【作者】 王莉；

【导师】 李炳志；

【作者基本信息】 天津大学 ， 制药工程， 2023， 博士

【摘要】 木质纤维素乙醇工业生产主流工艺包括四个方面:预处理、酶解、发酵和分离。其中预处理过程中产生的有毒物质以及水解产物中含量仅次于葡萄糖的单糖——木糖是阻碍乙醇生产的两个主要因素。多种多样的抑制剂对细胞产生协同抑制作用从而影响发酵效率,发酵菌株利用木糖的能力低进而导致木质纤维素生物质转化效率低,因此增强发酵菌株的抑制剂耐受性和木糖利用能力是解决抑制剂和木糖利用问题的关键。本研究基于全局调控策略,针对酿酒酵母对复合抑制剂(糠醛、乙酸和苯酚)的耐受问题和对木糖的利用问题,研究了工程化异源全局调控因子IrrE对菌株的调控效果和调控机制。针对原核生物调控因子IrrE在真核生物酿酒酵母中的调控机制问题,从转录水平上系统解析IrrE及其突变体I24的表达高酵母菌株复合抑制剂耐受性的分子机制,结合代谢物检测和关键基因过表达验证手段,证实了IrrE及其突变体I24在酿酒酵母中发挥着全局调控的作用。IrrE在细胞内的表达主要通过调控与活性氧清除、压力保护剂糖原和海藻糖积累、能量存储、ATP产生以及NADPH再生等相关的基因的转录水平,来增强菌株的抑制剂耐受性。I24主要通过调节和转录激活因子/转录因子、能量存储、细胞膜和细胞内环境稳定以及核糖体发生等相关的基因的转录水平,增强抑制剂环境下菌株的发酵效率。IrrE和I24在酿酒酵母中调控机制的差异暗示了定向进化赋予了I24不同的调控机制,同时也反映了IrrE的可塑性。对三个能明显增强酵母菌株耐受性的IrrE突变体进行单位点突变分析,结合定点突变策略,构建得到的突变体L65P,I103T,E119V,L160F,P162S,M169V,V204A,R244G,Base824 Deletion,V299A以及A300V对高菌株的耐受性有促进作用。针对酿酒酵母对复合抑制剂的耐受和对木糖的利用问题,基于真核生物相较于原核生物细胞结构区室化分布更加明显的特点,结合定位策略,研究了IrrE的亚细胞定位表达对菌株的调控效果和调控机制。构建得到的IrrE细胞质、线粒体和细胞核定位表达的三种菌株在木糖发酵环境中的木糖消耗速率是对照菌株的1.3倍,在加入30%FAP的木糖培养环境中的木糖消耗速率是对照菌株的1.2倍。转录组分析表明,木糖发酵环境比含有50%FAP的木糖发酵环境更能引发IrrE的广泛调节作用,在木糖培养环境中,三种定位形式的IrrE的表达在酿酒酵母细胞内引起的调控作用非常相似,IrrE的亚细胞定位表达增强了和木糖代谢、糖转运、膜蛋白、转运蛋白、转座子、转录因子、抗氧化相关的酶以及多种nc RNA相关的基因的转录水平。在含有50%FAP的木糖培养环境中,只有IrrE的细胞质表达对木糖代谢相关路径有一些调控作用,且IrrE的细胞质表达使细胞内和糖转运、膜蛋白、转运蛋白、热休克蛋白以及ATP合酶相关的基因的转录水平显著上调。进一步开展了IrrE在酿酒酵母中的组合亚细胞定位表达,高了细胞对木糖的利用能力和对复合抑制剂的耐受能力,使细胞在含有40%FAP的木糖平板上的存活率高了10~3倍。GRE策略和定位手段相结合筛选得到的突变体,在50%FAP的木糖平板上,与原始菌株Ywl33/IrrE-SV40相比,突变菌株Ywl33/I194-SV40的细胞存活率高了100倍;与原始菌株Ywl33/MLS-IrrE相比,突变菌株Ywl33/MLS-I125的细胞存活率高了100倍;对照菌在这种环境中不能存活。发酵能力表征发现,在50%FAP的木糖发酵环境中,最优细胞核定位突变菌Ywl33/I194-SV40的木糖消耗速率是对照菌株的1.49倍,最终乙醇滴度是对照菌株Ywl33/p RS416的4.14倍,乙醇生产速率是对照菌株Ywl33/p RS416的6.78倍,四个线粒体定位突变菌的木糖消耗速率是对照菌株的1.4倍左右,最终乙醇滴度是对照菌株Ywl33/p RS416的3倍以上,乙醇生产速率是对照菌株Ywl33/p RS416的6倍左右。进一步的突变位点分析暗示了三个结构域协同作用,赋予IrrE及其突变体强大的调控作用,增强酿酒酵母的木糖利用能力及复合抑制剂耐受性。IrrE对酿酒酵母的调控作用为高菌株抑制剂耐受性及木糖利用能力供指导。更多还原

