Simultaneous saccharification and fermentation of steam-pretreated bagasse using Saccharomyces cerevisiae TMB3400 and Pichia stipitis CBS6054

Sugarcane bagasse--a residue from sugar and ethanol production from sugar cane--is a potential raw material for lignocellulosic ethanol production. This material is high in xylan content. A prerequisite for bioethanol production from bagasse is therefore that xylose is efficiently fermented to ethanol. In the current study, ethanolic fermentation of steam-pretreated sugarcane bagasse was assessed in a simultaneous saccharification and fermentation (SSF) set-up using either Saccharomyces cerevisiae TMB3400, a recombinant xylose utilizing yeast strain, or Pichia stipitis CBS6054, a naturally xylose utilizing yeast strain. Commercial cellulolytic enzymes were used and the content of water insoluble solids (WIS) was 5% or 7.5%. S. cerevisiae TMB3400 consumed all glucose and large fraction of the xylose in SSF. Almost complete xylose conversion could be achieved at 5% WIS and 32 degrees C. Fermentation did not occur with P. stipitis CBS6054 at pH 5.0. However, at pH 6.0, complete glucose conversion and high xylose conversion (>70%) was obtained. Microaeration was required for P. stipitis CBS6054. This was not necessary for S. cerevisiae TMB3400.     

树干毕赤酵母和酿酒酵母混合糖发酵产乙醇

以树干毕赤酵母和酿酒酵母为发酵菌株,酸性蒸汽爆破玉米秸秆预水解液和纯糖模拟液为C源,采用固定化酵母细胞的方法,研究了酸爆玉米秸秆预水解液初始pH、N源种类及其浓度、3种发酵模式对树干毕赤酵母戊糖发酵的影响。结果表明:玉米秸秆预水解液适合发酵的初始pH范围为6.0～7.0;1.0 g/L的(NH4)2SO4作为N源,在40 g/L葡萄糖和25 g/L木糖培养基中发酵24 h,糖利用率达到99.47%,乙醇质量浓度为24.72 g/L,优于尿素和蛋白胨作为N源;3种模式的发酵体系中,以游离树干毕赤酵母和固定化酿酒酵母发酵性能最好,糖利用率和乙醇得率分别为99.43%和96.39%。     

树干毕赤酵母木糖还原酶基因的克隆及在大肠杆菌中的表达

根据NCBI中的木糖还原酶基因序列设计引物,利用高保真聚合酶克隆树干毕赤酵母木糖还原酶基因,加A后克隆到质粒pGM-T中,测序验证.然后将目的基因克隆到舍有强启动子的穿梭表达载体p424GPD中,构建含有XYL1基因的重组质粒p424GPD-XYL1.将p424GPD-XYL1转化到大肠杆菌中,提取总蛋白,聚丙烯酰胺凝胶电泳分析.酶活测定确定木糖还原酶基因XYL1在大肠杆菌中得到活性表达,表明表达载体构建成功.表达载体的成功构建为后续构建重组酿酒酵母利用木糖发酵奠定基础.     

Effect of the reversal of coenzyme specificity by expression of mutated Pichia stipitis xylitol dehydrogenase in recombinant Saccharomyces cerevisiae

AIMS: To determine the effects on xylitol accumulation and ethanol yield of expression of mutated Pichia stipitis xylitol dehydrogenase (XDH) with reversal of coenzyme specificity in recombinant Saccharomyces cerevisiae. METHODS AND RESULTS: The genes XYL2 (D207A/I208R/F209S) and XYL2 (S96C/S99C/Y102C/D207A/I208R/F209S) were introduced into S. cerevisiae, which already contained the P. stipitis XYL1 gene (encoding xylose reductase, XR) and the endogenously overexpressed XKS1 gene (encoding xylulokinase, XK). The specific activities of mutated XDH in both strains showed a distinct increase in NADP(+)-dependent activity in both strains with mutated XDH, reaching 0.782 and 0.698 U mg(-1). In xylose fermentation, the strain with XDH (D207A/I208R/F209S) had a large decrease in xylitol and glycerol yield, while the xylose consumption and ethanol yield were decreased. In the strain with XDH (S96C/S99C/Y102C/D207A/I208R/F209S), the xylose consumption and ethanol yield were also decreased, and the xylitol yield was increased, because of low XDH activity. CONCLUSIONS: Changing XDH coenzyme specificity was a sufficient method for reducing the production of xylitol, but high activity of XDH was also required for improved ethanol formation. SIGNIFICANCE AND IMPACT OF THE STUDY: The difference in coenzyme specificity was a vital parameter controlling ethanolic xylose fermentation but the XDH/XR ratio was also important.     

