Study On The Tolerance Mechanism Of Yeast To Furfural Inhibitors During Xylitol Fermentation

Lignocellulose is the most abundant and promising resource for the production of fuels and bio-based chemicals.In order to release the sugars from lignocellulose,the lignocellulose must be pretreated due to its high refractory nature to degradation.However,various degradation by-products including 2-furancarboxaldehyde(furfural),5-hydroxymethyl-2-furancarboxaldehyde,organic acids and phenols,which negatively affect microbial metabolism during fermentation,severely inhibit microbial growth and fermentation.Furfural is one of the most typical inhibitors of pretreated hemicellulose hydrolysis products,reducing yeast cell growth and xylitol production.In this study,a Candida tropicalis Y31-N strain capable of high furfural concentrations was obtained through a domestication method with a gradient increase in furfural concentration;and the response of the parental and tolerant strains to furfural inhibitors was compared at the transcriptional and metabolic levels to investigate the tolerance mechanism of the tolerant strain in response to furfural stress.Candida tropicalis was used as the original strain.A tolerant strain Y31-N was screened by the method of domestication with gradient increase of furfural concentration,which was able to tolerate 6 g/L furfural.The fermentation culture was carried out in high sugar medium containing 6 g/L furfural,and the ability of the strain to tolerate furfural was substantially increased compared with the original strain,and its final xylitol yield was not significantly affected,with a xylitol yield of 30.8 g/L.It degraded furfural completely within6 h,showing a comparable ability of furfural tolerance during fermentation.By comparing and analyzing the response of parental and tolerant strains to furfural stress at the transcriptional level and validating the transcriptome results by a quantitative real-time PCR(q PCR),it was found that furfural inhibitors caused oxidative stress,osmotic stress and unfolded protein stress in Candida tropicalis.Compared to the parental strain,the tolerant strain was able to resist furfural stress through enhanced transcript levels of genes related to proteasome and peroxisome,endoplasmic reticulum protein processing,ubiquitin-mediated protein hydrolysis processes,and biosynthesis of unsaturated fatty acids for rapid clearance of intracellular error protein accumulation and maintenance of cell membrane integrity for self-protection.In addition,the tolerant strain resist furfural stress through higher energy metabolic viability and a rapidly response to NAD(P)H and ATP depletion under stressful environmental conditions,giving it comparable ability of furfural tolerance during fermentation.In this study,In this study,comparative metabolomic studies of parental and tolerant strains in the presence and absence of furfural(5 g/L)stress showed that differences in metabolism between the tolerant strain Y31-N compared to the parental strain Y-3themselves determined,to some extent,the differences in their furfural tolerance.Under furfural stress,the highly up-regulated metabolic pathways of glutathione,glycine,serine and threonine in the tolerant strain Y31-N compared to the parental strain Y-3 could directly affect the cellular levels of NAD(P)~+,NAD(P)H and ATP,thus protecting cells from furfural damage;the key cellular pathways of valine,leucine,isoleucine,pantothenic acid and Co A biosynthesis could help regulate fatty acid saturation to control cell membrane fluidity,thus ensuring the integrity of cell membrane function against furfural stress;the higher TCA cycle,glycolytic and phenylalanine metabolic activity in the tolerant strain Y31-N facilitated adequate production of intermediate substrates and energy,which contributed to the greater furfural tolerance of the tolerant strain compared to the parental strain.