研究表明,毕赤酵母(Komagataella phaffii)的高渗甘油信号通路(high osmolarity glycerol pathway, HOG pathway)与酿酒酵母(Saccharomyces cerevisiae)有所不同,且毕赤酵母的高渗抗性较低,在毕赤酵母的发酵工业生产应用中造成了一定影响,因此构建一株耐高渗的毕赤酵母菌株有其实用价值。我们在前期研究中通过引入外源甘油合成基因3-磷酸甘油脱氢酶1(glycerol-3-phosphate dehydrogenase 1, ScGPD1),3-磷酸甘油磷酸酶(glycerol-3-phosphate phosphatase 2, ScGPP2)的策略,增强了毕赤酵母的甘油合成能力,提高了毕赤酵母的高渗抗性。在该研究中,通过敲除不利于甘油积累的甘油通道蛋白(glyceroporin)基因FPS1,甘油激酶(glycerol kinase)基因GUT1的策略,减少了胞内甘油的外排和代谢,显著提高了毕赤酵母的高渗抗性。之后将上述2种策略结合,构建了双策略菌株,进一步提高了毕赤酵母的高渗抗性。通过对比证明了毕赤酵母无法通过积累胞内甘油来抵抗高渗,主要是因为甘油的外排和代谢导致甘油难以积累,而不是合成能力不足。最后,通过HOG1的敲除,发现毕赤酵母Hog1对高渗抗性同时具有负面和正面2种作用,但总体上不利于毕赤酵母的高渗抗性。
It has been reported that the HOG (high osmolarity glycerol) pathway of Komagataella phaffii is different from that of Saccharomyces cerevisiae, and K. phaffii has weak resistance of hyperosmotic stress, which limits its application of fermentation in industrial production. Therefore, it has practical value to construct an engineered K. phaffii strain with resistance to hyperosmotic stress. In our previous study, the strategy of introducing the exogenous glycerol synthesis genes ScGPD1 (glycerol-3-phosphate dehydrogenase 1) and ScGPP2 (glycerol-3-phosphate phosphatase 2) into K. phaffii, was applied to enhance the ability of glycerol synthesis. As a result, the resistance to hyperosmotic stress was improved. In this study, another strategy, knocking out the glycerol accumulation negative genes glyceroporin FPS1 and glycerol kinase GUT1 in K. phaffii, was applied to reduce the efflux and metabolism of glycerol. Thus, the resistance to hyperosmotic stress was significantly improved. By combining the above two strategies, dual-strategy K. phaffii strains were constructed, and the resistance to hyperosmotic stress was further improved. It was proved that K. phaffii was not able to resist hyperosmotic stress by accumulating intracellular glycerol, mainly because the efflux and metabolism of glycerol made it difficult for accumulation, rather than insufficient glycerol synthesis ability. Finally, by knocking out HOG1, it was found that K. phaffii Hog1 had both negative and positive effects on the resistance to hyperosmotic stress, but it was collectively adverse to the resistance to hyperosmotic stress of K. phaffii.
[1] AHMAD M, HIRZ M, PICHLER H, et al.Protein expression in Pichia pastoris:Recent achievements and perspectives for heterologous protein production[J].Applied Microbiology and Biotechnology, 2014, 98(12):5 301-5 317.
[2] 王莲哲, 刘士俊, 刘佳乐, 等.树蛙抗菌肽Cathelicidin在毕赤酵母中的表达及抑菌活性分析[J].农业生物技术学报, 2021, 29(1):67-72.
WANG L Z, LIU S J, LIU J L, et al.Expression of tree frog (Rhacophorus) cathelicidin peptide in Pichia pastoris and its antibacterial activity analysis[J].Journal of Agricultural Biotechnology, 2021, 29(1):67-72.
[3] 杜加亮, 古琼, 刘悦越, 等.诺如病毒VP1蛋白病毒样颗粒在毕赤酵母中的分泌表达[J].中国生物制品学杂志, 2020, 33(10):1 097-1 103.
DU J L, GU Q, LIU Y Y, et al.Secretory expression of virus-like particles of Norovirus VP1 in Pichia pastoris[J].Chinese Journal of Biologicals, 2020, 33(10):1 097-1 103.
[4] 刘毓均, 卢春, 袁萍, 等.牛胃溶菌酶基因在毕赤酵母中的表达、发酵参数优化及抑菌活性研究[J].黑龙江畜牧兽医, 2020, 589(1):125-130;162.
