木质纤维素预处理抑制物产生及脱除方法的研究进展

利用纤维素酶将木质纤维素降解成可发酵性糖,然后发酵生产氢气、乙醇、丁醇等生物燃料及高附加值产品,是当今全球研究的热点。预处理是生物质转化过程中至关重要的步骤,而预处理过程中产生的抑制物对木质纤维素后续的酶解和发酵微生物有负面影响。因此了解预处理方法及其过程中产生的抑制物及脱除方法是能否高效转化生物质的基础。文中首先介绍了木质纤维素常用的两类预处理方法即化学法和物理化学法。随后阐述了不同抑制物的产生及其抑制机制,并重点介绍了多种脱毒方法。最后展望了脱除木质纤维素预处理抑制物的研究趋势:应用交联聚乙烯亚胺和金属有机骨架化合物等新型材料脱除抑制物或通过基因工程、代谢工程技术等构建抑制物耐受性菌株等。     

木质纤维素水解副产物对乙醇发酵的影响及应对措施

利用木质纤维素原料生产燃料乙醇,预处理是必需的环节,在预处理过程中会产生一些对微生物生长和发酵有抑制作用的化合物,这些抑制剂可分为3类:呋喃醛类化合物、酚类化合物和弱酸。近些年来,关于抑制剂的研究取得了一些重要的研究进展。介绍了各种抑制剂的产生、作用机理及近几年的相关研究成果;阐述了应对抑制剂影响的多种措施,包括采用新型的预处理方式在源头上控制抑制剂的产生、发酵前利用有效的脱毒方法减少抑制剂的浓度、耐受抑制剂的菌株的选育、通过发酵过程的控制有效减少抑制物的毒害作用等。目前,大量研究集中在抑制剂耐受性菌株的开发上,总结了通过诱变、驯化、代谢工程改造等方法以及这些方法的联用选育耐受性菌株的研究成果及进展,指出对微生物进行代谢工程改造是克服抑制剂对乙醇发酵影响的最有前途的方法,并对将来的研究方向进行了展望,以期为该领域研究人员提供方法的参考。     

大肠杆菌对木质纤维素水解液抑制物的胁迫耐受性

木质纤维素类生物质是前景广阔的化石原料替代品，其生物炼制可生产生物能源、生物基化学品和生物材料等多种产品，可降低碳排放，有助于实现“双碳”目标，因此受到越来越多的关注。然而，木质纤维素生物炼制需要经过预处理、微生物发酵和产物纯化等多个步骤，其中，预处理过程产生的多种化合物抑制微生物的细胞生长和发酵性能，是制约生物转化效率的瓶颈之一。大肠杆菌是木质纤维素生物炼制常用的宿主，被广泛应用于多种化合物的生产，研究其对木质纤维素水解液中抑制物的耐受性，对于提高木质纤维素生物炼制效率具有重要意义。本文首先介绍了木质纤维素的主要成分和基本结构，对木质纤维素的预处理方法以及预处理后水解液中的主要抑制物种类进行了简单阐述；随后，总结了木质纤维素水解液中几类主要抑制物呋喃类、羧酸类和酚类对大肠杆菌细胞的毒性，以及大肠杆菌对上述抑制物的胁迫响应机制和基于机制的菌株改造靶点；最后，综述了提高大肠杆菌对上述抑制物的胁迫耐受性的菌株改造策略，包括随机突变、实验室适应性进化和组学辅助的理性设计等，为利用代谢工程构建用于木质纤维素生物炼制的高效大肠杆菌菌株提供参考。     

适于二代燃料乙醇生产酿酒酵母的精准构制

以木质纤维素为原料的二代燃料乙醇工业生产对发酵微生物的基本要求,一是可对木质纤维素组分中的全糖发酵,二是对预处理过程产生的毒性物质具有高耐受性。酿酒酵母(Saccharomyces cerevisiae)是具有优良生产性能的传统乙醇发酵生产菌株,是适合包括二代燃料乙醇等生物基化合物转化的理想底盘细胞。近30年来,利用理性代谢工程改造、非理性适应性进化以及新兴起的合成生物学等策略,对酿酒酵母进行精准构制,极大地提高了其二代燃料乙醇生产的产业化性能。综述了适于二代燃料乙醇生产酿酒酵母精准构制过程中的己糖和戊糖代谢途径工程、辅酶工程、糖转运蛋白、抗性元件发掘以及产业化推进等方面的研究进展。     

