Huang Mengmeng, Gao Yanyun, Zhou Xiangshan, Zhang Yuanxing, Cai Menghao
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China.
Shanghai Collaborative Innovation Center for Biomanufacturing, 130 Meilong Road, Shanghai, 200237, China.
Bioprocess Biosyst Eng. 2017 Mar;40(3):341-350. doi: 10.1007/s00449-016-1701-y. Epub 2016 Oct 28.
Unfolded protein response (UPR) usually happens when expressing heterologous proteins in high level, which may help cells to facilitate protein processing. Here, we evaluated the effects of the UPR activator HAC1p on a raw-starch hydrolyzing α-amylase (Gs4j-amyA), so as to improve heterologous production of the enzyme in Pichia pastoris. The gene (amyA) encoding Gs4j-amyA was first codon-optimized and expressed in P. pastoris under the control of the AOX1 promoter. A high gene dosage (12 copies) of amyA facilitated amylase expression which produced an enzyme activity of 305 U/ml. A spliced HAC1 encoding an UPR activator HAC1p was then co-expressed and the dosage effects of HAC1 on amylase expression was investigated. Six copies of HAC1 driven by AOX1 promoter produced a high amylase activity of 2200 U/ml, further increasing by 621%. However, excessive gene dosages driven by the same promoter led to a titration effect of its transcription factors and decreased the amount of amyA transcripts. Thus, constitutive expression of HAC1 by GAP promotor was further involved and Gs4j-amyA activity reached 3700 U/ml finally, which was further increased by 68.2%. Moreover, Gs4j-amyA was glycosylated in P. pastoris which generated higher enzyme activity than that in E. coli. Generally, regulating HAC1p expression by different strategies enhanced amylase production by 11.1 folds, indicating a reference for expression of other proteins in P. pastoris.
未折叠蛋白反应（UPR）通常在高水平表达异源蛋白时发生，这可能有助于细胞促进蛋白质加工。在此，我们评估了UPR激活剂HAC1p对一种生淀粉水解α-淀粉酶（Gs4j-amyA）的影响，以便提高该酶在毕赤酵母中的异源产量。编码Gs4j-amyA的基因（amyA）首先进行密码子优化，并在AOX1启动子的控制下在毕赤酵母中表达。amyA的高基因剂量（12拷贝）促进了淀粉酶的表达，产生的酶活性为305 U/ml。然后共表达编码UPR激活剂HAC1p的剪接型HAC1，并研究HAC1对淀粉酶表达的剂量效应。由AOX1启动子驱动的6拷贝HAC1产生了2200 U/ml的高淀粉酶活性，进一步提高了621%。然而，由相同启动子驱动的过量基因剂量导致其转录因子的滴定效应，并减少了amyA转录本的数量。因此，进一步采用GAP启动子组成型表达HAC1，最终Gs4j-amyA活性达到3700 U/ml，进一步提高了68.2%。此外，Gs4j-amyA在毕赤酵母中发生了糖基化，产生的酶活性高于在大肠杆菌中的活性。一般来说，通过不同策略调节HAC1p的表达使淀粉酶产量提高了11.1倍，为在毕赤酵母中表达其他蛋白质提供了参考。
