不同宿主来源的重组酿酒酵母混合糖代谢比较

【目的】研究不同工业酿酒酵母宿主背景对重组酵母木糖利用效率的影响。【方法】将木糖利用途径的木糖还原酶(XR)、木糖醇脱氢酶(XDH)和木酮糖激酶(XK)编码基因串联后分别转入3株不同的工业酿酒酵母中,得到重组酵母ZQ1、ZQ5和ZQ7。分别对3个木糖途径代谢基因的表达水平、酶活和重组菌株的木糖发酵效率进行比较。【结果】重组菌株在木糖代谢基因转录、酶活性和木糖利用性能方面有很大差异,其中ZQ5木糖代谢能力最强,ZQ7其次,ZQ1木糖利用能力最弱。ZQ7在初始木糖浓度为20 g/L时木糖利用速率快于ZQ5,表明木糖浓度对重组菌发酵性能评价具有影响。【结论】不同菌株的遗传背景和木糖浓度对重组菌木糖利用的影响很大,评价重组酵母的木糖利用需考虑宿主的遗传背景和底物浓度的影响。     

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.     

Effects of acetic acid on the kinetics of xylose fermentation by an engineered, xylose-isomerase-based Saccharomyces cerevisiae strain

Acetic acid, an inhibitor released during hydrolysis of lignocellulosic feedstocks, has previously been shown to negatively affect the kinetics and stoichiometry of sugar fermentation by (engineered) Saccharomyces cerevisiae strains. This study investigates the effects of acetic acid on S. cerevisiae RWB 218, an engineered xylose-fermenting strain based on the Piromyces XylA (xylose isomerase) gene. Anaerobic batch cultures on synthetic medium supplemented with glucose–xylose mixtures were grown at pH 5 and 3.5, with and without addition of 3 g L⁻¹ acetic acid. In these cultures, consumption of the sugar mixtures followed a diauxic pattern. At pH 5, acetic acid addition caused increased glucose consumption rates, whereas specific xylose consumption rates were not significantly affected. In contrast, at pH 3.5 acetic acid had a strong and specific negative impact on xylose consumption rates, which, after glucose depletion, slowed down dramatically, leaving 50% of the xylose unused after 48 h of fermentation. Xylitol production was absent (<0.10 g L⁻¹) in all cultures. Xylose fermentation in acetic –acid-stressed cultures at pH 3.5 could be restored by applying a continuous, limiting glucose feed, consistent with a key role of ATP regeneration in acetic acid tolerance.     

Ethanol production from glucose and dilute-acid hydrolyzates by encapsulated S. cerevisiae

The performance of encapsulated Saccharomyces cerevisiae CBS 8066 in anaerobic cultivation of glucose, in the presence and absence of furfural as well as in dilute-acid hydrolyzates, was investigated. The cultivation of encapsulated cells in 10 sequential batches in synthetic media resulted in linear increase of biomass up to 106 g/L of capsule volume, while the ethanol productivity remained constant at 5.15 (+/-0.17) g/L x h (for batches 6-10). The cells had average ethanol and glycerol yields of 0.464 and 0.056 g/g in these 10 batches. Addition of 5 g/L furfural decreased the ethanol productivity to a value of 1.31 (+/-0.10) g/L x h with the encapsulated cells, but it was stable in this range for five consecutive batches. On the other hand, the furfural decreased the ethanol yield to 0.41-0.42 g/g and increased the yield of acetic acid drastically up to 0.068 g/g. No significant lag phase was observed in any of these experiments. The encapsulated cells were also used to cultivate two different types of dilute-acid hydrolyzates. While the free cells were not able to ferment the hydrolyzates within at least 24 h, the encapsulated yeast successfully converted glucose and mannose in both of the hydrolyzates in less than 10 h with no significant lag phase. However, since the hydrolyzates were too toxic, the encapsulated cells lost their activity gradually in sequential batches.     

Optimization of 1,2,4‐butanetriol production from xylose in Saccharomyces cerevisiae by metabolic engineering of NADH/NADPH balance

1,2,4‐Butanetriol (BT) is used as a precursor for the synthesis of various pharmaceuticals and the energetic plasticizer 1,2,4‐butanetriol trinitrate. In Saccharomyces cerevisiae, BT is biosynthesized from xylose via heterologous four enzymatic reactions catalyzed by xylose dehydrogenase, xylonate dehydratase, 2‐ketoacid decarboxylase, and alcohol dehydrogenase. We here aimed to improve the BT yield in S. cerevisiae by genetic engineering. First, the amount of the key intermediate 2‐keto‐3‐deoxy‐xylonate as described previously was successfully reduced in 41% by multiple integrations of Lactococcus lactis 2‐ketoacid decarboxylase gene kdcA into the yeast genome. Since the heterologous BT synthetic pathway is independent of yeast native metabolism, this manipulation has led to NADH/NADPH imbalance and deficiency during BT production. Overexpression of the NADH kinase POS5Δ17 lacking the mitochondrial targeting sequence to relieve NADH/NADPH imbalance resulted in the BT titer of 2.2 g/L (31% molar yield). Feeding low concentrations of glucose and xylose to support the supply of NADH resulted in BT titer of 6.6 g/L with (57% molar yield). Collectively, improving the NADH/NADPH ratio and supply from glucose are essential for the construction of a xylose pathway, such as the BT synthetic pathway, independent of native yeast metabolism.     

Comparative sugar recovery and fermentation data following pretreatment of poplar wood by leading technologies

Through a Biomass Refining Consortium for Applied Fundamentals and Innovation among Auburn University, Dartmouth College, Michigan State University, the National Renewable Energy Laboratory, Purdue University, Texas A&M University, the University of British Columbia, and the University of California at Riverside, leading pretreatment technologies based on ammonia fiber expansion, aqueous ammonia recycle, dilute sulfuric acid, lime, neutral pH, and sulfur dioxide were applied to a single source of poplar wood, and the remaining solids from each technology were hydrolyzed to sugars using the same enzymes. Identical analytical methods and a consistent material balance methodology were employed to develop comparative performance data for each combination of pretreatment and enzymes. Overall, compared to data with corn stover employed previously, the results showed that poplar was more recalcitrant to conversion to sugars and that sugar yields from the combined operations of pretreatment and enzymatic hydrolysis varied more among pretreatments. However, application of more severe pretreatment conditions gave good yields from sulfur dioxide and lime, and a recombinant yeast strain fermented the mixed stream of glucose and xylose sugars released by enzymatic hydrolysis of water washed solids from all pretreatments to ethanol with similarly high yields. An Agricultural and Industrial Advisory Board followed progress and helped steer the research to meet scientific and commercial needs. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.