【Abstract】 The industrial production process of lignocellulosic ethanol encompasses four key as-pects:pretreatment,enzymatic hydrolysis,fermentation,and separation.Among these,two primary factors that impede ethanol production are the toxic substances generated during the pretreatment process and the xylose present in the hydrolysis products,second only to glucose in terms of content.Various inhibitors have a synergistic inhibitory effect on cells,which detrimentally impacts fermentation efficiency.The limited ability of fermenting strains to utilize xylose results in low efficiency of lignocellulosic biomass conversion.Con-sequently,enhancing the inhibitor tolerance and xylose utilization ability of fermenting strains is crucial for resolving the issues associated with inhibitors and xylose utilization.Based on the global regulatory strategy,this study examined the regulatory impact and mechanism of the engineered heterologous global regulator IrrE on Saccharomyces cere-visiae(S.cerevisiae),with a focus on addressing the issues related to tolerance towards complex inhibitors(furfural,acetic acid,and phenol,FAP)and the utilization of xylose.With regards to elucidating the regulatory mechanism of the prokaryotic regulator IrrE in the eukaryotic organism S.cerevisiae,the transcriptome analysis was performed on the molecular mechanism that the yeast tolerance to complex inhibitor was enhanced by exog-enous global regulatory factor IrrE and its mutant I24.For further selection,metabolite de-tection and key genes overexpression verification experiments were also carried out.Ac-cordingly,the global regulatory role of IrrE and its mutant I24 in S.cerevisiae were con-firmed.Specifically,the transcription levels of genes associated with the reactive oxygen species(ROS)scavenging enzymes,glycogen and trehalose accumulation,energy storage,ATP production,and NADPH regeneration were found to be modulated by IrrE to enhance the inhibitor tolerance of BY4742.Unlike IrrE,I24 primarily enhances the fermentation efficiency of strains under inhibitory conditions by regulating the transcription levels of genes related to transcription activators/factors,energy storage,cell membrane,intracellular environment stability,and ribosome occurrence.The disparities in the regulatory mecha-nisms of IrrE and I24 in BY4742 indicate that directed evolution has endowed I24 with distinct regulatory mechanisms,while also reflecting the adaptability of IrrE.Single point mutation analysis found that,compared with the control strain,the constructed mutants L65P,I103T,E119V,L160F,P162S,M169V,V204A,R244G,Base 824 Deletion,V299A,and A300V showed improved inhibitor tolerance.Directing attention towards the challenge of cultivating increased tolerance to intricate inhibitory substances and optimizing the utilization of xylose within S.cerevisiae,an explo-ration was conducted on the regulatory impact and mechanism of the subcellular localiza-tion expression of IrrE in yeast.This investigation employed localization strategies that cap-italized on the more conspicuous and compartmentalized distribution of cellular structures in eukaryotes,as juxtaposed with their prokaryotic counterparts.Three strains expressing IrrE in the cytoplasm,mitochondria,and nucleus were created to study the effect of subcel-lular localization of IrrE on strain related performance.The xylose consumption rates of these three strains in xylose fermentation environments were 1.3 times higher than those of the control strain,and 1.2 times higher in the presence of 30%FAP xylose culture environ-ment.Transcriptome analysis revealed that IrrE can elicit more extensive regulatory effects when strains in the xylose fermentation environment than in the xylose fermentation envi-ronment containing 50%FAP.In the xylose fermentation environment,the regulatory ef-fects induced by the expression of three forms of IrrE are highly similar.The subcellular localization expression of IrrE augments the transcription level of genes associated with xylose metabolism,sugar transport,membrane proteins,transport proteins,transposons,transcription factors,antioxidant-related enzymes,and various nc RNAs.In the xylose fer-mentation environment with 50%FAP,solely the cytoplasmic expression of IrrE exerts some regulatory effects on the xylose metabolism-related pathways,and the cytoplasmic expression of IrrE significantly upregulates the transcription levels of genes related to sugar transport,membrane proteins,transport proteins,heat shock proteins,and ATPase.Furthermore,the combination of subcellular localization expressions of IrrE in yeast enhances the ability of cells to utilize xylose and withstand complex inhibitors,leading to a10~3-fold increase in cell viability on xylose plates containing 40%FAP.Several mutants swere obtained by the combination of GRE strategy and localization method.Among them,on xylose plates containing 50%FAP,in comparison to the original strain Ywl33/IrrE-SV40,the cell viability of the mutant strain Ywl33/I194-SV40 increased by 100 times;compared to the original strain Ywl33/MLS-IrrE,the cell viability of the mutant strain Ywl33/MLS-I125 increased by 100 times;the control strain cannot survive in this environment.Fermen-tation capacity characterization reveals that in the xylose fermentation environment contain-ing 50%FAP,the optimal nuclear-localized mutant strain Ywl33/I194-SV40 exhibits an xylose consumption rate 1.49 times higher than that of the control strain,an final ethanol titer 4.14 times higher than that of the control strain,and an ethanol production rate 6.78times higher than that of the control strain Ywl33/p RS416.The xylose consumption rates of the four mitochondrial-localized mutant strains were about 1.4 times higher than that of the control strain Ywl33/p RS416,the final ethanol titer surpasses 3 times that of the control strain,with an ethanol production rate approximately 6 times higher than that of the control strain.Further analysis of mutation sites suggests the synergistic action of three structural domains,providing IrrE and its mutants with robust regulatory effects,enhancing the xylose utilization ability and multiple inhibitors tolerance of yeast.The regulatory role of IrrE in S.cerevisiae offers guidance for improving strain inhibitor tolerance and xylose utilization.更多还原