Improved bioconversion of lignocellulosic biomass by Saccharomyces cerevisiae engineered for tolerance to acetic acid

Lignocellulosic biomass has considerable potential for the production of fuels and chemicals as a promising alternative to conventional fossil fuels. However, the bioconversion of lignocellulosic biomass to desired products must be improved to reach economic viability. One of the main technical hurdles is the presence of inhibitors in biomass hydrolysates, which hampers the bioconversion efficiency by biorefinery microbial platforms such as Saccharomyces cerevisiae in terms of both production yields and rates. In particular, acetic acid, a major inhibitor derived from lignocellulosic biomass, severely restrains the performance of engineered xylose‐utilizing S. cerevisiae strains, resulting in decreased cell growth, xylose utilization rate, and product yield. In this study, the robustness of XUSE, one of the best xylose‐utilizing strains, was improved for the efficient conversion of lignocellulosic biomass into bioethanol under the inhibitory condition of acetic acid stress. Through adaptive laboratory evolution, we successfully developed the evolved strain XUSAE57, which efficiently converted xylose to ethanol with high yields of 0.43–0.50 g ethanol/g xylose even under 2–5 g/L of acetic stress. XUSAE57 not only achieved twofold higher ethanol yields but also improved the xylose utilization rate by more than twofold compared to those of XUSE in the presence of 4 g/L of acetic acid. During fermentation of lignocellulosic hydrolysate, XUSAE57 simultaneously converted glucose and xylose with the highest ethanol yield reported to date (0.49 g ethanol/g sugars). This study demonstrates that the bioconversion of lignocellulosic biomass by an engineered strain could be significantly improved through adaptive laboratory evolution for acetate tolerance, which could help realize the development of an economically feasible lignocellulosic biorefinery to produce fuels and chemicals.     

Stress tolerance of pressure-shocked Saccharomyces cerevisiae

Pressure shock treatment induced synthesis of heat shock protein (hsp104) and tolerance against various stresses such as high temperature, high pressure and high concentration of ethanol in Saccharomyces cerevisiae. The optimum pressures that induced maximal tolerance against these stresses were in the range of 50–75 MPa and depended on the type of stress. However, pressure shock did not stimulate trehalose production in the cells. © Rapid Science Ltd. 1998     

Efficient screening of environmental isolates for Saccharomyces cerevisiae strains that are suitable for brewing

We developed an efficient screening method for Saccharomyces cerevisiae strains from environmental isolates. MultiPlex PCR was performed targeting four brewing S. cerevisiae genes (SSU1, AWA1, BIO6, and FLO1). At least three genes among the four were amplified from all S. cerevisiae strains. The use of this method allowed us to successfully obtain S. cerevisiae strains.     

Isoprene hydrocarbons production upon heterologous transformation of Saccharomyces cerevisiae

Aims: Isoprene (2‐methyl‐1,3‐butadiene; C₅H₈) is naturally produced by photosynthesis and emitted in the atmosphere by the leaves of many herbaceous, deciduous and woody plants. Fermentative yeast and fungi (Ascomycota) are not genetically endowed with the isoprene production process. The work investigated whether Ascomycota can be genetically modified and endowed with the property of constitutive isoprene production. Methods and Results: Two different strategies for expression of the IspS gene in Saccharomyces cerevisiae were employed: (i) optimization of codon usage of the IspS gene for specific expression in S. cerevisiae and (ii) multiple independent integrations of the IspS gene in the rDNA loci of the yeast genome. Copy number analysis showed that IspS transgenes were on the average incorporated within about 25% of the endogenous rDNA. Codon use optimization of the Pueraria montana (kudzu vine) IspS gene (SckIspS) for S. cerevisiae showed fivefold greater expression of the IspS protein compared with that of nonoptimized IspS (kIspS). With the strategies mentioned earlier, heterologous expression of the kudzu isoprene synthase gene (kIspS) in S. cerevisiae was tested for stability and as a potential platform of fermentative isoprene production. The multi‐copy IspS transgenes were stably integrated and expressed for over 100 generations of yeast cell growth and constitutively produced volatile isoprene hydrocarbons. Secondary chemical modification of isoprene to a number of hydroxylated isoprene derivatives in the sealed reactor was also observed. Conclusion: Transformation of S. cerevisiae with the Pueraria montana var. lobata (kudzu vine) isoprene synthase gene (IspS) conferred to the yeast cells constitutive isoprene hydrocarbons production in the absence of adverse or toxic effects. Significance and Impact of the Study: First‐time demonstration of constitutive isoprene hydrocarbons production in a fermentative eukaryote operated through the mevalonic acid pathway. The work provides concept validation for the utilization of S. cerevisiae, as a platform for the production of volatile hydrocarbon biofuels and chemicals.     