抑制剂耐受性酵母底盘细胞的设计与构建

纤维素乙醇是一种低碳清洁的绿色能源,可与传统石油基液体燃料混合使用,具备广阔的应用前景.纤维素乙醇的生产历经木质纤维素预处理、糖化和酿酒酵母发酵等工艺,而预处理过程会产生多种副产物,显著抑制酵母细胞的生长速率和发酵性能.因此,构建抑制剂耐受性酵母底盘细胞,有助于提高纤维素乙醇的生产效率,降低生产成本.针对抑制剂耐受性酵...     

过表达长链鞘氨醇激酶基因LCB4提高酿酒酵母抑制物耐受性

木质纤维素预处理过程中产生的有毒副产物严重影响了纤维素乙醇发酵,提高酿酒酵母抑制物耐受性是提高纤维素乙醇发酵效率的有效方法。文中通过过表达LCB4基因,研究了重组菌株S288C-LCB4在乙酸、糠醛和香草醛胁迫下的细胞生长和乙醇发酵性能。结果表明,LCB4过表达菌株在分别含有10 g/L乙酸、1.5 g/L糠醛和1 g/L香草醛的平板中生长均优于对照菌株;在分别含有10 g/L乙酸、3 g/L糠醛和2 g/L香草醛的液体乙醇发酵过程中,重组菌株S288C-LCB4乙醇发酵产率分别为0.85 g/(L·h)、0.76 g/(L·h)和1.12 g/(L·h),比对照菌株提高了34.9%、85.4%和330.8%;且糠醛和香草醛胁迫下发酵时间分别缩短了30 h和44 h。根据发酵终点发酵液代谢物分析发现重组菌株比对照菌株产生了更多甘油、海藻糖和琥珀酸,这些物质有利于增强菌株的抑制物耐受性。综上所述,LCB4基因过表达可显著提高酿酒酵母S288C在乙酸、糠醛和香草醛胁迫下的乙醇发酵性能。     

过表达tRNA基因tL(CAA)K提高酿酒酵母乙酸耐受性

乙酸是木质纤维素类生物质水解液中的常见毒性抑制物,选育乙酸耐受性好的酿酒酵母菌株,有利于高效利用木质纤维素类生物质,发酵生产生物燃料和生物基化学品。目前对酿酒酵母抗逆性的研究多集中在转录水平,但对转运RNA (Transfer RNA,tRNA) 在耐受性中的作用研究较少。在对酿酒酵母抗逆性研究过程中发现,一些转运RNA基因在耐受性好的酿酒酵母菌株中转录明显上调。本文深入分析了精氨酸tRNA基因tR(ACG)D和亮氨酸tRNA基因tL(CAA)K过表达对酿酒酵母耐受木质纤维素水解液的影响。结果表明,在4.2 g/L乙酸胁迫条件下进行乙醇发酵时,过表达tL(CAA)K的菌株生长和发酵性能均优于对照酵母菌株,乙醇生产强度比对照菌株提高了29.41%,但过表达tR(ACG)D基因的菌株生长和代谢能力较对照菌株明显降低,体现了不同tRNA的不同调控作用。进一步分析发现,过表达tL(CAA)K的重组酵母菌株乙酸耐受性调控相关基因HAA1、MSN2和MSN4等胁迫耐受性相关转录因子编码基因的转录水平上调。本文的研究为选育高效利用木质纤维素资源进行生物炼制的酵母菌株提供了新的改造策略,也为进一步揭示酿酒酵母tRNA基因表达调控对抗逆性的影响提供了基础。     

过表达MRP8提高酿酒酵母乙酸耐性及乙醇发酵效率

乙酸是木质纤维素水解液中含量较多的抑制物,因此提高酿酒酵母菌株对乙酸的耐受性有助于提高纤维素乙醇生产效率。本文中,笔者利用基于CRISPR/Cas9系统的基因组编辑技术过表达了酿酒酵母(Saccharomyces cerevisiae)S288c线粒体核糖体蛋白编码基因MRP8,并比较了过表达MRP8的菌株与对照菌株的生长和发酵特性。平板耐性检测发现,MRP8过表达明显提高了菌株的乙酸胁迫耐受性;乙醇发酵结果表明,在4.8 g/L乙酸胁迫条件下,过表达菌株MRP8-3在51 h消耗全部的葡萄糖,发酵时间缩短了25 h,显著优于相同时间的对照菌株。本研究结果为构建高效纤维素乙醇发酵的酿酒酵母菌株提供了新思路。     

