Bioconversion of lignocellulose-derived sugars to ethanol by engineered Saccharomyces cerevisiae

Lignocellulosic biomass from agricultural and agro-industrial residues represents one of the most important renewable resources that can be utilized for the biological production of ethanol. The yeast Saccharomyces cerevisiae is widely used for the commercial production of bioethanol from sucrose or starch-derived glucose. While glucose and other hexose sugars like galactose and mannose can be fermented to ethanol by S. cerevisiae, the major pentose sugars D-xylose and L-arabinose remain unutilized. Nevertheless, D-xylulose, the keto isomer of xylose, can be fermented slowly by the yeast and thus, the incorporation of functional routes for the conversion of xylose and arabinose to xylulose or xylulose-5-phosphate in Saccharomyces cerevisiae can help to improve the ethanol productivity and make the fermentation process more cost-effective. Other crucial bottlenecks in pentose fermentation include low activity of the pentose phosphate pathway enzymes and competitive inhibition of xylose and arabinose transport into the cell cytoplasm by glucose and other hexose sugars. Along with a brief introduction of the pretreatment of lignocellulose and detoxification of the hydrolysate, this review provides an updated overview of (a) the key steps involved in the uptake and metabolism of the hexose sugars: glucose, galactose, and mannose, together with the pentose sugars: xylose and arabinose, (b) various factors that play a major role in the efficient fermentation of pentose sugars along with hexose sugars, and (c) the approaches used to overcome the metabolic constraints in the production of bioethanol from lignocellulose-derived sugars by developing recombinant S. cerevisiae strains.     

酿酒酵母代谢工程技术

自20世纪90年代初期诞生以来,代谢工程历经了30年的快速发展。作为代谢工程的首选底盘细胞之一,酿酒酵母细胞工厂已被广泛应用于大量大宗化学品和新型高附加值生物活性物质的生物制造,在能源、医药和环境等领域取得了巨大的突破。近年来,合成生物学、生物信息学以及机器学习等相关技术也极大地促进了代谢工程的技术发展和应用。文中回顾了近30年来酿酒酵母代谢工程重要的技术发展,首先总结了经典代谢工程的常用方法和策略,以及在此基础上发展而来的系统代谢工程和合成生物学驱动的代谢工程技术。最后结合最新技术发展趋势,展望了未来酿酒酵母代谢工程发展的新方向。     

Control of xylose consumption by xylose transport in recombinant Saccharomyces cerevisiae

Saccharomyces cerevisiae TMB3001 has previously been engineered to utilize xylose by integrating the genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) and overexpressing the native xylulokinase (XK) gene. The resulting strain is able to metabolize xylose, but its xylose utilization rate is low compared to that of natural xylose utilizing yeasts, like Pichia stipitis or Candida shehatae. One difference between S. cerevisiae and the latter species is that these possess specific xylose transporters, while S. cerevisiae takes up xylose via the high-affinity hexose transporters. For this reason, in part, it has been suggested that xylose transport in S. cerevisiae may limit the xylose utilization.We investigated the control exercised by the transport over the specific xylose utilization rate in two recombinant S. cerevisiae strains, one with low XR activity, TMB3001, and one with high XR activity, TMB3260. The strains were grown in aerobic sugar-limited chemostat and the specific xylose uptake rate was modulated by changing the xylose concentration in the feed, which allowed determination of the flux response coefficients. Separate measurements of xylose transport kinetics allowed determination of the elasticity coefficients of transport with respect to extracellular xylose concentration. The flux control coefficient, C(J) (transp), for the xylose transport was calculated from the response and elasticity coefficients. The value of C(J) (transp) for both strains was found to be < 0.1 at extracellular xylose concentrations > 7.5 g L(-1). However, for strain TMB3260 the flux control coefficient was higher than 0.5 at xylose concentrations < 0.6 g L(-1), while C(J) (transp) stayed below 0.2 for strain TMB3001 irrespective of xylose concentration.     

