Key Glycosylation Sites Of Invertase Suc2 In Saccharomyces Cerevisiae

Jerusalem artichoke is a high-quality economic crop with strong resistance.It is one of the preferred sources of biomass for industrial production of bioethanol.However,most of the wild Saccharomyces cerevisiae strains lack inulinase which can efficiently degrade inulin,and it is difficult to directly produce bioethanol by using inulin as a raw material.In the previous study,our group screened a wild-type S.cerevisiae L610 which can efficiently degrade inulin.It was found that its highly expressed invertase Suc2 is the key enzyme for degrading inulin.Previous studies have shown that there is a large difference in the enzyme activity of Suc2 heterologously expressed by different host strain.Using mass spectrometry,it was found that these Suc2 samples have different degrees of glycosylation modification.Therefore,it is speculated that glycosylation modification may affect the enzyme activity of Suc2.In order to further validate this hypothesis,the S.cerevisiae recombinant strain BY-BS overexpressing invertase Suc2 was used as a starting strain to investigate the effect of glycosylation modification of Suc2 on inulin catabolic the ability of S.cerevisiae.In this work,13 S.cerevisiae mutants with different Suc2 deglycosylation modification were constructed by site-directed mutagenesis.The enzyme activity of Suc2 in the mutant strain,the ethanol yield of the mutant strain,and the product composition were detected.The analysis revealed some key glycosylation site for the sucrose invertase Suc2.In order to obtain S.cerevisiae mutants with different mutation sites site-directed mutagenesis was used to mutate asparagine(Asn)at 13 glycosylation sites on Suc2 in S.cerevisiae to structurally similar aspartic acid(Asp).respectively.The enzyme activities of Suc2 in 13 mutant strains with inulin and sucrose as substrates were separately examined.The results showed that in all the mutant strains,the inulinase activities of BY-N4 D,BY-N78 D and BY-N146 D were increased by 3.95,1.92,1.62 times and the invertase activity was increased by 1.57,1.60,1.57 respectively,compared with the starting strain BY-BS;The enzymatic activity of BY-N45 D was significantly reduced,and its inulinase activity and sucrase activity were 0.47 and 0.59 times of BY-BS,respectively.By examining the effect of deglycosylation modification on the production of ethanol using inulin as a carbon source,we found that the trend is almost in consistent with changes that of inulinase activity.Based on the results of ethanol production and enzyme activity,seven glycosylation sites have a greater impact on Suc2 enzyme activity,namely N4,N45,N78,N92,N146,N350,N379.The ion chromatography detection of the inulin fermentation broth of the above seven glycosylation modified mutant strains and BY-BS showed that the mutant BY-N4 D could degrade the inulin DP less than 10 more effectively than BY-BS,the degradation ability of BY-N45 D to inulin DP less than 10 is weaker than that of BY-BS.The research results obtained in this work lay the foundation for rational transformation of S.cerevisiae and construction of industrial strains producing inulin-based ethanol.