[Improving the expression of recombinant xylanase in Pichia pastoris with episomal expression plasmid].

Pichia pastoris is a versatile protein expression system. At present, the expression plasmids are integrated plasmids whereas the episomal plasmids are rarely used. In this study, the autonomously replicating sequence derived from yeast itself was ligated into the integrative expression vector pGAP to generate an autonomously replicative expression vector pGAPZαA-PARS. When the vector was used to express the xylanase (XynA) gene in P. pastoris, the highest enzyme activity reached 343 U/mL with glycerol as the carbon source, which was 45.9% higher than that of the integrative expression. At the same time, the specific enzyme activity of XynA was increased by 81.2%. We further studied the expression level of recombinant strains with different carbon sources such as glycerol, glucose, sucrose and mixed carbon source (sucrose︰glycerol=1︰2). The highest expression level was achieved with glycerol and the lowest with sucrose. The episomal expression vector pGAPZαA-PARS greatly promotes the application of P. pastoris in the food industry because GAP promoter does not require methanol as induction material. Meanwhile, the episomal vector can greatly improve the expression level, which lays the foundation for further research to improve the high expression of GAP promoter.巴斯德毕赤酵母是用途广泛的蛋白表达系统。目前用于毕赤酵母的质粒主要以整合型质粒为主，很少见到游离的质粒用于外源基因的表达。文中通过将来源于酵母自身的自主复制序列连接到酵母整合型表达载体pGAP 中构成自主复制的游离型表达载体pGAPZαA-PARS，将该载体用于表达木聚糖酶基因。转化毕赤酵母后同传统的整合型表达菌株相比，以甘油为碳源时最高酶活达到343 U/mL，比整合型表达提高了45.9%。同时游离载体表达重组酶比活相对整合表达提高了81.2%。为了节约发酵成本，进一步研究了分别以甘油、葡萄糖、蔗糖、混合碳源 (蔗糖︰甘油=1︰2) 等不同碳源下游离型重组菌株的表达水平。发现甘油表达水平最高，蔗糖最低，但是以工业葡萄糖为碳源时产酶成本最低。由于pGAP 载体不需要以甲醇为碳源，因而文中所构建的游离载体pGAPZαA-PARS 极大促进了毕赤酵母在食品行业中的应用。同时，游离型载体可大幅度提高表达水平，为进一步研究提高GAP 启动子的高效表达奠定了基础。.