酵母对木质纤维素酸解物中抑制物的应答及菌株开发

木质纤维素稀酸预处理过程中产生的抑制物会干扰酵母细胞的生长和发酵。根据酵母对抑制物应答的特点,开发那些能够对抑制物原位脱毒的高耐受性菌株,是生物质乙醇转化工业可持续发展的关键。综述了木质纤维素稀酸预处理过程中抑制物的产生、分类、对酵母的影响以及酵母对其应答的特点,结合系统生物学和基因工程方法从酵母耐受的角度探讨了耐受性优势酵母菌株的开发。     

酿酒酵母纤维素乙醇统合加工(CBP)的策略及研究进展

木质纤维素乙醇的统合生物加工过程(Consolidated bioprocessing,CBP)是将纤维素酶和半纤维素酶生产、纤维素水解和乙醇发酵过程组合或部分组合,通过一种微生物完成。统合生物加工过程有利于降低生物转化过程的成本,越来越受到研究者的普遍关注。酿酒酵母Saccharomyces cerevisiae是传统的乙醇发酵菌株。介绍了影响外源基因在酿酒酵母中表达水平的因素,纤维素酶和半纤维素酶在酿酒酵母中表达研究进展及利用酿酒酵母统合加工纤维素乙醇的策略。     

Metabolic responses to ethanol in Saccharomyces cerevisiae using a gas chromatography tandem mass spectrometry-based metabolomics approach

During the fermentation process, Saccharomyces cerevisiae cells are often inhibited by the accumulated ethanol, and the mechanism of the S. cerevisiae response to ethanol is not fully understood. In the current study, a systematic analytical approach was used to investigate the changes in the S. cerevisiae cell metabolome that were elicited by treatment with various concentrations of ethanol. Gas chromatography-mass spectrometry and a multivariate analysis were employed to investigate the ethanol-associated intracellular biochemical changes in S. cerevisiae. The intracellular metabolite profiles that were found upon treatment of the cells with different concentrations of ethanol were unique and could be distinguished with the aid of principal component analysis. Furthermore, partial least-squares-discriminant analysis revealed a group classification and pairwise discrimination between the control without ethanol and ethanol treated groups, and 29 differential metabolites with variable importance in the projection value greater than 1 were identified, which was also confirmed by the subsequent hierarchical cluster analysis. The metabolic relevance of these compounds in the response of S. cerevisiae to ethanol stress was investigated. Under ethanol stress, the glycolysis was inhibited and the use of carbon sources for fermentation was diminished, which might account for the growth inhibition of S. cerevisiae cells. It was suggested that S. cerevisiae cells change the levels of fatty acids, e.g., hexadecanoic, octadecanoic and palmitelaidic acids, to maintain the integrity of their plasma membrane through decreasing membrane fluidity in the medium containing ethanol. Moreover, the increased levels of some amino acids idemtified in the cells of ethanol-treated experimental group might also confer ethanol tolerance to S. cerevisiae. These results reveal that the metabolomics strategy is a powerful tool to gain insight into the molecular mechanism of a microorganism's cellular response to environmental stress factors.     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation.

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

Determination of the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation

Considerable controversy exists concerning the intracellular concentration of ethanol in Saccharomyces cerevisiae during fermentation. This controversy results from problems in the measurement of the intracellular concentration of compounds like ethanol, which are being produced rapidly by metabolism and potentially diffuse rapidly from the cell. We used a new method for the determination of intracellular ethanol based on the exclusion of [14C]sorbitol to estimate the aqueous cell volume. This method avoided many of the technical problems in previous reports. Our results indicate that the extracellular concentrations of ethanol in fermenting suspensions of S. cerevisiae are less than or equal to those in the intracellular environment and do not increase to the high levels previously reported even during the most active stages of batch fermentation.     

