Inhibitory Mechanism Of Pichia Stipitis To Three Inhibitors In Lignocellulose Hydrolysates

With the depletion of non-renewable resources such as petroleum,fuel ethanol produced from lignocellulosic materials has become a hot topic.The key techniques contain: pretreatment of lignocellulose,enzyme hydrolysis,and sugars fermentation to ethanol.In terms of sugars fermentation,the main problem is pentose fermentation.The pentose fermentation process is complicated,and the fermentation time and ethanol yield are low.Additionally,during the process of pretreatment of lignocellulose,inhibitors which are produced and will inhibit the enzymatic hydrolysis and ethanol fermentation.Therefore,in this study the complex among different ferementation stages during yeast strain pentose fermentation were analyzed on metabolite,protein,mRNA,and lipid levels.The aims of current work were to evaluate how the intracellular metabolites of P.stipitis were affected under three representative inhibitors(acetic acid 2.0 g/L,5-HMF 1.0 g/L,and vanillin 0.5 g/L).The main results are as follows:(1)When the three combined inhibitors were added the lag phase of the yeast prolonged to 28 h,14 times longer than the yeast without inhibitors.Inhibitors slowed down the growth rate of yeast cells.Comparative metabolomics analysis revealed that three conbined inhibitors has the synergistic effect on the cell growth.The metabolomic analysis during lag and middle exponential phase revealed that,metabolites particularly those related fatty acids and amino acids changed dramatically.(2)The response mechanism of P.stipitis to three combined inhibitors was studied by proteomics technology.The combination of the three inhibitors reduced the glycolytic activity,which may be associated with the inhibitory effects of the three inhibitors on yeast growth and produce more energy to resist the inhibitors.Yeast cells adaptively increased the expression of the TCA cycle proteins in the presence of the three inhibitors.Proteins related to energy metabolism was increased obviously in the presence of three combined inhibitors.In addition,protein expression associated with unfolded protein was increased.Moreover,most unfoldable proteins are heat shock proteins,indicating that heat shock protein protected the translation process.(3)Transcriptome analysis was performed on the response of P.stipitis to three combined inhibitors.Genes involved in carbohydrate metabolism were observed to be significantly down-regulated in the presence of three combined inhibitors in both lag and middle exponential phases.In order to survive under stress conditions,the yeast cells activated oxidative phosphorylation of the ATP synthesis pathway.The synthesis and metabolism of lipids were inhibited dramatically.INO1,YBO9,FAA24,FAA4,and FAA22,involved in long chain fatty acids synthesis,were upregulated in the presence of inhibitors.Most of genes associated with ribosomal synthesis were up-regulated after the adding of three combined inhibitors.However,the expression of ribosomal genens were down-regulated when in the inhibitor free condotions.(4)Phospholipids extracts obtained from yeast cells were analyzed both in negative and positive modes.A total of 80 phospholipids were identified,and most the potential biomarkers were unsaturated lipids with long chain length,which indicated that under inhibitory stress P.stipitis tends to form long chain and unsaturated lipids for self-protection.However,samples from inhibitor-plus condition during the lag phase possessed the lowest PI/PS value about 1.4%.In other words,the changes in the PC/PE ratios with inhibitors were coincidence with the changes of in xylose utilization.The change in the saturation levels of phospholipids in the presence of stressors increases membrane fluidity during the lag phase.Which make it easier for the inhibitor to throught into the yeast cells and inhibit the cell.