休哈塔假丝酵母HDYXHT-01利用木糖生产乙醇的发酵工艺优化

采用Plackett-Burman (PB) 方法和中心组合设计 (Ccentral composit design,CCD) 对休哈塔假丝酵母Candida shehataeHDYXHT-01利用木糖发酵生产乙醇的工艺进行优化。PB试验设计与分析结果表明：硫酸铵、磷酸二氢钾、酵母粉和接种量是影响木糖乙醇发酵的4个关键因素,以乙醇产量为响应目标,采用CCD和响应面分析法 (Response surface methodology,RSM),确定了木糖乙醇发酵的最佳工艺为：硫酸铵1.73 g/L、磷酸二氢钾3.56 g/L、酵母粉2.62 g/L和接种量5.66％,其他发酵条件为：木糖80 g/L,MgSO4·7H2O 0.1 g/L,pH 5.0,培养温度30 ℃,装液量100 mL/250 mL,摇床转速140 r/min,发酵时间48 h,在该条件下发酵液中乙醇产量可以达到26.18 g/L,比未优化前提高了1.15倍。     

休哈塔假丝酵母转化木糖和葡萄糖为乙醇的发酵转录谱

[目的]探究木糖发酵典型菌株休哈塔假丝酵母在己糖和戊糖发酵中的转录谱及差异,筛选出与木糖利用和乙醇发酵代谢途径及调控相关的关键性酶和功能蛋白质基因.[方法]应用新一代高通量测序技术454 GS FLX Titanium分别构建了休哈塔假丝酵母木糖、葡萄糖发酵的cDNA文库,并进行De novo转录组的表达序列标签大规模测序和序列比较分析,进而挖掘出该酵母中参与木糖代谢和乙醇发酵的相关基因.[结果]分别对木糖和葡萄糖发酵样本进行二分之一RUN测序并各自得到60万条reads,序列平均长度400 bp.共拼接得到7250条(木糖)和7168条(葡萄糖)contigs,并利用BLAST对木糖样品和葡萄糖样品中的2421个基因(contig)和2456个基因(contig)进行了功能注释和GO分类.通过两个文库间的序列对比分析,共发现158个基因属于差异表达状态(P＜0.05).基于经典的糖酵解及乙醇发酵途径筛选出与木糖乙醇发酵相关的候选基因,并且比较分析其转录水平的差异.[结论]基于大规模转录谱测序和比较分析首次筛选出休哈塔假丝酵母中参与木糖代谢和乙醇发酵的基因群,可为后续的分子生物学及代谢调控研究提供基础数据.     

Identification of required nutrient components of yeast nitrogen base for Candida shehatae ATTCC 22984 fermenting xylose to ethanol

Summary Five components of Yeast Nutrient Base (YNB) of Difco have been identified as required nutrients for Candida shehatae ATCC 22984 in fermenting xylose to ethanol with ammonium sulfate as the nitrogen source. They are potassium phosphate monobasic, magnesium sulfate, zinc sulfate, thiamine hydrochloride, and biotin. The fermentation results in the minimum medium containing only the five required nutrient components plus xylose and ammonium sulfate have been shown to be comparable to those in the full YNB plus xylose.     

Kinetic modeling of Candida shehatae ATCC 22984 on xylose and glucose for ethanol production

Candida shehatae ATCC 22984, a xylose-fermenting yeast, showed an ability to produce ethanol in both glucose and xylose medium. Maximum ethanol produced by the yeast was 48.8?g/L in xylose and 52.6?g/L in glucose medium with ethanol yields that varied between 0.3 and 0.4?g/g depended on initial sugar concentrations. Xylitol was a coproduct of ethanol production using xylose as substrate, and glycerol was detected in both glucose and xylose media. Kinetic model equations indicated that growth, substrate consumption, and product formation of C. shehatae were governed by substrate limitation and inhibition by ethanol. The model suggested that cell growth was totally inhibited at 40?g/L of ethanol and ethanol production capacity of the yeast was 52?g/L, which were in good agreement with experimental results. The developed model could be used to explain C. shehatae fermentation in glucose and xylose media from 20 to 170?g/L sugar concentrations.     