海藻糖与热激蛋白在酿酒酵母耐受乙醇胁迫中的作用

在燃料乙醇发酵生产过程中,酿酒酵母经常会受到高浓度乙醇的胁迫,导致乙醇转化率和产量降低。面对高浓度乙醇的胁迫,酿酒酵母也具有应对胁迫的应激机制。在对这种应激机制进行了解的基础上,如能提高酿酒酵母对乙醇的耐受性,对于燃料乙醇生产具有重要意义。在高浓度乙醇胁迫下,酿酒酵母细胞会产生一系列保护性物质,如海藻糖、热激蛋白、脯氨酸等,这些物质能够提高酿酒酵母细胞对乙醇的耐受性。海藻糖作为一种重要的碳源、能量贮藏物质,不仅能稳定细胞膜、蛋白质和核酸等大分子物质,还可增强酿酒酵母对高浓度乙醇的耐受性。此外,酿酒酵母还可以产生大量的热激蛋白,增强酿酒酵母的抗逆性。从海藻糖和热激蛋白在乙醇胁迫下对酿酒酵母细胞保护作用的研究方面进行了综述,并对存在的问题进行了讨论与展望。     

酿酒酵母在纤维素乙醇生产中对毒性化合物的耐受机理研究进展

利用木质纤维素生产燃料乙醇的过程中,前期预处理所产生的抑制剂会影响酵母的正常生长和后续的发酵过程。为减小抑制剂的影响所采取的一些脱毒策略往往造成糖的损失和生产成本的增加,这在实际生产与经济上是不可行的。因此,具有强的抑制剂耐受性的酿酒酵母菌株对于提高纤维素乙醇产率是十分重要的。近十年来,对于酿酒酵母胁迫耐受机制的研究取得了一些重要的进展,着重介绍目前酿酒酵母对抑制剂耐受机制的研究现状,包括一些关键性基因的表达及代谢通路过程分析等。同时也介绍一些应对抑制剂提高酵母发酵能力的措施。     

酿酒酵母X330高浓度发酵时耐酒精性能的初步研究

在完全合成培养基条件下,就渗透压保护剂和营养物质对一株产高浓度酒精的酿酒酵母X330高浓度发酵时耐酒精性能的影响进行了初步研究。结果表明,与渗透压相比,营养缺乏对酿酒酵母高浓度发酵时酒精耐受性能可能起着更为关键和重要的作用。发酵培养基中各营养元素对耐酒精性能的影响不同,由高到低的顺序是酵母抽提物>蛋白胨>硫酸镁>维生素C=磷酸二氢钾>氯化钙=硫酸铵。渗透压保护剂(甘氨酸和脯氨酸)能有效提高菌体酒精耐受性能。当甘氨酸添加浓度为20mmol/L或脯氨酸添加浓度为10mmol/L时,发酵终点酒精浓度最高,菌体于30℃在18%(V/V)酒精冲击下的存活率最大,且均高于对照组(未添加甘氨酸且未添加脯氨酸)水平,但甘氨酸的促进作用强于脯氨酸。     

Fermentation of cellodextrins to ethanol using mixed-culture fermentations

The potential for enhancing ethanol production from cellodextrins by employing mixed-culture (Candida wickerhamii-Saccharomyces cerevisiae) fermentations was investigated. Initially, ethanol production was monitored in fermentation medium containing 50 g/L glucose plus 45 g/L cellobiose. Inoculum levels and times of inoculum addition were varied. Of the conditions tested, the most rapid rates of ethanol formation occurred in fermentations in which either C. wickerhamii and S. cerevisiae were coinoculated at a ratio of 57 : 1 cell/mL or in fermentations in which a 10-fold-greater S. cerevisiae inoculum was added to a pure culture C. wickerhamii fermentation after 1 day incubation. These conditions were used to attempt to enhance fermentations in which cellodextrins produced by trifluoroacetic acid hydrolysis of cellulose served as the sole carbon source. Cellodextrins that were not further purified after cellulose hydrolysis contained compounds that were slightly inhibitory to C. wickerhamii. In this case the mixed-culture fermentations produced 12-45% more ethanol than a pure culture C. wickerhamii fermentation. However, if the substrate was treated with Darco G-60 charcoal, the toxic materials were apparently removed and the pure culture C. wickerhamii fermentations performed as well as the mixed-culture fermentations.     

