Screening Of Fusarium Oxysporum For Ethanol Production From Bagasse By Simultaneous Saccharification And Study Of Key Enzymes For Pentose Conversion

In this study,Fusarium oxysporum was used as the target and strains with high cellulose degradation ability were screened.At the same time,the degradation effect,key enzyme activity and alcohol production capacity of the fermenting monosaccharides of the high ethanol yielding strain were measured,and the mechanism of high ethanol production was investigated from the perspective of molecular biology by combining the transcriptome data of the strain.The results are as follows The main findings of the study are as follows.(1)Screening of Fusarium oxysporum strains for strong cellulose degradationFourteen strains of Fusarium oxysporum were subjected to triple tests of Congo red plate staining,filter paper disintegration and total cellulose enzyme activity assay to determine the cellulose degradation ability of each strain in turn.The results of the Congo red plate staining test showed that the hydrolysis circles of each strain ranged from 3.2 to 4.5 cm in diameter,with seven strains including mh2 having hydrolysis circles of 4 cm or more.The disintegration effect of the filter paper sheets was classified into four classes and mh2 was found to be the most effective in degrading the filter paper sheets into a paste.Finally,the cellulase activity assay showed that mh2 had the highest enzymatic activity compared to other strains,and thus mh2 was identified as the strongest cellulose degrading strain for this test.(2)A preliminary study on the efficacy and mechanism of simultaneous saccharification fermentation of sugarcane bagasse by Fusarium oxysporum for ethanol productionThe fermentation efficiency of strain mh2 was measured by simultaneous saccharification and fermentation tests using bagasse as substrate.The mechanism of high ethanol production was investigated by using mh5 and Cs20 as positive and negative controls,respectively.The results showed that mh2 could produce ethanol by synchronous saccharification fermentation of bagasse without initial sugar at a yield of 40 g/kg,and up to98 g/kg by synchronous saccharification fermentation of bagasse containing residual sugar.mh2’s cellulase activity,cellulose degradation rate and ethanol conversion rate of fermented glucose and xylose were measured during the fermentation process,and the results showed that The enzyme activities and cellulose degradation rates of mh2 were higher than those of the control strain,with the degradation rate reaching 25.60%.Under the condition of higher enzyme activities,the synergistic effects among the cellulases promoted the hydrolysis of cellulose,which in turn provided a large source of monosaccharides for the production of ethanol;the ethanol conversion rates of mh2 fermented glucose and xylose were significantly higher than those of the control,reaching 0.443 g/g and 0.213 g/g,which can rapidly convert the monosaccharides in the hydrolysis stage and achieve high ethanol production.(3)Initial functional investigation of the key enzyme of xylose metabolism in high ethanol-producing strains of Fusarium oxysporum based on transcriptomeBased on transcriptome data,bioinformatics analysis was performed to predict the function of the key enzymes of the xylose metabolism pathway(EC:1.1.1.14)in high and low ethanol producing strains.Phylogenetic analysis revealed a total of 64 genes annotated in the xylose metabolism pathway in mh2 and Cs20,of which eight members were annotated as(EC:1.1.1.14)with a relatively stable phylogenetic process and high homology.Upon structural domain analysis,eight members were conserved in their evolutionary process.(EC:1.1.1.14)is an L-aidulose alcohol dehydrogenase,a zinc-dependent polyol dehydrogenase that can be activated by monosaccharide stacking and is rapidly involved in the interconversion of alcohols,aldehydes and ketones during the sugar conversion phase,driving the metabolic flow from glucose and xylose to ethanol.Sequence and motif analysis revealed that all eight members have a substrate binding catalytic domain and a coenzyme binding domain that catalyse the reversible oxidation of ethanol to the corresponding acetaldehyde or ketone while carrying out L-aidulose alcohol dehydrogenase synthesis.Structural analysis revealed a tetrameric structure of L-aidoosterol dehydrogenase,each containing multiple α-helix and β-fold structures,which allows for stable expression of the enzyme gene.