Furfural Tolerant ZM CP4 Based On Error-PCR Whole Genome Shuffling

Bioethanol from lignocellulose materials grabs a great attention because of the dramatic reduction of fossil fuels and global warming.Bioethanol is produced from lignocellulose that needs for a pretreatment to release fermentable sugars as it is recalcitrant to degradation.However,inhibitors will be introduced during the pretreatment,especially furfural,which will adversely affect the growth and fermentation of microbes.Furfural will also have an inhibited effect on the growth and fermentation of Zymomonas mobilis which possesses several appealing characteristics for ethanol production.Thus,development of furfural-tolerant strains is essential for production of fuels from lignocellulose.In this study,Zymomonas mobilis CP4 was subjected to error-PCR whole genome shuffling,and stable mutant F211 and F27 tolerating 3g/L furfural was obtained after two rounds of selection.In the presence of 3 g/L furfural,the final cell density and ethanol yields of mutants were 140–149 % and 115-118 % higher than CP4,respectively.When furfural concentration was decreased to 1 g/L,all strains began to rapidly grow and ferment,indicating the inhibition concentration.When cultured in the medium with 15-20 %?w/v?glucose,mutants showed significantly higher growth and ethanol production rate than CP4,indicating higher tolerance to high concentration glucose of mutants.Furfural reductase activity in mutants was higher than that in CP4.Genome resequencing revealed that F211 and F27 had 12 and 13 mutations,respectively.No mutation related to furfural reductase gene was found.Knockout of gene transcriptional regulator?ZCP4₁431?led to higher furfural tolerance and reductase activity than control,indicating that increased tolerance of mutants may result from increased furfural reductase activity,which causes the acceleration of furfural detoxification process.In summary,this study provides excellent furfural mutants and valuable clues for clarification of furfural tolerance and strain development using inverse metabolic engineer strategy.