酿酒酵母乙酸耐性分子机制的功能基因组进展

提高工业酿酒酵母对高浓度代谢产物及原料中的毒性底物等环境胁迫因素的耐受性,对提高工业生产效率具有重要的意义。乙酸是纤维素原料水解产生的主要毒性副产物之一,其对酵母细胞的生长和代谢都具有较强的抑制作用,因此,对酿酒酵母乙酸耐性分子机制的研究可为选育优良菌种提供理论依据。近年来,通过细胞全局基因表达分析和代谢组分析,以及对单基因敲除的所有突变体的表型组研究,对酿酒酵母乙酸耐性的分子机制有了更多新的认识,揭示了很多新的与乙酸毒性适应性反应和乙酸耐性提高相关的基因。综述了近年来酿酒酵母乙酸耐性的基因组规模的研究进展,以及在此基础上构建乙酸耐性提高的工业酵母菌的代谢工程操作。结合本课题组的研究,对金属离子锌在酿酒酵母乙酸耐性中的作用进行了深入分析。未来对酿酒酵母乙酸耐性分子机理的认识及改造将深入到翻译后修饰和合成生物学等新的水平,所获得的认知,将为选育可高效进行纤维素原料生物转化、高效生产生物燃料和生物基化学品的工业酿酒酵母的菌株奠定理论基础。     

酿酒酵母乙酸耐受性相关的微卫星分子标记筛选

【目的】筛选与酵母乙酸耐受性状紧密相关的微卫星分子标记。【方法】以两株表型差异菌株YHA和YLA作为亲本构建F2代菌株共计160株,选取15个微卫星位点通过PCR方法在40株子代中扩增产物,利用SPSS 11.5软件分析耐酸性状与微卫星序列间的相关性。【结果】找到3个与乙酸耐受性性状相关的微卫星位点,其中位点14P2与酵母乙酸耐受性状有极显著的正相关性(P<0.01),15P2和15P3与酵母乙酸耐受性具有显著的负相关性(P<0.01和P<0.05);此外对于微卫星位点14P2,耐酸亲本YHA在该位点的基因片段(344 bp)在子代耐酸菌株中出现频率达到70.6%,而不耐酸亲本YLA的基因片段(331 bp)在子代不耐酸菌株中出现的频率达91.3%。【结论】微卫星14P2的等位基因在子代菌株中的遗传具有明显的偏好性,该微卫星位点与某种耐酸基因存在一定的连锁遗传,为酵母分子标记辅助育种提供了有价值的遗传标记。     

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.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Ethanol and acetic acid tolerance in free and immobilized cells of Saccharomyces cerevisiae and Acetobacter aceti

Saccharomyces cerevisiae and Acetobacter aceti cells were immobilized by entrapment in Ca-alginate or by adsorption on to preformed cellulose beads and were treated with 0-20% (v/v) ethanol and 0-10% (v/v) acetic acid. At 20% (v/v) ethanol, lethal for free yeast cells, 62-72% of the immobilized cells survived. In 10% (v/v) acetic acid, free and adsorbed Acetobacter aceti cells ceased to grow but 69% of entrapped cells survived. Cells released from the carrier showed an intermediate survival (20-60%).     

Induction of acetic acid tolerance and trehalose accumulation by added and produced ethanol in Saccharomyces cerevisiae

Both added (49.6 g/l) and produced ethanol (46.2 g/l) caused an increase in the acetic acid tolerance of Saccharomyces cerevisiaegrown in an anaerobic chemostat; added ethanol, however, to a less extent than produced ethanol. The ethanol induced acetic acid tolerance of the cells was linked with an accumulation of trehalose within the cells. These results indicate that trehalose plays a role in the ethanol induced acetic acid tolerance of S. cerevisiae.     

Development of increased acetic acid tolerance in anaerobic homoacetogens through induced mutagenesis and continuous selection

Effective methods for large scale mutagenesis of strictly anaerobic, thermophilic microorganisms were developed. Mutagenesis and selection were carried out under highly reduced conditions without using plating techniques. Ultraviolet light (u.v.) or ethyl methanesulphonate (EMS) produced similar improvements in acetic acid tolerance of Clostridium thermoaceticum. An additive effect was observed when u.v. and EMS were applied simultaneously. N-Methyl-N-nitro-N-nitrosoguanidine was ineffective.     