LIU Y J, LU C, YUAN P, et al.Study on expression of Bovine stomach Lysozyme gene in Pichia Pastoris, optimization of fermentation parameters and antibacterial activity[J].Heilongjiang Animal Science and Veterinary Medicine, 2020, 589(1):125-130;162.
[5] 王颢霖. 普鲁兰酶基因在毕赤酵母中的表达研究[J].河南农业, 2020(21):52-54.
WANG H L.Study on the expression of Pullulanase gene in Pichia pastoris[J].Henan Agriculture, 2020(21):52-54.
[6] DUNAYEVICH P, BALTANÁS R, CLEMENTE J A, et al.Heat-stress triggers MAPK crosstalk to turn on the hyperosmotic response pathway[J].Scientific Reports, 2018,8:15 168.
[7] SAITO H, TATEBAYASHI K.Regulation of the osmoregulatory HOG MAPK cascade in yeast[J].Journal of Biochemistry, 2004,136(3):267-272.
[8] TATEBAYASHI K, YAMAMOTO K, NAGOYA M, et al.Osmosensing and scaffolding functions of the oligomeric four-transmembrane domain osmosensor Sho1[J].Nature Communications, 2015, 6:6 975.
[9] TATEBAYASHI K, YAMAMOTO K, TOMIDA T, et al.Osmostress enhances activating phosphorylation of Hog1 MAP kinase by mono-Phosphorylated Pbs2 MAP2K[J].The EMBO Journal, 2020, 39(5):e103444.
[10] CAPALDI A P, KAPLAN T, LIU Y, et al.Structure and function of a transcriptional network activated by the MAPK Hog1[J].Nature Genetics, 2008, 40(11):1 300-1 306.
[11] HOHMANN S, KRANTZ M, NORDLANDER B.Yeast Osmoregulation, Osmosensing and Osmosignaling[M].Manhattan:Academic Press,2007:29-45.
[12] XIANG L, YANKUN Y, CHUNJUN Z, et al.Transcriptional analysis of impacts of glycerol transporter 1 on methanol and glycerol metabolism in Pichia pastoris[J].FEMS Yeast Research, 2017.DOI:10.1093/femsyr/fox081.
[13] BAI C, TESKER M, ENGELBERG D.The yeast Hot1 transcription factor is critical for activating a single target gene, STL1[J].Molecular Biology of the Cell, 2015, 26(12):2 357-2 374.
[14] ANSELL R, GRANATH K, HOHMANN S, et al.The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation[J].The EMBO Journal, 1997, 16(9):2 179-2 187.
[15] REP M, ALBERTYN J, THEVELEIN J M, et al.Different signalling pathways contribute to the control of GPD1 gene expression by osmotic stress in Saccharomyces cerevisiae[J].Microbiology, 1999, 145(Pt3):715-727.
[16] WANG R B, ZHAO T Y, ZHUO J L, et al.MAPK/HOG signaling pathway induced stress-responsive damage repair is a mechanism for Pichia pastoris to survive from hyperosmotic stress[J].Journal of Chemical Technology & Biotechnology, 2021, 96(2):412-422.
[17] TAMÁS M J,HOHMANN S.The Osmotic Stress Response of Saccharomyces cerevisiae[M].Berlin:Springer Berlin Heidelberg, 2003:121-200.
[18] LEE J, REITER W, DOHNAL I, et al.MAPK Hog1 closes the S.cerevisiae glycerol channel Fps1 by phosphorylating and displacing its positive regulators[J].Genes & Development, 2013, 27(23):2 590-2 601.
[19] 王荣斌, 赵天宇, 卓俊林, 等.巴斯德毕赤酵母MAPK/HOG信号通路的分子互作研究[J].生物学杂志, 2020, 37(3):7-11.
WANG R B, ZHAO T Y, ZHUO J L, et al.The interactions of MAPK/HOG signal pathway factors in Pichia pastoris[J].Journal of Biology, 2020, 37(3):7-11.
[20] TANAKA K, TATEBAYASHI K, NISHIMURA A, et al.Yeast osmosensors Hkr1 and Msb2 activate the Hog1 MAPK cascade by different mechanisms[J].Science Signaling, 2014, 7(314):21.