Ethanol production from hardwood spent sulfite liquor using an adapted strain of Pichia stipitis

Conditions have been optimized for fermentation of pretreated hardwood spent sulfite liquor (HSSL) using an adapted strain of Pichia stipitis. The pretreatments, consisting of boiling and overliming with Ca(OH)₂ of HSSL, to partially remove inhibitors, and adaptation of the yeast strain to HSSL, were both critical for a successful fermentation. Ethanol concentration was increased from 6.7 to 20.2 g l⁻¹ using adapted P. stipitis (A) and pretreated HSSL. The maximum ethanol yield (Y _p/s) and productivity (Q _p) were 0.41 g g⁻¹ and 0.44 g l⁻¹ h⁻¹, respectively, at an oxygen transfer rate of 2.0 mmol O₂ l⁻¹ h⁻¹. The optimized results with this strain were compared to those of other xylose-fermenting yeasts and Saccharomyces cerevisiae (SSL-acclimatized) currently used at an industrial plant for the fermentation of spent sulfite liquor. Journal of Industrial Microbiology & Biotechnology (2001) 26, 145–150. Received 23 June 2000/ Accepted in revised form 21 October 2000     

固定化酵母乙醇发酵及固化小球微生态的研究

比较了SA-PVA-SiO₂固定化酿酒酵母和游离酿酒酵母的乙醇发酵能力,采用批次发酵试验研究固定化酿酒酵母的发酵稳定性。结果表明,SA-PVA-SiO₂固定化酿酒酵母的发酵速度比游离酿酒酵母快,发酵周期短;发酵稳定性很好,30℃,橡胶塞、90 r/min摇床培养24 h时,乙醇体积分数均在3%~3.5％之间,连续发酵14批次后,固化小球的形态依然完好,不发粘。通过扫描电镜对酿酒酵母包埋微生态环境进行了分析,图像表明固定化小球的内部环境非常有利于酵母细胞的厌氧发酵产乙醇,充分证明了SA-PVA-SiO₂固定化酿酒酵母乙醇发酵的优越性。     

Irreversible formation of pseudohyphae by haploid Saccharomyces cerevisiae

Abstract Culturing haploid strains of Saccharomyces cerevisiae in liquid minimal medium with 2% ethanol and 2% leucine resulted in the formation of long anucleate pseudohyphae. This occurred only with the combination of ethanol as carbon source and leucine as nitrogen source and was independent of mating type. The transition to a pseudohyphal form observed under these conditions appears to be irreversible. These findings further extend our view of the developmental alternatives in this important model eukaryote.     

酿酒酵母乙醇耐受性机理研究进展

酿酒酵母(Sacchromyces cerevisiae)一直是主要的生物乙醇和酿酒业发酵菌株, 具有发酵速度快、乙醇产量高特性。然而, 产物乙醇积累造成的毒性效应是限制乙醇产量的主要因素之一, 研究酿酒酵母乙醇耐受性为解决这一工业难题奠定了理论基础。本文从乙醇对酵母细胞生理、细胞结构和组分的影响, 以及酿酒酵母乙醇耐受性的遗传基础方面综述了酿酒酵母乙醇耐受性机理的研究进展。     

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris

A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae alpha-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant.     

The expression of a Pichia stipitis xylose reductase mutant with higher K(M) for NADPH increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

Xylose fermentation by Saccharomyces cerevisiae requires the introduction of a xylose pathway, either similar to that found in the natural xylose-utilizing yeasts Pichia stipitis and Candida shehatae or similar to the bacterial pathway. The use of NAD(P)H-dependent XR and NAD(+)-dependent XDH from P. stipitis creates a cofactor imbalance resulting in xylitol formation. The effect of replacing the native P. stipitis XR with a mutated XR with increased K(M) for NADPH was investigated for xylose fermentation to ethanol by recombinant S. cerevisiae strains. Enhanced ethanol yields accompanied by decreased xylitol yields were obtained in strains carrying the mutated XR. Flux analysis showed that strains harboring the mutated XR utilized a larger fraction of NADH for xylose reduction. The overproduction of the mutated XR resulted in an ethanol yield of 0.40 g per gram of sugar and a xylose consumption rate of 0.16 g per gram of biomass per hour in chemostat culture (0.06/h) with 10 g/L glucose and 10 g/L xylose as carbon source.     

Comparison of corn steep liquor with other nutrients in the fermentation of D-Xylose by Pichia stipitis CBS 6054

Summary Pichia stipitis CBS 6054 ferments D-Xylose to ethanol in a medium containing corn steep liquor as the only source of nitrogen, amino acids, vitamins and other nutrients. The ethanol yield and fermentation rate compare favorably to those obtained with media containing more expensive sources of nitrogen, vitamins and amino acids. Corn steep liquor is a good source of nutrients that can support growth and fermentation activity of this xylose fermenting yeast.     

Stress-related challenges in pentose fermentation to ethanol by the yeast Saccharomyces cerevisiae

Conversion of agricultural residues, energy crops and forest residues into bioethanol requires hydrolysis of the biomass and fermentation of the released sugars. During the hydrolysis of the hemicellulose fraction, substantial amounts of pentose sugars, in particular xylose, are released. Fermentation of these pentose sugars to ethanol by engineered Saccharomyces cerevisiae under industrial process conditions is the subject of this review. First, fermentation challenges originating from the main steps of ethanol production from lignocellulosic feedstocks are discussed, followed by genetic modifications that have been implemented in S. cerevisiae to obtain xylose and arabinose fermenting capacity per se. Finally, the fermentation of a real lignocellulosic medium is discussed in terms of inhibitory effects of furaldehydes, phenolics and weak acids and the presence of contaminating microbiota.     