Metabolic flux analysis of Candida tropicalis growing on xylose in an oxygen-limited chemostat

We have studied the metabolism of xylose by Candida tropicalis in oxygen-limited chemostat. In vitro enzyme assays indicated that glycolytic and gluconeogenetic enzymes are expressed simultaneously facilitating substrate cycling. Enhancing the redox imbalance by cofeeding of formate increased xylose and oxygen consumption rates and ethanol, xylitol, glycerol and CO2 production rates at steady state. Metabolic flux analysis (MFA) indicated that fructose 6-phosphate is replenished from the pentose phosphate pathway in sufficient amounts without contribution of the gluconeogenetic pathway. Substrate cycling between pyruvate kinase, pyruvate carboxylase and phospho-enol-pyruvate kinase increased ATP turnover. Cofeeding of formate increased the ATP yield. The ATP yields of xylose and xylose-formate cultivation were 6.9 and 8.7 mol ATP/C-mol CDW, respectively, as calculated from the MFA.     

Effect of controlled oxygen limitation on Candida shehatae physiology for ethanol production from xylose and glucose

Carbon distribution and kinetics of Candida shehatae were studied in fed-batch fermentation with xylose or glucose (separately) as the carbon source in mineral medium. The fermentations were carried out in two phases, an aerobic phase dedicated to growth followed by an oxygen limitation phase dedicated to ethanol production. Oxygen limitation was quantified with an average specific oxygen uptake rate (OUR) varying between 0.30 and 2.48 mmolO₂ g dry cell weight (DCW)^?1 h^?1, the maximum value before the aerobic shift. The relations among respiration, growth, ethanol production and polyol production were investigated. It appeared that ethanol was produced to provide energy, and polyols (arabitol, ribitol, glycerol and xylitol) were produced to reoxidize NADH from assimilatory reactions and from the co-factor imbalance of the two-first enzymatic steps of xylose uptake. Hence, to manage carbon flux to ethanol production, oxygen limitation was a major controlled parameter; an oxygen limitation corresponding to an average specific OUR of 1.19 mmolO₂ g DCW^?1 h^?1 allowed maximization of the ethanol yield over xylose (0.327 g g^?1), the average productivity (2.2 g l^?1 h^?1) and the ethanol final titer (48.81 g l^?1). For glucose fermentation, the ethanol yield over glucose was the highest (0.411 g g^?1) when the specific OUR was low, corresponding to an average specific OUR of 0.30 mmolO₂ g DCW^?1 h^?1, whereas the average ethanol productivity and ethanol final titer reached the maximum values of 1.81 g l^?1 h^?1 and 54.19 g l^?1 when the specific OUR was the highest.     

Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose.

The efficient diversion of pyruvate from normal fermentative pathways to ethanol production in Klebsiella oxytoca M5A1 requires the expression of Zymomonas mobilis genes encoding both pyruvate decarboxylase and alcohol dehydrogenase. Final ethanol concentrations obtained with the best recombinant, strain M5A1 (pLOI555), were in excess of 40 g/liter with an efficiency of 0.48 g of ethanol (xylose) and 0.50 g of ethanol (glucose) per g of sugar, as compared with a theoretical maximum of 0.51 g of ethanol per g of sugar. The maximal volumetric productivity per hour for both sugars was 2.0 g/liter. This volumetric productivity with xylose is almost twice that previously obtained with ethanologenic Escherichia coli. Succinate was also produced as a minor product during fermentation.     

Metabolic engineering of Klebsiella oxytoca M5A1 for ethanol production from xylose and glucose.

The efficient diversion of pyruvate from normal fermentative pathways to ethanol production in Klebsiella oxytoca M5A1 requires the expression of Zymomonas mobilis genes encoding both pyruvate decarboxylase and alcohol dehydrogenase. Final ethanol concentrations obtained with the best recombinant, strain M5A1 (pLOI555), were in excess of 40 g/liter with an efficiency of 0.48 g of ethanol (xylose) and 0.50 g of ethanol (glucose) per g of sugar, as compared with a theoretical maximum of 0.51 g of ethanol per g of sugar. The maximal volumetric productivity per hour for both sugars was 2.0 g/liter. This volumetric productivity with xylose is almost twice that previously obtained with ethanologenic Escherichia coli. Succinate was also produced as a minor product during fermentation.     

Metabolic engineering of the initial stages of xylose catabolism in yeast for the purpose of constructing efficient producers of ethanol from lignocellulosics

Plant biomass possesses huge potential as a source for the production of biofuels. Glucose and the five-carbon sugar xylose are the principal constituents of biomass. The yeast Saccharomyces cerevisiae, which is used for industrial production of ethanol from glucose is not capable of fermenting xylose. Thus, it is necessary to find in Nature or to create microorganisms capable of achieving efficient fermentation of glucose and xylose, as a means of achieving economically feasible biomass conversion into ethanol. Active fermentation of xylose may be achieved if the initial stages of metabolism are efficiently performed [1]. In this review, the enzymes of the initial stages of xylose metabolism in yeast (xylose reductase, xylitol dehydrogenase, and xylulokinase) and bacteria (xylose isomerase and xylulokinase) are characterized. The ways for constructing yeast strains capable of achieving efficient alcoholic xylose fermentation are discussed.     

