杏鲍菇麦角硫因生物合成基因挖掘及在酵母中的组合表达

Discovery of ergothioneine biosynthesis genes from Pleurotus eryngii and its combinatorial expression in Saccharomyces cerevisiae

【目的】麦角硫因(EGT)是一种组氨酸衍生而来的天然氨基酸,具有抗氧化、抗炎症、抗癌以及预防神经系统类疾病等多种生理功能。从杏鲍菇中挖掘出EGT合成酶基因并在酵母中进行表达及功能鉴定,阐明杏鲍菇EGT的合成途径,为高效合成EGT提供基因资源及培育高产EGT的杏鲍菇新品种提供依据。【方法】运用生物信息学方法从杏鲍菇基因组中克隆了4个EGT合成酶基因PeEgt1、PeEgt2-1、PeEgt2-2、PeEgt2-3,并以酿酒酵母IMX581菌株为表达宿主对该基因进行异源表达,采用高效液相色谱法测定EGT产量,鉴定基因功能。【结果】分别构建了单基因、双基因和三基因的酿酒酵母IMX581工程菌株。整合了PeEgt1的单基因酿酒酵母工程菌株可合成EGT;与单基因工程菌株相比,整合了PeEgt1、PeEgt2-1或PeEgt1、PeEgt2-2的双基因酿酒酵母工程菌株的EGT产量均有显著提高,而整合了PeEgt1、PeEgt2-3的双基因酿酒酵母工程菌株的EGT产量没有提高。表明PeEgt1、PeEgt2-1、PeEgt2-2基因有功能,表达的酶有活性,而PeEgt2-3基因没有功能。整合了3个基因的PeEgt1-PeEgt2-1-PeEgt2-2酿酒酵母工程菌株的EGT产量较双基因工程菌株没有显著提高,推测可能是PeEgt1合成的组氨酸甜菜碱半胱氨酸亚砜底物不足以提供PeEgt2-1、PeEgt2-2两个酶的反应,限制了酿酒酵母EGT的进一步合成。【结论】在杏鲍菇中克隆的EGT合成酶基因PeEgt1、PeEgt2-1、PeEgt2-2有功能,表达的酶有活性,且PeEgt2-1、PeEgt2-2可能是同工酶;杏鲍菇EGT的生物合成途径由PeEgt1、PeEgt2两个基因组成。

【Objective】Ergothioneine(EGT),a type of natural amino acids evolved from histidine,boasted functions like antioxidant,anti-inflammatory,antitumorous effects and prevention against nervous system disease.In the study,EGT synthetase genes in Pleurotus eryngii were discovered and expressed in Saccharomyces cerevisiae for functional identification,aiming to provide gene resources for the effective synthesis of EGT in P.eryngii and breed of new line.【Method】Four EGT synthetase genes,PeEgt1,PeEgt2-1,PeEgt2-2 and PeEgt2-3,were cloned from P.eryngii by bioinfomatics methods,and heterologously expressed in S.cerevisiae strain IMX581,followed by EGT yield quantification via high performance liquid chromatography(HPLC)and gene function identification.【Result】Various engineered strains with single,double and triple genes expressed were successfully constructed.Strain which only integrated PeEgt1(IMX581-PeEgt1)was capable of producing EGT;strains with double genes expressed,either PeEgt1/PeEgt2-1 or PeEgt1/PeEgt2-2,had higher EGT yields than that of IMX581-PeEgt1,while that of strain with PeEgt1/PeEgt2-3 expressed was not improved,implying that genes PeEgt1,PeEgt2-1 and PeEgt2-2 can produce proteins with enzymatic activity,while PeEgt2-3 can not.Meanwhile,the EGT yields of engineered strain with triple genes PeEgt1,PeEgt2-1 and PeEgt2-2 expressed were not significantly higher than those of the double genes,suggesting that the hercynynlcysteine sulfoxide(HcySul)substrate,synthesized by PeEgt1 enzyme,may be insufficient for the reaction of PeEgt2-1 and PeEgt2-2 enzymes,which limited the further synthesis of EGT in brewing yeast.【Conclusion】Genes PeEgt1,PeEgt2-1 and PeEgt2-2 had functions of producing proteins with enzymatic activity,and PeEgt2-1 and PeEgt2-2 are prone to be isozymes which play some roles in the biosynthetic pathway of EGT synthesis.