米黑根毛霉脂肪酶在毕赤酵母中的高效表达与机制研究

Rhizomucor miehei lipase (RML) is an important food enzyme, which is widely used in oil processing and food flavor improvement. Because domestic research has not broken through for a long time, it can only rely on imports with expensive price. In our preliminary study, a recombinant strain of Pichia pastoris with the production of extracellular RML protein reached to domestic maximum 0.64 g/L was obtained, but its enzyme activity was not synchronously increased. Further study found that the transcription level of genes HAC1, PDI, KAR2, MON2 and VPS10 related to RML folding and secretion in this strain were significantly changed. This indicated that the pressure of protein folding and secretion produced in the strain. And a huge space was provided for synchronously improving protein yield and activity of RML by reducing protein folding and secretion pressure in the host. Therefore, this project intends to overexpress or delete these five genes in this strain, in order to obtain the "double-high-yield" strain with simultaneously improvement of RML protein yield and enzyme activity. At the same time, the mechanism of "double-high-yield" will be explored by detecting transcriptional level of key genes related to the steps in the pathway of protein synthesis and secretion, as well as analyzing the spatial conformation, secretion efficiency and enzymatic properties of RML. This study can not only provide references for the efficient expression of other foreign protein, but also play a positive role in reducing the production cost of food related to RML in China, which has important theoretical significance and application value.

米黑根毛霉脂肪酶（RML）是一种重要的食品酶，广泛用于油脂加工和食品风味改良，其价格昂贵且只能依赖进口，国内研究长期又未有突破。申请人前期获得一株高产RML的巴斯德毕赤酵母重组菌株，其蛋白产量达到国内最高值0.64g/L，但其活性未同步提高。进一步研究发现菌中与RML折叠和分泌相关的HAC1、PDI、KAR2、MON2和VPS10基因转录水平变化显著，说明菌中有蛋白折叠和分泌压力，这为改造菌株实现RML蛋白产量和活性的同步提高提供巨大空间。因此，本项目拟在该菌中过表达或缺失这5个基因，获得高产和高活同步提高的“双高产”RML生产菌株；并分析菌中与RML折叠和分泌相关步骤关键基因的转录水平变化，结合RML的空间构象、分泌效率和酶学性质等，阐明菌株“双高产”的机制。本研究不仅能为其它外源蛋白的高效表达提供参考，还为降低我国与RML相关食品领域的生产成本有积极作用，具有重要的理论意义和应用价值。

本项目在前期研究的基础上，进行进一步探索，首先通过在2-拷贝（未产生蛋白折叠和分泌阻力）和4-拷贝菌株（产生蛋白折叠和分泌阻力）中分别过表达与RML折叠和分泌相关的HAC1、PDI、KAR2、MON2基因，成功筛选到8株过表达菌株。对这8株过表达菌株摇瓶发酵，检测细胞生长、胞外酶活及酶活分泌效率，发现在未产生蛋白折叠和分泌阻力的2-拷贝菌株中过表达HAC1、PDI、KAR2、MON2时，均未使胞外酶活得到提升，且过表达KAR2时胞外酶活出现显著下降。而在产生蛋白折叠和分泌阻力的4-拷贝菌株中过表达HAC1和PDI时，RML的胞外酶活分别提高到原来的1.38倍和1.32倍，获得了两株高产菌株。进一步对这两株高产菌株中参与UPR反应的基因进行转录水平变化检测，发现这两株RML高表达菌株中内质网对蛋白的折叠能力均增强。且对过表达PDI后的菌株所分泌的RML纯化，分析其酶学性质，发现其比活提高1.2-1.7倍，证明过表达PDI基因后，菌株通过增强对RML蛋白的折叠使其恢复原有的催化能力，初步揭示RML高产的机制。最后，还将RML作为主导酶，建立了超声波辅助的一步法组合酶催化水解猪板油制备脂肪酸的绿色工艺，经反应条件优化，反应6小时猪板油的水解率可达到97%。