System-level insights into yeast metabolism by thermodynamic analysis of elementary flux modes

One of the most obvious phenotypes of a cell is its metabolic activity, which is defined by the fluxes in the metabolic network. Although experimental methods to determine intracellular fluxes are well established, only a limited number of fluxes can be resolved. Especially in eukaryotes such as yeast, compartmentalization and the existence of many parallel routes render exact flux analysis impossible using current methods. To gain more insight into the metabolic operation of S. cerevisiae we developed a new computational approach where we characterize the flux solution space by determining elementary flux modes (EFMs) that are subsequently classified as thermodynamically feasible or infeasible on the basis of experimental metabolome data. This allows us to provably rule out the contribution of certain EFMs to the in vivo flux distribution. From the 71 million EFMs in a medium size metabolic network of S. cerevisiae, we classified 54% as thermodynamically feasible. By comparing the thermodynamically feasible and infeasible EFMs, we could identify reaction combinations that span the cytosol and mitochondrion and, as a system, cannot operate under the investigated glucose batch conditions. Besides conclusions on single reactions, we found that thermodynamic constraints prevent the import of redox cofactor equivalents into the mitochondrion due to limits on compartmental cofactor concentrations. Our novel approach of incorporating quantitative metabolite concentrations into the analysis of the space of all stoichiometrically feasible flux distributions allows generating new insights into the system-level operation of the intracellular fluxes without making assumptions on metabolic objectives of the cell.     

The conversion of human complement component C5 into fragment C5b by the alternative-pathway C5 convertase.

The cleavage of human complement component C5 to fragment C5b by the alternative pathway C5 convertase was studied. The alternative-pathway C5 convertase on zymosan can be represented by the empirical formula zymosan--C3b2BbP. Both properdin-stabilized C3 and C5 convertase activities decay with a half life of 34 min correlating with the loss of the Bb subunit. The C5 convertase functions in a stepwise fashion: first, C5 binds to C3b and this is followed by cleavage of C5 to C5b. The capacity to bind C3b is a stable feature of component C5, as C5b also has this binding capacity. Component C5, unlike component C3, does not form covalent bonds with zymosan after activation, and C5 is not inhibited by amines. Therefore C5, although similar in structure to C3, does not appear to contain the internal thioester group reported for C3 and C4.     

Dynamic flux balance analysis for microbial conversion of glycerol into 1,3-propanediol by Klebsiella pneumoniae

Bioprocess and Biosystems Engineering - To investigate the relationship between the yield of 1,3-propanediol (1,3-PD) and the flux variation in metabolic pathways of Klebsiella pneumoniae, an...     

酿酒酵母W5及休哈塔假丝酵母20335原生质体制备条件的确定

确定了酿酒酵母W5及休哈塔假丝酵母20335原生质体制备的最佳条件。选取不同脱壁预处理时间及不同酶解时间,对酿酒酵母W5、休哈塔假丝酵母20335进行原生质体制备和再生,比较制备率和再生率。确定脱壁预处理30 min后,以终浓度2%的蜗牛酶,30℃、100 r/min酶解处理15 min为双亲株原生质体制备的最佳条件。利用原生质体融合的方法,以酿酒酵母W5和休哈塔假丝酵母20335为亲本株,构建可以利用木糖生产生物乙醇的新型酿酒酵母融合株,该前期工作为W5、20335原生质体融合工作奠定了重要的基础,对于将木质纤维素原料转化为生物乙醇的研究具有极其重要的意义。     

Metabolic and bioprocess engineering of the yeast Candida famata for FAD production