木糖代谢基因表达水平对酿酒酵母重组菌株产物形成的影响

以Ｅ．ｃｏｌｉ－Ｓ．ｃｅｒｅｖｉｓｉａｅ穿梭质粒ＹＥｐ２４为骨架,将树干毕赤酵母（Ｐｉｃｈｉａ ｓｔｉｐｉｔｉｓ ＣＢＳ６０５４）的木糖还原酶（ＸＲ）基因ＸＹＬ１及木糖醇脱氢酶（ＸＤＨ）基因ＸＹＬ１分别以不同的相对表达方向置于酿酒酵母的乙醇脱氢酶Ｉ（ＡＤＨ１）启动子和磷酸甘油激酶（ＰＧＫ）启动子下,构建不同ＸＹＬ１及ＸＹＬ２的重组质粒。这些重组质粒分别转化酿酒酵母（Ｈ１５８）受体菌。得到的重组菌株     

spt15突变基因对酿酒酵母木糖利用的影响

利用基因工程手段得到重组菌YPH499-3中的spt15有效突变基因,通过表达载体pYX212转化入酿酒酵母原始菌株YPH499中,重新获得酿酒酵母重组菌株。对其性状进行研究,结果表明该菌株能有效利用木糖并共发酵木糖和葡萄糖。在30oC、200r/min,发酵72h时,50g/L木糖的利用率为82.0%,乙醇产率为28.4%;当木糖和葡萄糖以质量比1:1混合发酵时,木糖和葡萄糖的利用率分别为80.4%和100%,乙醇产率为31.4%;同时发现木糖醇的含量极低。从而验证了有效突变基因spt15-10对酿酒酵母共发酵木糖和葡萄糖产酒精的影响。     

Identification and functional expression of a new xylose isomerase from the goat rumen microbiome in Saccharomyces cerevisiae

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.     

酿酒酵母木糖发酵酒精途径工程的研究进展

途径工程(Pathway engineering),被称为第三代基因工程,改变代谢流向,开辟新的代谢途径是途径工程的主要目的。利用途径工程理念,对酿酒酵母（Saccharomyces cerevisiae）代谢途径进行理性设计,以拓展这一传统酒精生产菌的底物范围,使其充分利用可再生纤维质水解物中的各种糖分,是酿酒酵母酒精途径工程的研究热点之一。这里介绍了近年来酿酒酵母以木糖为底物的酒精途径工程的研究进展。     

Oscillatory metabolism of Saccharomyces cerevisiae in continuous culture

Short-period (40-50 min) synchronized metabolic oscillation was found in a continuous culture of yeast Saccharomyces cerevisiae under aerobic conditions at low-dilution rates. During oscillation, many parameters changed cyclically, such as dissolved oxygen concentration, respiration rate, ethanol and acetate concentrations in the culture, glycogen, ATP, NADH, pyruvate and acetate concentrations in the cells. These changes were considered to be associated with glycogen metabolism. When glycogen was degraded, the respiro-fermentative phase was observed, in which ethanol was produced and the respiration rate decreased. In this phase, the levels of intracellular pyruvate and acetate became minimum, ATP became high and intracellular pH at its lowest level. When glycogen metabolism changed from degradation to accumulation, the respiratory phase started, during which ethanol was re-assimilated from the culture and the respiration rate increased. Intracellular pyruvate and acetate became maximum, ATP decreased and the intracellular pH appeared high. These findings may indicate new aspects of the control mechanism of glycogen metabolism and how respiration and ethanol fermentation are regulated together under aerobic conditions.     

酿酒酵母转化木糖生产化学品的研究进展

木糖的有效利用是木质纤维素生产生物燃料或化学品经济性转化的基础.30年来,通过理性代谢改造和适应性进化等工程策略,显著提高了传统乙醇发酵微生物——酿酒酵母Saccharomyces cerevisiae的木糖代谢能力.因此,近年来在酿酒酵母中利用木糖生产化学品的研究逐步展开.研究发现,酿酒酵母分别以木糖和葡萄糖为碳源时...     