Improvement of xylose fermentation in respiratory-deficient xylose-fermenting Saccharomyces cerevisiae

Effective conversion of xylose in lignocelluloses is expected to reduce the production cost of second-generation biofuels significantly. The factors affecting xylose fermentation in Saccharomyces cerevisiae that express xylose reductase-xylitol dehydrogenase (XR-XDH) are studied. Although overproduction of non-oxidative pentose phosphate pathway significantly increased the aerobic-specific growth rate on xylose and slightly improved conversion of xylose to ethanol under oxygen-limited conditions, the elimination of respiration by deleting cytochrome C oxidase subunit IV gene impeded aerobic growth on xylose. However, the adaptive evolution of the respiratory-deficient strain with an NADP(+)-preferring XDH mutant in xylose media dramatically improved its xylose-fermenting ability. The specific growth rate, ethanol yield, and xylitol yield of the evolved strain on xylose were 0.06h(-1), 0.39gg(-1), and 0.13gg(-1) consumed xylose, respectively. Similar to anaerobic fermentation, the evolved strain exhibited accumulated ethanol rather than recycled it under aerobic conditions.     

Xylulose fermentation by Saccharomyces cerevisiae and xylose-fermenting yeast strains

Xylulose fermentation by four strains of Saccharomyces cerevisiae and two strains of xylose-fermenting yeasts, Pichia stipitis CBS 6054 and Candida shehatae NJ 23, was compared using a mineral medium at a cell concentration of 10 g (dry weight)/l. When xylulose was the sole carbon source and fermentation was anaerobic, S. cerevisiae ATCC 24860 and CBS 8066 showed a substrate consumption rate of 0.035 g g cells^–1 h^–1 compared with 0.833 g g cells^–1 h^–1 for glucose. Bakers' yeast and S. cerevisiae isolate 3 consumed xylulose at a much lower rate although they fermented glucose as rapidly as the ATCC and the CBS strains. While P. stipitis CBS 6054 consumed both xylulose and glucose very slowly under anaerobic conditions, C. shehatae NJ 23 fermented xylulose at a rate of 0.345 g g cells^–1 h^–1, compared with 0.575 g g cells^–1 h^–1 for glucose. For all six strains, the addition of glucose to the xylulose medium did not enhance the consumption of xylulose, but increased the cell biomass concentrations. When fermentation was performed under oxygen-limited conditions, less xylulose was consumed by S. cerevisiae ATCC 24860 and C. shehatae NJ 23, and 50%–65% of the assimilated carbon could not be accounted for in the products determined.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.     

Fermentation performance of engineered and evolved xylose-fermenting Saccharomyces cerevisiae strains

Lignocellulose hydrolysate is an abundant substrate for bioethanol production. The ideal microorganism for such a fermentation process should combine rapid and efficient conversion of the available carbon sources to ethanol with high tolerance to ethanol and to inhibitory components in the hydrolysate. A particular biological problem are the pentoses, which are not naturally metabolized by the main industrial ethanol producer Saccharomyces cerevisiae. Several recombinant, mutated, and evolved xylose fermenting S. cerevisiae strains have been developed recently. We compare here the fermentation performance and robustness of eight recombinant strains and two evolved populations on glucose/xylose mixtures in defined and lignocellulose hydrolysate-containing medium. Generally, the polyploid industrial strains depleted xylose faster and were more resistant to the hydrolysate than the laboratory strains. The industrial strains accumulated, however, up to 30% more xylitol and therefore produced less ethanol than the haploid strains. The three most attractive strains were the mutated and selected, extremely rapid xylose consumer TMB3400, the evolved C5 strain with the highest achieved ethanol titer, and the engineered industrial F12 strain with by far the highest robustness to the lignocellulosic hydrolysate.     