Structural visualization of de novo transcription initiation by Saccharomyces cerevisiae RNA polymerase II

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酿酒酵母戊糖转运蛋白及C6/C5共代谢菌株的研究进展

充分利用木质纤维素中的糖分是提高以此类生物质为原料生产二代燃料乙醇经济盈利性的基本要求,也是实现其他生物基化学品规模化生产的基础。传统的乙醇生产微生物酿酒酵母Saccharomyces cerevisiae具有独特的生产性能及内在优势,是备受关注的底盘细胞,但其不能有效地利用戊糖。利用代谢工程、合成生物学策略,对二代燃料乙醇生产专用酿酒酵母的精准构制持续研究了30余年,已明显改善了其对木糖/葡萄糖的乙醇共发酵能力。近年来关注点集中在早期忽略的限速步骤即糖转运环节的研究上,以期实现不同糖分各行其道、高效专一性转运蛋白各行其责的二代燃料乙醇生产特种酿酒酵母所需的糖转运理想状态。文中主要综述了酿酒酵母戊糖转运蛋白的研究进展,及酿酒酵母的木糖和L-阿拉伯糖代谢工程的研究现状。     

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。     

Comparisons of five Saccharomyces cerevisiae strains for ethanol production from SPORL‐pretreated lodgepole pine

The performances of five yeast strains under three levels of toxicity were evaluated using hydrolysates from lodgepole pine pretreated by Sulfite Pretreatment to Overcome the Recalcitrance of Lignocelluloses (SPORL). The highest level of toxicity was represented by the whole pretreated biomass slurry, while intermediate toxicity was represented by the hydrolysate with partial loading of pretreatment spent liquor. The zero toxicity was represented using the enzymatic hydrolysate produced from thoroughly washed SPORL lodgepole pine solids. The results indicate that strains D5A and YRH400 can tolerate the whole pretreated biomass slurry to produce 90.1 and 73.5% theoretical ethanol yield. Strains Y1528, YRH403, and FPL450 did not grow in whole hydrolysate cultures and were observed to have lower ethanol productivities than D5A and YRH400 on the hydrolysate with intermediate toxicity. Both YRH400 and YRH403 were genetically engineered for xylose fermentation but were not able to consume xylose efficiently in hydrolysate. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1076–1083, 2014     

Effects of different growth forms of Mucor indicus on cultivation on dilute-acid lignocellulosic hydrolyzate, inhibitor tolerance, and cell wall composition

The dimorphic fungus Mucor indicus was grown in different forms classified as purely filamentous, mostly filamentous, mostly yeast-like and purely yeast-like, and the relationship between morphology and metabolite production, inhibitor tolerance and the cell wall composition was investigated. Low concentrations of spores in the inoculum with subsequent aeration promoted filamentous growth, whereas higher spore concentrations and anaerobic conditions promoted yeast-like growth. Ethanol was the main metabolite with glycerol next under all conditions tested. The yields of ethanol from glucose were between 0.39 and 0.42 g g⁻¹ with productivities of 3.2–5.0 g l⁻¹ h⁻¹. The ethanol productivity of mostly filamentous cells was increased from 3.9 to 5.0 g l⁻¹ h⁻¹ by the presence of oxygen, whereas aeration of purely yeast-like cells showed no such effect. All growth forms were able to tolerate 4.6 g l⁻¹ furfural and 10 g l⁻¹ acetic acid and assimilate the sugars, although with different consumption rates. The cell wall content of the fungus measured as alkali insoluble materials (AIM) of the purely yeast-like cells was 26% of the biomass, compared to 8% of the pure filaments. However, the chitosan concentration of the filaments was 29% of the AIM, compared to 6% of the yeast-like cells.     

肝脏发育过程中转录因子的调控

肝脏的发育经历了一系列内胚层和中胚层之间复杂的相互作用,其中转录因子扮演着重要角色。肝脏发育主要可分为两个阶段,首先是前肠内胚层感受心脏中胚层的信号而建立响应态(competence),肝向特化基因逐渐表达并形成新生肝芽。此阶段受到转录调控网络的控制,其中FoxA家族,锌指结构转录因子GATA4/6,同源结构域因子Hhex、Onecut1、Onecut2和Prox1发挥了重要的作用。其次是肝脏内细胞群体如肝细胞和胆管细胞的分化成熟阶段。这个过程的完成主要受肝富集转录因子HNF1α、HNF4、HNF6和C/EBPα的调控。本文概述了肝脏发育中复杂的转录调控网络及其发挥的作用。     

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...     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy-brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

植物含油量相关转录因子WRINKLED1的研究进展

植物油具有重要的食用和工业价值,对其需求越来越大。提高作物种子含油量育种一直在进行,与通过单一限速酶的遗传工程法相比,利用转相关转录因子基因改造植物脂肪代谢过程是更好选择之一。介绍植物含油量相关转录因子WRINKLED1的发现、结构、功能、调控、基因工程和聚类分析等,并对其应用前景进行展望。     

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hybrid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.