Effect Of Different XR/XDH Ratio Levels On Ethanol Production Of Recombinant Saccharomyces Cerevisiae

Currently,most factories take grains as substrate resources to produce ethanol,which not only wastes food resources,but also restricts the sustainable development of the fuel ethanol industry.Cellulosic biomass is abundant in nature,and it can be converted to a mass of xylose.Some reports show that Saccharormyces cerevisiae can establish a D-xylose-utilizing pathway after modified by genetic engineering method.However,the cofactor imbalances of xylose utilization genes limit the ethanol production,as well as the utilization of lignocellulose.In this study,the expression plasmids with genes of XR and XDH from different hosts have been constructed and transformed into Saccharomyces cerevisiae.The change of xylose utilization and ethanol production under the different ratio of XR and XDH enzyme activity in the recombinants has been showed and one recombinant which can produce ethanol efficiently has been screened.The xylose reductase gene(xyll)and xylitol dehydrogenase gene(xyl2)were cloned from the Pichia stipitis.CICC1960 and Candida shehatae 20335 by genetic engineering method.Then the two genes were inserted into the integrated plasmids that contain URA3 or rDNA.Finally the plasmids were transformed into the Saccharomyces cerevisiae W5.Four kinds of recombinants with the different xylose metabolism genes were constructed,named as SUN-?(P.stipitis CICC1960,xyll and xyl2).SUN-?(C.shehatae 20335,xyll and xy12)?sSUN-?(C.shehatae 20335,xyl1;P.stipitis CICC1960,xyl2)?SUN-?(P.stipitis CICC1960,xyl1;C.shehatae 20335,xyl2).The four kinds of recombinants were screended by high concentration G418 resistance in order to obtain high-copy recombinants and ten ones of each kind were selected randomly from the screening results for the test of fermentation and enzyme activity.The results showed that the ferment ability of four kinds of recombinants were different along with the change of ratio of XR and XDH enzyme activity.It also showed that SUN-? would produce more ethanol when the ratio of XR and XDH enzyme activity is between 0.003 and 0.037 meanwhile the ratio of NADH and NADPH cofactors activity for utilization of XR is betweent 2.056 and 3.643,the recombinant of SUN-?-9 had the highest yield of ethanol,0.06 g/g,when it utilized xylose to produce ethanol.SUN-?would produce more ethanol when the ratio of XR and XDH enzyme activity is between 0.013 and 0.076 meanwhile the ratio of NADH and NADPH is betweent 2.250 and 5.073;the recombinant of SUN-?-8 had the highest yield of ethanol,0.09 g/g,when it utilized xylose to produce ethanol;SUN-? would produce more ethanol when the ratio of XR and XDH enzyme activity is between 0.004 and 0.022 meanwhile the ratio of NADH and NADPH is betweent 5.190 and 8.386,and this group showed the most higher xylose utilization,10.7g/L,the recombinant of SUN-?-2 had the highest yield of ethanol,0.06 g/g,when it utilized xylose to produce ethanol;SUN-? would produce more ethanol when the ratio of XR and XDH enzyme activity is between 0.049 and 0.109 meanwhile the ratio of NADH and NADPH is betweent 0.714 and 1.860.the recombinant of SUN-?-1 had the highest yield of ethanol,0.09 g/g,when it utilized xylose to produce ethanol.In addition,it also showed that the yield of ethanol of the SUN-? group were higher.The reason perhaps was that the combined pattern of genes was better for ethanol production.The results of fermentation in fermenter showed that the yield of ethanol of SUN-?-1 which has the highest ethanol yield using xylose and glucose as hybrid carbon source could be up to 0.54 g/g at the condition of 30? and 300 rpm.Conclusion,in addition to XR/XDH ratio can affect the ethanol yield for the recombinants,the change of NADH/NADPH ratio can also effect xylose utilization and ethanol yield for the recombinants;on the one hand,the XR with the higher affinity for NADH can increase the xylose utilization for the recombinants,on the other hand,the XR with the lower affinity for NADH can take the recombinants more inclined to product ethanol;meanwhile,the difference about NADH/NADPH ratio can effect the optimal ratio of XR/XDH directly.In this study,the recombinants of Saccharomyces cerevisiae which contain homologous and heterologous xylose metabolism genes were successfully constructed,and the coenzyme preference in different recombinants was analyzed as well.This study can lay the foundation on further improvement to suitable proportion of cofactor ratio and the effects of heterologous expression of xylose metabolism genes.