Nonhomologous end joining and homologous recombination DNA repair pathways in integration mutagenesis in the xylose-fermenting yeast Pichia stipitis

Pichia stipitis integrates linear homologous DNA fragments mainly ectopically. High rates of randomly occurring integration allow tagging mutagenesis with high efficiency using simply PCR amplificates of suitable selection markers from the P. stipitis genome. Linearization of an autonomously replicating vector caused a distinct increase of the transformation efficiency compared with the circular molecule. Cotransformation of a restriction endonuclease further enhanced the transformation efficiency. This effect was also observed with integrative vector DNA. In most cases vector integration in chromosomal targets did not depend on microhomologies, indicating that restriction-enzyme-mediated integration (REMI) does not play an essential role in P. stipitis. Small deletions were observed at the ends of the integrated vectors and in the target sites. Disruption of the PsKU80 gene increased the frequency of homologous integration considerably but resulted in a remarkable decrease of the transformation efficiency. These results suggest that in P. stipitis the nonhomologous end joining (NHEJ) pathway obviously predominates the homologous recombination pathway of double-strand break repair.     

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。     

Effects of xylulokinase activity on ethanol production from D-xylulose by recombinant Saccharomyces cerevisiae

AIMS: Recombinant Saccharomyces cerevisiae strains harbouring different levels of xylulokinase (XK) activity and effects of XK activity on utilization of xylulose were studied in batch and fed-batch cultures. METHODS AND RESULTS: The cloned xylulokinase gene (XKS1) from S. cerevisiae was expressed under the control of the glyceraldehyde 3-phosphate dehydrogenase promoter and terminator. Specific xylulose consumption rate was enhanced by the increased specific XK activity, resulting from the introduction of the XKS1 into S. cerevisiae. In batch and fed-batch cultivations, the recombinant strains resulted in twofold higher ethanol concentration and 5.3- to six-fold improvement in the ethanol production rate compared with the host strain S. cerevisiae. CONCLUSIONS: An effective conversion of xylulose to xylulose 5-phosphate catalysed by XK in S. cerevisiae was considered to be essential for the development of an efficient and accelerated ethanol fermentation process from xylulose. SIGNIFICANCE AND IMPACT OF THE STUDY: Overexpression of the XKS1 gene made xylulose fermentation process accelerated to produce ethanol through the pentose phosphate pathway.     

Determination of kinetic parameters of fermentation processes by a continuous unsteady-state method: application to the alcoholic fermentation of D-xylose by Pichia stipitis

A quick technique for determination of kinetic parameters of fermentation processes is proposed and applied to the transformation of D-xylose into ethanol by Pichia stipitis. The commonly used method to evaluate these parameters is based on achieving several steady states. In the proposed procedure, mu(m) and K(s) can be determined from only one steady state, by provoking a disturbance over it, after allowing the system to return to the original conditions. The main difference between the steady and unsteady state methods is the required fermentation time; while the former method lasted 350 h, the latter required a period 25 times lower. Kinetic and stoichiometric parameters were determined with both methods under anoxic and limited oxygen concentration conditions. Results from the two methods were compared, giving only 2% and 4.5% differences in the values of K(s) and mu(m) and a little over 4% for mu(m) were the deviations under the latter ones. (c) 1993 Wiley & Sons, Inc.     

Mutagenesis of the glucoamylase signal peptide of Saccharomyces diastaticus and functional analysis in Saccharomyces cerevisiae

To improve the efficiency of the glucoamylase signal peptide (GSP) of Saccharomyces diastaticus for the secretion of foreign proteins, hybrid plasmids containing one of four types of GSP mutant (m1, Pro(-18)-->Leu(-18); m2, Tyr(-13)-->Leu(-13); m3, Ser(-9)-->Leu(-9); m4, Asn(-5)-->Pro(-5)) were constructed and evaluated in Saccharomyces cerevisiae using Bacillus endo-1,4-beta-D-glucanase (CMCase) as a reporter gene. CMCase secretion by m1, m2 and m3 GSP mutants was increased, likely resulting from a higher probability of the modified GSP to assume an alpha-helical structure. Especially in the case of m3, the substitution of Leu for a polar residue, Ser(-9), in the hydrophobic region resulted in approximately a twofold increase in extracellular CMCase activity. In mutant 4, which disrupts the alpha-helix of GSP, CMCase was less efficiently secreted.     

Influence of magnesium concentration on growth,ethanol and xylitol production by Pichia stipitis NRRL Y-7124

Summary The effect of Mg⁺² on Pichia stipitis growth and ethanol production was studied under condition of constant oxygen uptake rate (OUR) . Biomass/xylose and biomass/Mg⁺² yields increased with Mg⁺² concentration with a maximum value at Mg⁺² 4mM, ethanol being the main product obtained. At low Mg⁺² levels (ImM) 49 % of carbon flux to ethanol was redirected to xylitol production, accomplished through NADH intracellular accumulation.