Breeding Of Yeast Strain For Efficient Ethanol Fermentation From Xylose

For efficient bioconversion of lignocellulosic biomass in fuel ethanol production,yeast strains with improved productivity in the utilization of xylose are highly appreciated.Three xylose-fermentating yeast strains,Candida shehatae,Candida tropicalis,Pachysolen tannophilus, were evaluated during a series of determination steps.As a result,Candida shehatae was selected out for further study because of good fermentation performance and high ethanol adaptation ability.The optimizing fermentation medium contained xylose 50 g·L^-1,peptone 3 g·L^-1,yeast extract powder 3 g·L^-1under the condition of 30℃and rotational speed 100 r·min^-1.Next,xylose-fermentating Candida shehatae was first subjected to ethanol adaptation, and the strain with improved ethanol tolerance was further mutagenized by UV irradiation. One respiration deficient mutant Rd-5 with enhanced xylose fermenting ability was selected. Protoplasts of Rd-5 were inactivated by UV treatment before the PEG-mediated protoplast fusion with a Saccharomyces cerevisiae strain with excellent ethanol-fermenting capability.The protoplasts were regenerated on xylose plates,from which fusant F-6 was demonstrated to ferment xylose to ethanol with the highest efficiency among the fusants obtained.Comparing with the parent Candida shehatae stain,the maximum ethanol production by fusant F-6 was increased by 28%,with the production level of 18.75 g·L^-1from 50 g·L^-1xylose,and the ethanol yield was calculated to be 0.375.Simulating the real lignocellulosic acid hydrolysates, the mixture contained xylose and glucose was established.The result indicated that the fermentibility of fusant was a little better than its parents.After that,Candida shehatae stain was treated by using genome shuffling technology in order to get superior capabilities for fermentation application.Mutagenized by UV irradiation, multiple mutants were obtained as parent strains to produce complex progeny.This is impor- tant for the marked acceleration of directed evolution realized through genome shuffling.A practical combination of classical breeding such as protoplast fusion and shuffling should thus provide a means rapidly to breed populations of organisms to produce combinatorial libraries of complex progeny.