基于开源和节流两种策略的耐高渗毕赤酵母菌株的构建

Construction of high osmolarity resistant Komagataella phaffii strains based on two strategies of opening up the sources and reducing the efflux

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.