Xylose assimilation enhances the production of isobutanol in engineered Saccharomyces cerevisiae

Bioconversion of xylose—the second most abundant sugar in nature—into high-value fuels and chemicals by engineered Saccharomyces cerevisiae has been a long-term goal of the metabolic engineering community. Although most efforts have heavily focused on the production of ethanol by engineered S. cerevisiae, yields and productivities of ethanol produced from xylose have remained inferior as compared with ethanol produced from glucose. However, this entrenched focus on ethanol has concealed the fact that many aspects of xylose metabolism favor the production of nonethanol products. Through reduced overall metabolic flux, a more respiratory nature of consumption, and evading glucose signaling pathways, the bioconversion of xylose can be more amenable to redirecting flux away from ethanol towards the desired target product. In this report, we show that coupling xylose consumption via the oxidoreductive pathway with a mitochondrially-targeted isobutanol biosynthesis pathway leads to enhanced product yields and titers as compared to cultures utilizing glucose or galactose as a carbon source. Through the optimization of culture conditions, we achieve 2.6 g/L of isobutanol in the fed-batch flask and bioreactor fermentations. These results suggest that there may be synergistic benefits of coupling xylose assimilation with the production of nonethanol value-added products.     

不同启动子控制下木酮糖激酶的差异表达及其对酿酒酵母木糖代谢的影响

[目的]以不同强度的启动子控制表达木酮糖激酶基因,并研究其引起的不同木酮糖激酶活性水平对木糖利用酿酒酵母(Saccharomyces cerevisiae)代谢流向的影响.[方法]以酿酒酵母CEN.PK 113-5D为出发菌株,选择酿酒酵母内源启动子TEF1p,PGK1p和HXK2p,利用Cre-loxP无标记同源重组系统,置换染色体上木酮糖激酶基因XKS1的启动子(XKS1p)序列;并通过附加体质粒引入木糖代谢上游途径,构建不同水平表达木酮糖激酶的木糖利用工程菌株;从木酮糖激酶的转录水平、酶活水平、胞内的ATP浓度及木糖代谢等性状,对各菌株进行评价.[结果]转录及酶活测定结果显示,与天然状态相比,所选择的启动子对木酮糖激酶均表现出更强的启动效率.菌株体内表达木酮糖激酶活性水平由高至低的顺序为其基因XKS1在启动子PGK1p、TEF1p、HXK2p和XKS1p控制下.随着木酮糖激酶的活性的提高,胞内的ATP水平下降,而转化木糖生成乙醇的能力上升.最高乙醇产率为0.35g/g消耗的总糖,此时副产物木糖醇产率最低,为0.18g/g消耗的木糖.[结论]通过在染色体上置换启动子,提高了木酮糖激酶的表达水平.在一定范围内,木酮糖激酶的高活性有利于木糖向乙醇的转化.     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因, 我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析, 发现酵母在对数生长中期的表达谱非常稳定, 而一旦进入对数生长后期, 则出现明显的代谢重构现象。许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调; 而许多涉及酵母转座和DNA重组的基因则表达下调; 一些中心代谢途径也发生了代谢重构, 包括: 琥珀酸和a-酮戊二酸生成途径基因的一致上调, 都与氨基酸合成和代谢相关基因表达的结果相吻合。结果表明: 由于氨基酸合成的需求量增加, 进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环, 导致酒精的生产速率降低。     

Redirection of the Glycolytic Flux Enhances Isoprenoid Production in Saccharomyces cerevisiae

Sufficient supply of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is a prerequisite of the overproduction of isoprenoids and related bioproducts in Saccharomyces cerevisiae. Although S. cerevisiae highly depends on the oxidative pentose phosphate (PP) pathway to produce NADPH, its metabolic flux toward the oxidative PP pathway is limited due to the rigid glycolysis flux. To maximize NADPH supply for the isoprenoid production in yeast, upper glycolytic metabolic fluxes are reduced by introducing mutations into phosphofructokinase (PFK) along with overexpression of ZWF1 encoding glucose‐6‐phosphate (G6P) dehydrogenase. The PFK mutations (Pfk1 S724D and Pfk2 S718D) result in less glycerol production and more accumulation of G6P, which is a gateway metabolite toward the oxidative PP pathway. When combined with the PFK mutations, overexpression of ZWF1 caused substantial increases of [NADPH]/[NADP⁺] ratios whereas the effect of ZWF1 overexpression alone in the wild‐type strain is not noticeable. Also, the introduction of ZWF1 overexpression and the PFK mutations into engineered yeast overexpressing acetyl‐CoA C‐acetyltransferase (ERG10), truncated HMG‐CoA reductase isozyme 1 (tHMG1), and amorphadiene synthase (ADS) leads to a titer of 497 mg L^–1 of amorphadiene (3.7‐fold over the parental strain). These results suggest that perturbation of upper glycolytic fluxes, in addition to ZWF1 overexpression, is necessary for efficient NADPH supply through the oxidative PP pathway and enhanced production of isoprenoids by engineered S. cerevisiae.     