Flavins in the form of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) play an important role in metabolism as cofactors for oxidoreductases and other enzymes. Flavin nucleotides have applications in the food industry and medicine; FAD supplements have been efficiently used for treatment of some inheritable diseases. FAD is produced biotechnologically; however, this compound is much more expensive than riboflavin. Flavinogenic yeast Candida famata synthesizes FAD from FMN and ATP in the reaction catalyzed by FAD synthetase, a product of the FAD1 gene. Expression of FAD1 from the strong constitutive promoter TEF1 resulted in 7- to 15-fold increase in FAD synthetase activity, FAD overproduction, and secretion to the culture medium. The effectiveness of FAD production under different growth conditions by one of these recombinant strains, C. famata T-FD-FM 27, was evaluated. First, the two-level Plackett–Burman design was performed to screen medium components that significantly influence FAD production. Second, central composite design was adopted to investigate the optimum value of the selected factors for achieving maximum FAD yield. FAD production varied most significantly in response to concentrations of adenine, KH₂PO₄, glycine, and (NH₄)₂SO₄. Implementation of these optimization strategies resulted in 65-fold increase in FAD production when compared to the non-optimized control conditions. Recombinant strain that has been cultivated for 40 h under optimized conditions achieved a FAD accumulation of 451 mg/l. So, for the first time yeast strains overproducing FAD were obtained, and the growth media composition for maximum production of this nucleotide was designed.     

Conversion of D-xylose into ethanol by the yeast Pachysolen tannophilus

Summary The yeast Pachysolen tannophilus has been identified as being able to convert an aldopentose, D-xylose, into ethanol. A feature of the conversion is that it can take place under aerobic conditions.Issued as N.R.C.C. Publication No. 19095.     

Direct and quantitative conversion of starch to ethanol by the yeast Schwanniomyces alluvius

Summary The yeast Schwanniomyces alluvius ferments soluble starch to ethanol at a conversion efficiency of greater than 95%. Only trace amounts of side products are detectable.NRCC publication no. 20435.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

Metabolic engineering of Synechocystis sp. PCC 6803 for enhanced ethanol production based on flux balance analysis

Synechocystis sp. PCC 6803 is an attractive host for bio-ethanol production due to its ability to directly convert atmospheric carbon dioxide into ethanol using photosystems. To enhance ethanol production in Synechocystis sp. PCC 6803, metabolic engineering was performed based on in silico simulations, using the genome-scale metabolic model. Comprehensive reaction knockout simulations by flux balance analysis predicted that the knockout of NAD(P)H dehydrogenase enhanced ethanol production under photoautotrophic conditions, where ammonium is the nitrogen source. This deletion inhibits the re-oxidation of NAD(P)H, which is generated by ferredoxin-NADP⁺ reductase and imposes re-oxidation in the ethanol synthesis pathway. The effect of deleting the ndhF1 gene, which encodes NADH dehydrogenase subunit 5, on ethanol production was experimentally evaluated using ethanol-producing strains of Synechocystis sp. PCC 6803. The ethanol titer of the ethanol-producing ∆ndhF1 strain increased by 145%, compared with that of the control strain.

     

Metabolic flux ratio analysis by parallel 13C labeling of isoprenoid biosynthesis in Rhodobacter sphaeroides

Metabolic engineering for increased isoprenoid production often benefits from the simultaneous expression of the two naturally available isoprenoid metabolic routes, namely the 2-methyl-D-erythritol 4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. Quantification of the contribution of these pathways to the overall isoprenoid production can help to obtain a better understanding of the metabolism within a microbial cell factory. Such type of investigation can benefit from ¹³C metabolic flux ratio studies. Here, we designed a method based on parallel labeling experiments (PLEs), using [1-¹³C]- and [4-¹³C]glucose as tracers to quantify the metabolic flux ratios in the glycolytic and isoprenoid pathways. By just analyzing a reporter isoprenoid molecule and employing only four equations, we could describe the metabolism involved from substrate catabolism to product formation. These equations infer ¹³C atom incorporation into the universal isoprenoid building blocks, isopentenyl-pyrophosphate (IPP) and dimethylallyl-pyrophosphate (DMAPP). Therefore, this renders the method applicable to the study of any of isoprenoid of interest. As proof of principle, we applied it to study amorpha-4,11-diene biosynthesis in the bacterium Rhodobacter sphaeroides. We confirmed that in this species the Entner-Doudoroff pathway is the major pathway for glucose catabolism, while the Embden-Meyerhof-Parnas pathway contributes to a lesser extent. Additionally, we demonstrated that co-expression of the MEP and MVA pathways caused a mutual enhancement of their metabolic flux capacity. Surprisingly, we also observed that the isoprenoid flux ratio remains constant under exponential growth conditions, independently from the expression level of the MVA pathway. Apart from proposing and applying a tool for studying isoprenoid biosynthesis within a microbial cell factory, our work reveals important insights from the co-expression of MEP and MVA pathways, including the existence of a yet unclear interaction between them.