Effect Of Cofactor Biosynthesis On Isobutanol Yield In Saccharomyces Cerevisiae

With the decreasing of fossil fuel,biofuels have been received more and more attentions in academics.Comparing to ethanol that is the first generation biofuel,isobutanol has lower hygroscopicity and volatility,higher-octane value and energy density.As a substitute for ethanol,isobutanol can be blended with gasoline to improve fuel performance.At present stage,most microbes can't produce isobutanol and have lower tolerance to isobutanol;and production of isobutanol is difficult to improve.While Saccharomyces cerevisiae has received more attention because it can produce low amount of isobutanol and has higher tolerance to isobutanol.In addition,as a model organism in eukaryotes,S.cerevisiae has been completed gene sequencing and has mature recombinant DNA technology.Therefore,in this study,we chose S.cerevisiae as the parental strain and used recombinant DNA technologies to improve isobutanol production.W303-1A was used as original strain in this study.By analysing isobutanol biosynthesis pathway in S.cerevisiae and using recombinant DNA technology,we constructed engineered strains with higher isobutanol production.On the basis of previous works in our laboratory,the main works of this research were as follows: Firstly,we constructed plasmid YCplac22-ZWF1 and strains carrying overexpressed ZWF1 that encoded glucose-6-phosphate dehydrogenase,which could improve NADPH biosynthesis to maintain cofactor balance to improve isobutanol production.And fermentation characters of these engineered strains were detected.Secondly,we constructed the plasmid YEplac112-PGK1p-ILV5-GFP-CYC1 to overexpress ILV5 that encoded acetohydroxyacid reductoisomerase.Engineered strains that overexpressing ILV5 were constructed by converting plasmid YEplac112-PGK1p-ILV5-GFP-CYC1 into strains overexpressing ILV2,ARO10 and BAT2.And characters of these engineered strains were also determined.Thirdly,we constructed plasmid YCplac33-TDH3p-COX4-ADH7-GFP-CYC1 to overexpress ADH7 that encoded NADPH-dependent alcohol dehydrogenase.Diploid yeast strains with overexpressing ILV2,ILV3,ILV5,ARO10,ADH7 and ZWF1 were constructed and fermentation characters of these diploid strains were also detected.Fourthly,W303-1A was dealt with EMS to screen mutant strains with higher tolerance to isobutanol.In this work,we constructed 20 engineered strains and tested micro-anaerobic fermentation characters of these engineered strains.The growth rate,glucose consumption rate,ethanol and isobutanol yield were detected.Compared to the control strain,isobutanol production of engineered strain HZAL-7 pILV2 pLV3 22-ZWF1 was increased by 4.7-fold.This result showed that overexpressing ZWF1 could increase isobutanol production by increasing NADPH biosynthesis,which was needed in isobutanol biosynthetic pathway.Isobutanol yield of HZAL-7 pARO10 pILV2 pILV5 was increased by 6.7-fold.This result shows that overexpressing ILV5 in strain carrying overexpressed ILV2 and ARO10 could further increase isobutanol production.In addition,although isobutanol production of the diploid strains constructed in this work didn't increase obviously.But these results also provide us important reference values and directions for further researches.In conclution,our works opens up a method to increase isobutanol production and lays a theoretical foundation for further increasing isobutanol production in S.cerevisiae.Meanwhile,EMS was used to mutate yeast strain W303-1A.And four mutant strains that could tolerate 3% isobutanol were isolated.These mutant strains were carried out micro-anaerobic fermentation experiments.We found that mutant ems-39 has a strong ability of suger consumption.This part of study will lay a foundation for the study of molecular mechanisms of isobutanol tolerance in S.cerevisiae.