Impact of systems biology on metabolic engineering of Saccharomyces cerevisiae

Industrial biotechnology is a rapidly growing field. With the increasing shift towards a bio-based economy, there is rising demand for developing efficient cell factories that can produce fuels, chemicals, pharmaceuticals, materials, nutraceuticals, and even food ingredients. The yeast Saccharomyces cerevisiae is extremely well suited for this objective. As one of the most intensely studied eukaryotic model organisms, a rich density of knowledge detailing its genetics, biochemistry, physiology, and large-scale fermentation performance can be capitalized upon to enable a substantial increase in the industrial application of this yeast. Developments in genomics and high-throughput systems biology tools are enhancing one's ability to rapidly characterize cellular behaviour, which is valuable in the field of metabolic engineering where strain characterization is often the bottleneck in strain development programmes. Here, the impact of systems biology on metabolic engineering is reviewed and perspectives on the role of systems biology in the design of cell factories are given.     

酿酒酵母对数生长后期代谢重构的全基因组表达谱芯片分析

为了探讨酵母进入对数生长后期以后酒精生产速度降低的原因,我们利用酵母表达谱芯片技术对酿酒酵母细胞从对数生长中期进入对数生长后期时的全基因组表达谱进行了分析,发现酵母在对数生长中期的表达谱非常稳定,而一旦进入对数生长后期.则出现明显的代谢重构现象.许多氨基酸合成和代谢相关的基因、离子转移以及与能量的生成和储存等功能相关的基因出现了不同程度的上调;而许多涉及酵母转座和DNA重组的基因则表达下调;一些中心代谢途径也发生了代谢重构.包括:琥珀酸和α-酮戊二酸生成途径基因的一致上调,都与氨基酸合成和代谢相关基因表达的结果相吻合.结果表明:由于氨基酸合成的需求量增加,进入对数生长后期酵母的代谢转向TCA循环和乙醛酸循环,导致酒精的生产速率降低.     

系列过表达非氧化磷酸戊糖途径基因对重组酿酒酵母菌株木糖代谢的影响

【目的】通过系统研究一个、两个及多个非氧化磷酸戊糖(PP)途径基因组合过表达对酿酒酵母木糖代谢的影响,以优化重组菌株的构建过程,构建高效的木糖代谢酿酒酵母菌株。【方法】在酿酒酵母中双拷贝过表达上游代谢途径的关键酶(木糖还原酶XR,木糖醇脱氢酶XDH,木酮糖激酶XKS),在此基础上构建了一系列PP途径基因过表达菌株,并对其木糖发酵性能进行比较研究。【结果】木糖发酵结果显示,不同组合过表达PP途径基因能不同程度改善重组菌株的木糖发酵性能。其中,过表达PP途径全部基因(RKI1,RPE1,TAL1和TKL1)使菌株的发酵性能最优,其乙醇产率和产量较对照菌株分别提高了39.25%和12.57%,同时较其他基因组合过表达菌株也有不同程度的改善。【结论】通过构建PP途径基因不同组合过表达酿酒酵母菌株,首次对PP途径基因对酿酒酵母木糖代谢的影响进行了系统研究,结果表明,不同组合强化PP途径基因对重组菌株木糖代谢的影响存在差异,相对于其他基因过表达组合,同步过表达PP途径全部基因最有利于碳通量流向乙醇。     

Display of Clostridium cellulovorans xylose isomerase on the cell surface of Saccharomyces cerevisiae and its direct application to xylose fermentation

Xylose isomerase (XI) is a key enzyme in the conversion of d ‐xylose, which is a major component of lignocellulosic biomass, to d ‐xylulose. Genomic analysis of the bacterium Clostridium cellulovorans revealed the presence of XI‐related genes. In this study, XI derived from C. cellulovorans was produced and displayed using the yeast cell‐surface display system, and the xylose assimilation and fermentation properties of this XI‐displaying yeast were examined. XI‐displaying yeast grew well in medium containing xylose as the sole carbon source and directly produced ethanol from xylose under anaerobic conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 346–351, 2013