Optimization of ethanol production in Saccharomyces cerevisiae by metabolic engineering of the ammonium assimilation

Ethanol is still one of the most important products originating from the biotechnological industry with respect to both value and amount. In addition to ethanol, a number of byproducts are formed during an anaerobic fermentation of Saccharomyces cerevisiae. One of the most important of these compounds, glycerol, is produced by yeast to reoxidize NADH, formed in synthesis of biomass and secondary fermentation products, to NAD+. The purpose of this study was to evaluate whether a reduced formation of surplus NADH and an increased consumption of ATP in biosynthesis would result in a decreased glycerol yield and an increased ethanol yield in anaerobic cultivations of S. cerevisiae. A yeast strain was constructed in which GLN1, encoding glutamine synthetase, and GLT1, encoding glutamate synthase, were overexpressed, and GDH1, encoding the NADPH-dependent glutamate dehydrogenase, was deleted. Hereby the normal NADPH-consuming synthesis of glutamate from ammonium and 2-oxoglutarate was substituted by a new pathway in which ATP and NADH were consumed. The resulting strain TN19 (gdh1-A1 PGK1p-GLT1 PGK1p-GLN1) had a 10% higher ethanol yield and a 38% lower glycerol yield compared to the wild type in anaerobic batch fermentations. The maximum specific growth rate of strain TN19 was slightly lower than the wild-type value, but earlier results suggest that this can be circumvented by increasing the specific activities of Gln1p and Glt1p even more. Thus, the results verify the proposed concept of increasing the ethanol yield in S. cerevisiae by metabolic engineering of pathways involved in biomass synthesis.     

Thermotolerant Kluyveromyces marxianus and Saccharomyces cerevisiae strains representing potentials for bioethanol production from Jerusalem artichoke by consolidated bioprocessing

Thermotolerant inulin-utilizing yeast strains are desirable for ethanol production from Jerusalem artichoke tubers by consolidated bioprocessing (CBP). To obtain such strains, 21 naturally occurring yeast strains isolated by using an enrichment method and 65 previously isolated Saccharomyces cerevisiae strains were investigated in inulin utilization, extracellular inulinase activity, and ethanol fermentation from inulin and Jerusalem artichoke tuber flour at 40?°C. The strains Kluyveromyces marxianus PT-1 (CGMCC AS2.4515) and S. cerevisiae JZ1C (CGMCC AS2.3878) presented the highest extracellular inulinase activity and ethanol yield in this study. The highest ethanol concentration in Jerusalem artichoke tuber flour fermentation (200?g?L(-1)) at 40?°C achieved by K. marxianus PT-1 and S. cerevisiae JZ1C was 73.6 and 65.2?g?L(-1), which corresponded to the theoretical ethanol yield of 90.0 and 79.7?%, respectively. In the range of 30 to 40?°C, temperature did not have a significant effect on ethanol production for both strains. This study displayed the distinctive superiority of K. marxianus PT-1 and S. cerevisiae JZ1C in the thermotolerance and utilization of inulin-type oligosaccharides reserved in Jerusalem artichoke tubers. It is proposed that both K. marxianus and S. cerevisiae have considerable potential in ethanol production from Jerusalem artichoke tubers by a high temperature CBP.     

Deletion of the GRE3 aldose reductase gene and its influence on xylose metabolism in recombinant strains of Saccharomyces cerevisiae expressing the xylA and XKS1 genes.

Saccharomyces cerevisiae ferments hexoses efficiently but is unable to ferment xylose. When the bacterial enzyme xylose isomerase (XI) from Thermus thermophilus was produced in S. cerevisiae, xylose utilization and ethanol formation were demonstrated. In addition, xylitol and acetate were formed. An unspecific aldose reductase (AR) capable of reducing xylose to xylitol has been identified in S. cerevisiae. The GRE3 gene, encoding the AR enzyme, was deleted in S. cerevisiae CEN.PK2-1C, yielding YUSM1009a. XI from T. thermophilus was produced, and endogenous xylulokinase from S. cerevisiae was overproduced in S. cerevisiae CEN.PK2-1C and YUSM1009a. In recombinant strains from which the GRE3 gene was deleted, xylitol formation decreased twofold. Deletion of the GRE3 gene combined with expression of the xylA gene from T. thermophilus on a replicative plasmid generated recombinant xylose utilizing S. cerevisiae strain TMB3102, which produced ethanol from xylose with a yield of 0.28 mmol of C from ethanol/mmol of C from xylose. None of the recombinant strains grew on xylose.