Li Cheng, Lin Ying, Zheng Xueyun, Pang Nuo, Liao Xihao, Liu Xiaoxiao, Huang Yuanyuan, Liang Shuli
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
Guangdong Research Center of Industrial Enzyme and Green Manufacturing Technology, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, P. R. China.
BMC Biotechnol. 2015 Sep 26;15:88. doi: 10.1186/s12896-015-0204-2.
Phytase is used as an animal feed additive that degrades phytic acid and reduces feeding costs and pollution caused by fecal excretion of phosphorus. Some phytases have been expressed in Pichia pastoris, among which the phytase from Citrobacter amalonaticus CGMCC 1696 had high specific activity (3548 U/mg). Improvement of the phytase expression level will contribute to facilitate its industrial applications.
To improve the phytase expression, we use modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum. The genetic stability and fermentation in 10-L scaled-up fed-batch fermenter was performed to prepare for the industrial production.
The phytase gene from C. amalonaticus CGMCC 1696 was cloned under the control of the AOX1 promoter (P AOX1 ) and expressed in P. pastoris. The phytase activity achieved was 414 U/mL. Modifications of P AOX1 and the α-factor signal peptide increased the phytase yield by 35 and 12%, respectively. Next, on increasing the copy number of the Phy gene to six, the phytase yield was 141% higher than in the strain containing only a single gene copy. Furthermore, on overexpression of HAC1 (i) (i indicating induced), a gene encoding Hac1p that regulates the unfolded protein response, the phytase yield achieved was 0.75 g/L with an activity of 2119 U/mL, 412% higher than for the original strain. The plasmids in this high-phytase expression strain were stable during incubation at 30 °C in Yeast Extract Peptone Dextrose (YPD) Medium. In a 10-L scaled-up fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL.
The production of a secreted protein will reach its limit at a specific gene copy number where further increases in transcription and translation due to the higher abundance of gene copies will not enhance the secretion process any further. Enhancement of protein folding in the ER can alleviate bottlenecks in the folding and secretion pathways during the overexpression of heterologous proteins in P. pastoris.
Using modification of P AOX1 and the α-factor signal peptide, increasing the gene copy number, and overexpressing HAC1 (i) to enhance folding and secretion of the protein in the endoplasmic reticulum, we have successfully increased the phytase yield 412% relative to the original strain. In a 10-L fed-batch fermenter, the phytase yield achieved was 9.58 g/L with an activity of 35,032 U/mL. Large-scale production of phytase can be applied towards different biocatalytic and feed additive applications.
植酸酶用作动物饲料添加剂，可降解植酸，降低饲料成本，并减少因粪便中磷排泄造成的污染。一些植酸酶已在毕赤酵母中表达，其中来自丙二酸柠檬酸杆菌CGMCC 1696的植酸酶具有较高的比活性（3548 U/mg）。提高植酸酶的表达水平将有助于其工业应用。
为提高植酸酶表达量，我们对PAOX1和α因子信号肽进行修饰，增加基因拷贝数，并过表达HAC1（i）以增强蛋白质在内质网中的折叠和分泌。在10 L规模的补料分批发酵罐中进行遗传稳定性和发酵，为工业生产做准备。
将来自丙二酸柠檬酸杆菌CGMCC 1696的植酸酶基因克隆到AOX1启动子（PAOX1）控制下，并在毕赤酵母中表达。获得的植酸酶活性为414 U/mL。对PAOX1和α因子信号肽的修饰分别使植酸酶产量提高了35%和12%。接下来，将Phy基因拷贝数增加到6个时，植酸酶产量比仅含单个基因拷贝的菌株高141%。此外，过表达HAC1（i）（i表示诱导），即编码调节未折叠蛋白反应的Hac1p的基因，获得的植酸酶产量为0.75 g/L，活性为2119 U/mL，比原始菌株高412%。该高植酸酶表达菌株中的质粒在酵母提取物蛋白胨葡萄糖（YPD）培养基中于30℃培养期间保持稳定。在10 L规模的补料分批发酵罐中，获得的植酸酶产量为9.58 g/L，活性为35032 U/mL。
分泌蛋白的产量在特定基因拷贝数时会达到极限，此时由于基因拷贝数增加导致转录和翻译进一步增加，不会进一步增强分泌过程。在内质网中增强蛋白质折叠可以缓解在毕赤酵母中过表达异源蛋白时折叠和分泌途径中的瓶颈。
通过对PAOX1和α因子信号肽进行修饰，增加基因拷贝数，并过表达HAC1（i）以增强蛋白质在内质网中的折叠和分泌，我们成功地使植酸酶产量相对于原始菌株提高了412%。在10 L补料分批发酵罐中，获得的植酸酶产量为9.58 g/L，活性为35032 U/mL。植酸酶的大规模生产可应用于不同的生物催化和饲料添加剂应用。