转醛醇酶基因差异表达影响酵母发酵木糖及耐受乙酸性能

【目的】木糖发酵是纤维素燃料乙醇生产的一个关键瓶颈,同时木质纤维素水解液中的乙酸严重抑制酿酒酵母的木糖发酵过程,因此通过基因工程手段提高菌株对木糖的利用以及对乙酸的耐受性具有重要意义。本研究以非氧化磷酸戊糖途径(PPP途径)中关键基因转醛醇酶基因(TAL1)为研究对象,探讨了3种不同启动子PTDH3、PAHP1和PUBI4,控制其表达对菌株利用木糖和耐受乙酸的影响。【方法】通过同源重组用3种启动子替换酿酒酵母基因工程菌NAPX37的TAL1基因的启动子PTAL1,再通过孢子分离和单倍体交配构建了纯合子,利用批次发酵比较了在以木糖为唯一碳源和混合糖(葡萄糖和木糖)为碳源条件下,3种启动子控制TAL1基因表达导致的发酵和乙酸耐受能力的差异。【结果】启动子PTDH3、PAHP1和PUBI4在不同程度上提高了TAL1基因的转录水平,提高了菌株对木糖的利用速率及乙酸耐受能力,提高了菌株在60 mmol/L乙酸条件下的葡萄糖利用速率。在以木糖为唯一碳源且无乙酸存在、以及混合糖为碳源的条件下,PAHP1启动子控制TAL1表达菌株的发酵结果优于PTDH3和PUBI4启动子的菌株,PAHP1启动子控制的TAL1基因的转录水平比较合适。在木糖为唯一碳源且乙酸为30 mmol/L时,PUBI4启动子控制TAL1基因表达的菌株发酵结果则优于PAHP1和PTDH3启动子菌株,此时PUBI4启动子控制的TAL1的转录水平比较合适。【结论】启动子PTDH3、PAHP1和PUBI4不同程度地提高TAL1基因的表达,在不同程度上改善了酵母菌株的木糖发酵速率和耐受乙酸性能,改善程度受发酵条件的影响。     

High enthalpy and low enthalpy death in Saccharomyces cerevisiae induced by acetic acid

Acetic acid at concentrations as may occur during vinification and other alcoholic yeast fermentations induced death of glucose-grown cell populations of Saccharomyces cerevisiae IGC 4072 at temperatures at which thermal death was not detectable. The Arrhenius plots of specific death rates with various concentrations of acetic acid (0-2%, w/v) pH 3.3 were linear and could be decomposed into two distinct families of parallel straight lines, indicating that acetic acid induced two types of death: (1) High enthalpy death (HED) predominated at lower acetic acid concentrations (> 0.5%, w/v) and higher temperatures; its enthalpy of activation (DeltaH( not equal)) approached that of thermal death (12.4 x 10(4) cal/mol); (2) Low enthalpy death (LED) predominated at higher acetic acid concentrations and lower temperatures with DeltaH( not equal) of 3.9 x 10(4) cal/mol. While the DeltaH( not equal) values for HED induced by acetic acid were similar with those reported earlier for HED induced by other fermentation endproducts, the values for the entropy coefficients were different: 127-168 entropy units mol(-1)L for acetic acid as compared with 3.6-5.1 entropy units mol(-1)L for ethanol, which agreed with experimental results indicating that acetic acid is over 30-times more toxic than ethanol with respect to yeast cell viability at high process temperatures.     

Copper tolerance in Saccharomyces cerevisiae

A commercial strain of Saccharomyces cerevisiae was serially cultured in media containing copper up to a final concentration of 10 mmol l^-1. This copper-tolerant subculture was assessed for its capacity to accumulate further quantities of copper. It was found that after Cu²⁺ accumulation the total copper content of this yeast was lower than the parent culture when exposed to similar conditions, indicating that the subculture was copper-resistant owing to reduced copper bioaccumulation properties. Although a low mass copper binding compound was isolated from the copper-tolerant subculture, no metallothionein was found. Scanning electron microscopy of S. cerevisiae showed the cell surface to be smooth except for bud scars. After exposure to copper ion-containing solutions the surface of copper-tolerant yeast became convoluted, the cell was generally more spherical and somewhat smaller.     

Effects of acetic acid on growth and fermentative activity of Saccharomyces cerevisiae

The effect of acetic acid on the growth and the fermentative activity of S. cerevisiae was analysed comparatively with the pH. This study showed that the pH does not affect these two activities. On the contrary, the acetic acid has an inhibition effect. This effect was modelised by the relation of Levenspiel. Finally, it was shown that the quantities of acetic acid produced by Brettanomyces were not sufficient to explain the inhibition of Saccharomyces.