同时利用AOX1和GAP启动子表达SAM合成酶促进重组毕赤酵母中S-腺苷甲硫氨酸积累

Combined use of AOX1 and GAP promoter to express SAM synthetase and enhance S-adenosylmethionine accumulation in Pichia pastoris

利用重组毕赤酵母(Pichia pastoris)强化表达S-腺苷甲硫氨酸(S-adenosyl methionine,SAM)合成酶(SAM synthetase,SAMS),发酵生产SAM时,胞内SAMS活性和ATP水平是影响SAM合成的重要因素。为了同时保持较高的SAMS活性和ATP供应,我们构建了一株既含有AOX1诱导型表达单元,又含有GAP组成型表达单元的双SAMS表达单元P.pastoris重组菌株Gd。它既能受甲醇调控表达SAMS,又能以甘油为碳源表达SAMS。在培养过程中,先是以甲醇诱导SAMS表达,得到较高的酶活,然后在发酵中后期再将碳源换为甘油。这样不但可以维持较高的酶活,而且可以提高胞内ATP含量。实验结果显示,对于同时利用AOX1和GAP启动子表达SAMS的重组菌,可采取甲醇和甘油两阶段补料,该重组菌的SAMS比产率高于组成型重组菌Xg,Gd进入甘油补料期后胞内ATP含量高于诱导型重组菌Ga。双表达单元菌株Gd的SAM产量达到1.41g/L,比诱导型表达SAMS的重组菌株提高了76.3%,比组成型表达SAMS的重组菌株提高了60.2%,。

The S-adenosylmethionine synthetase （SAMS） activity and ATP level are key factors for the synthesis of S-adenosylmethionine in the engineered Pi chia pastoris （ P. pastoris） in which the exprssion of SAMS was enhanced. In order to keep high levels of both SAMS activity and ATP, a recombinant P. pastoris （Gd） was constructed with the two expression cassettes of SAMS driven by AOX1 and GAP promoter respectively. With the combined use of AOX1 and GAP promoters, the SAMS could be expressed no matter methanol or glycerol was used as a carbon source. During the fermentation process, methanol was first fed to induce the AOXl-driven SAMS expression so that a high SAMS activity could be obtained. Then glycerol was substituted for methanol as the carbon source. With this feeding strategy, not only a high enzyme activity was kept, but also intracellular ATP content was improved. It showed that with the two-phase feeding strategy from methanol to glycerol, a higher specific SAMS productivity was obtained in Gd than in recombinant strain （Xg） with only the GAP-driven SAMS expression cassette, and a higher ATP content was detected in Gd than in recombinant strain （Ga） with only the AOX1-driven SAMS expression cassette. Overall, the final SAM concentration of Gd （1.41 g/L） was increased by 76.3% and 60.2% compared to Ga and Xg respectively.