Development Of Yeast Strains For Ethanol Production From Jerusalem Artichoke By Consolidated Bioprocessing

Fuel ethanol produced from renewable biomass resources is an alternative to fossil fuels.Consolidated bioprocessing(CBP)integrates enzyme production,biomass hydrolysis and microbial fermentation,which is promising for more efficient ethanol production.Jerusalem artichoke(JA)can grow well in marginal lands with high biomass yield,and thus is a candidate for energy crop,but report for ethanol production from JA is limited.Tubers(JAT)and stalk(JAS)are major biomass from JA.While JAT is feedstock for extracting value-added inulin,JAS can be used to produce ethanol.This research is to study ethanol production from JAT and JAS,with a focus on developing CBP strains from Saccharomyces cerevisiae,and optimizing the operating units with the process.Firstly,ethanol production from lignocellulosic JAS was investigated.Experimental results showed that the pretreatment with 2.0%(w/v)NaOH could remove lignin effectively and consequently increased cellulose content from 39.4%to 58.2%with 13.1%inulin remained.Batch-feeding of the pretreated JAS and cellulase was validated to be a good strategy for efficient saccharification of the cellulose component,which produced 115.8 g/L total sugars from 20%JAS.Furthermore,CBP strains were developed from industrial S.cerevisiae Angel by engineering it with inulinase expression through cell surface display for ethanol production,and S.cerevisiae MK01 was selected to ferment the JAS hydrolysate by the CBP strategy,which produced 38.3 g/L ethanol,making the ethanol yield of 0.361 g/g total sugars consumed,about 71%of the theoretical yield of 0.511.Secondly,S.cerevisiae MK01 was employed for ethanol production from JAT by the CBP strategy to address the challenge that might be raised when JA is planted at large scale,but market demand for inulin is limited.The fermentation was performed at elevated temperatures from 38 ? to 42 ?.No significant difference in growth was observed for the engineered strain when temperature was controlled at 38 ? and 40 ?,respectively,but inulinase activity was enhanced at 40 ?.As a result,the enzymatic hydrolysis of inulin was facilitated and ethanol production was consequently improved with 89.3 g/L ethanol produced within 72 h from 198.2 g/L total inulin sugars consumed.Similar results were also observed in ethanol production from JAT with 85.2 g/L ethanol produced within 72 h from 185.7 g/L total sugars consumed,and ethanol yield as high as 0.459,about 90%of the theoretical yield of 0.511,was achieved.Finally,the CBP strain with surface display of cellulase was further engineered with artificial transcription factors for thermotolerance to benefit ethanol fermentation at elevated temperature preferred by industrial production,and mechanism underlying the thermo-tolerance was explored.Compared to the parental strain,the engineered strain was capable of growing at 43 ?,and significant difference was detected in intracellular trehalose,accumulation of reactive oxygen species and superoxide dismutase activity,which correlated with the transcription of genes encoding the key enzymes.The degradation of phosphoric acid swollen cellulose by the thermotolerant strain was enhanced by 45.8%,which consequently facilitated ethanol production.As a result,the time for ethanol fermentation from JAS was shortened by 24 h at 43 ? in addition to improved ethanol production.Comparative transcriptomic analysis found significant up regulation of genes encoding key enzymes related to the cell wall integrity,PKC1-MPK1 pathway,and GABA shunt,indicating that alterations in membrane compositions might be behind the improved thermal tolerance of the strain.Overall,the progress with this research is significant for ethanol production from JA biomass,either stalk or tubers.