Han Minghai, Wang Weixian, Gong Xun, Zhou Jianli, Xu Cunbin, Li Yinfeng
College of Food and Pharmaceutical Engineering, Guizhou Institute of Technology, Guiyang, P.R. China.
Guangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, South China Normal University, Guangzhou, P.R. China.
Protein Pept Lett. 2021;28(12):1434-1441. doi: 10.2174/0929866528666211105111155.
Pichia pastoris is one of the most popular eukaryotic hosts for producing heterologous proteins, while increasing the secretion of target proteins is still a top priority for their application in industrial fields. Recently, the research effort to enhance protein production has focused on up-regulating the unfolded protein response (UPR).
We evaluated the effects of activated UPR via Hac1p co-expression with the promoter AOX1 (PAOX1) or GAP (PGAP) on the expression of recombinant chitosanase (rCBS) in P. pastoris.
The DNA sequence encoding the chitosanase was chemically synthesized and cloned into pPICZαA, and the resulting pPICZαA/rCBS was transformed into P. pastoris for expressing rCBS. The P. pastorisHAC1 cDNA was chemically synthesized and cloned into pPIC3.5K to give pPIC3.5K/Hac1p. The HAC1 cDNA was cloned into PGAPZB and then inserted with the HIS4 gene from pAO815 to construct the vector PGAPZB/Hac1p/HIS4. For co-expression of Hac1p, the two plasmids pPIC3.5K/Hac1p and PGAPZB/Hac1p/HIS4 were transformed into P. pastoris harboring the CBS gene. The rCBS was assessed based on chitosanase activity and analyzed by SDSPAGE. The enhanced Kar2p was detected with western blotting to evaluate UPR.
Hac1p co-expression with PAOX enhanced rCBS secretion by 41% at 28°C. Although the level of UPR resulting from Hac1p co-expression with PAOX was equivalent to that with PGAP in terms of the quantity of Kar2p (a hallmark of the UPR), substitution of PGAP for PAOX further increased rCBS production by 21%. The methanol-utilizing phenotype of P. pastoris did not affect rCBS secretion with or without co-expression of Hac1p. Finally, Hac1p co-expression withPAOX or PGAP promoted rCBS secretion from 22 to 30°C and raised the optimum induction temperature.
The study indicated that Hac1p co-expression with PAOX or PGAP is an effective strategy to trigger UPR of P. pastoris and a feasible means for improving the production of rCBS therein.
巴斯德毕赤酵母是生产异源蛋白最常用的真核宿主之一，而提高目标蛋白的分泌量仍是其在工业领域应用的首要任务。最近，提高蛋白产量的研究工作主要集中在上调未折叠蛋白反应（UPR）。
我们评估了通过与启动子AOX1（PAOX1）或GAP（PGAP）共表达Hac1p激活UPR对重组壳聚糖酶（rCBS）在巴斯德毕赤酵母中表达的影响。
化学合成编码壳聚糖酶的DNA序列并克隆到pPICZαA中，将得到的pPICZαA/rCBS转化到巴斯德毕赤酵母中以表达rCBS。化学合成巴斯德毕赤酵母HAC1 cDNA并克隆到pPIC3.5K中，得到pPIC3.5K/Hac1p。将HAC1 cDNA克隆到PGAPZB中，然后插入来自pAO815的HIS4基因，构建载体PGAPZB/Hac1p/HIS4。为了共表达Hac1p，将两个质粒pPIC3.5K/Hac1p和PGAPZB/Hac1p/HIS4转化到含有CBS基因的巴斯德毕赤酵母中。基于壳聚糖酶活性评估rCBS，并通过SDS-PAGE进行分析。用蛋白质印迹法检测增强的Kar2p以评估UPR。
在28°C时，与PAOX共表达Hac1p可使rCBS分泌量提高41%。尽管就Kar2p的量而言（UPR的一个标志），与PAOX共表达Hac1p产生的UPR水平与与PGAP共表达时相当，但用PGAP替代PAOX可使rCBS产量进一步提高21%。有无Hac1p共表达时，巴斯德毕赤酵母利用甲醇的表型均不影响rCBS的分泌。最后，与PAOX或PGAP共表达Hac1p可在22至30°C促进rCBS分泌，并提高最佳诱导温度。
该研究表明，与PAOX或PGAP共表达Hac1p是触发巴斯德毕赤酵母UPR的有效策略，也是提高其中rCBS产量的可行方法。
