外诱变构建高产小牛胰凝乳酶的多拷贝乳酸克鲁维酵母

Construction of a Kluyveromyces lactis strain with multi-copy integration for enhanced bovine chymosin production by CRISPR/Cas9 and UV mutagenesis

摘要:

小牛胰凝乳酶(bovine chymosin)作为重要的食品酶，广泛应用于乳制品行业中奶酪的生产制造。本研究将密码子优化后的小牛胰凝乳酶酶原基因作为目的基因，构建了分泌表达框，通过基因组整合至乳酸克鲁维酵母(Kluyveromyces lactis)，单拷贝菌株的摇瓶发酵活力达到了40 U/mL；基于单拷贝菌株，采用CRISPR/Cas9技术敲除筛选基因amdS，构建了多拷贝整合菌株，双拷贝和三拷贝整合菌株的摇瓶发酵活力分别增加到70 U/mL和78 U/mL。基于双拷贝整合菌株KLUcym^D，采用紫外诱变育种技术，在多轮诱变育种后筛选得到一株高产小牛胰凝乳酶的重组菌株KLUcym^D-M2，摇瓶发酵酶活力达到270 U/mL；5 L罐中发酵76 h，酶活力达到最高值600 U/mL。综上，本研究成功构建了一株高产小牛胰凝乳酶的乳酸克鲁维酵母重组菌株，为进一步工业化放大生产奠定了基础。

关键词:小牛胰凝乳酶;乳酸克鲁维酵母;合成生物学;多拷贝整合;紫外诱变育种

Abstract:

Bovine chymosin is an essential food enzyme widely used in cheese production in the dairy industry. This study used a codon-optimized prochymosin gene to construct an expression cassette for extracellular expression of bovine chymosin in Kluyveromyces lactis. After integration of the prochymosin gene into the host cell genome, the single-copy integration strain KLUcym showed the clotting activity of 40 U/mL in a shake flask. The CRISPR/Cas9 system was employed to delete amdS and construct the double-copy integration strain and triple-copy integration strain, which achieved the clotting activities of 70 U/mL and 78 U/mL in shake flasks, separately. Subsequently, multiple rounds of UV mutagenesis were performed on the double-copy strain KLUcym^D, and a recombinant K. lactis strain with a high yield of bovine chymosin was obtained. This strain achieved the clotting activity of 270 U/mL in a shake flask and 600 U/mL in a 5 L bioreactor after 76 h. In summary, we construct a strain KLUcym^D-M2 for high production of bovine chymosin, which lays a foundation of industrial fermentation.

Key words:bovine chymosin;Kluyveromyces lactis;synthetic biology;multi-copy integration;UV mutagenesis