Production of ethanol from wood hemicellulose hydrolyzates by a xylose-fermenting yeast mutant,Candida sp. XF 217

Summary Ethanol was produced from wood chip hemicellulose hydrolyzate by a xylose-fermenting yeast mutant, Candida sp. XF 217. The rates of D-xylose consumption and ethanol production were greater under aerobic than fermentative conditions. The slow rate of fermentation under fermentative conditions could be overcome by supplementing the broth with D-xylose isomerase (glucose isomerase). The ethanol yield, as based on the sugar consumed, was approximately 90% of the theoretical value.     

Quantitative production of xylitol from D-xylose by a high-xylitol producing yeast mutant Candida tropicalis HXP2

Summary Xylitol was produced as a metabolic by-product by a number of yeasts when grown on medium containing D-xylose as carbon and energy sources. Among the yeast strains tested, a mutant strain of Candida tropicalis (HXP2) was found to produce xylitol from D-xylose with a high yield (>90%). Ethanol was also produced by HXP2 when D-glucose, D-fructose, or sucrose were used as substrates. The high-xylitol-producing yeast mutant is a good organism for the production of xylitol from biomass that contains D-xylose.     

D-xylose catabolizing enzymes in Neurospora crassa and their relationship to D-xylose fermentation

Summary The induction of xylose reductase (XR) and xylitol dehydrogenase (XD) activities by D-xylose under different fermentation conditions was investigated in Neurospora crassa. The induction of NADPH-linked XR preceded NADH-linked XR and the ratio of NADH to NADPH-linked XR activity displayed variation from 0.02 to 0.2 suggesting the presence of two separate enzymes. Aerobic conditions were required by N. crassa for cell growth but not for ethanol production. Maximum ethanol of 0.3 g/g of D-xylose was produced when shifted to semiaerobic condition, where high NADH-linked XR and NAD-linked XD activities were observed.     

Engineering redox cofactor regeneration for improved pentose fermentation in Saccharomyces cerevisiae

Pentose fermentation to ethanol with recombinant Saccharomyces cerevisiae is slow and has a low yield. A likely reason for this is that the catabolism of the pentoses D-xylose and L-arabinose through the corresponding fungal pathways creates an imbalance of redox cofactors. The process, although redox neutral, requires NADPH and NAD+, which have to be regenerated in separate processes. NADPH is normally generated through the oxidative part of the pentose phosphate pathway by the action of glucose-6-phosphate dehydrogenase (ZWF1). To facilitate NADPH regeneration, we expressed the recently discovered gene GDP1, which codes for a fungal NADP+-dependent D-glyceraldehyde-3-phosphate dehydrogenase (NADP-GAPDH) (EC 1.2.1.13), in an S. cerevisiae strain with the D-xylose pathway. NADPH regeneration through an NADP-GAPDH is not linked to CO2 production. The resulting strain fermented D-xylose to ethanol with a higher rate and yield than the corresponding strain without GDP1; i.e., the levels of the unwanted side products xylitol and CO2 were lowered. The oxidative part of the pentose phosphate pathway is the main natural path for NADPH regeneration. However, use of this pathway causes wasteful CO2 production and creates a redox imbalance on the path of anaerobic pentose fermentation to ethanol because it does not regenerate NAD+. The deletion of the gene ZWF1 (which codes for glucose-6-phosphate dehydrogenase), in combination with overexpression of GDP1 further stimulated D-xylose fermentation with respect to rate and yield. Through genetic engineering of the redox reactions, the yeast strain was converted from a strain that produced mainly xylitol and CO2 from D-xylose to a strain that produced mainly ethanol under anaerobic conditions.     

Microbial conversion of xylose into useful bioproducts

Microorganisms can produce a number of different bioproducts from the sugars in plant biomass. One challenge is devising processes that utilize all of the sugars in lignocellulosic hydrolysates. D-xylose is the second most abundant sugar in these hydrolysates. The microbial conversion of D-xylose to ethanol has been studied extensively; only recently, however, has conversion to bioproducts other than ethanol been explored. Moreover, in the case of yeast, D-xylose may provide a better feedstock for the production of bioproducts other than ethanol, because the relevant pathways are not subject to glucose-dependent repression. In this review, we discuss how different microorganisms are being used to produce novel bioproducts from D-xylose. We also discuss how D-xylose could be potentially used instead of glucose for the production of value-added bioproducts.

     

Specific Detection of Xylella fastidiosa Pierce's Disease Strains

Pierce's disease (PD, Xylella fastidiosa) of grapevine is the primary pathogen limiting vinifera grape production in Florida and other regions of the southeastern United States. Quick and accurate detection of PD strains is essential for PD studies and control. A unique random amplified polymorphic DNA (PD1-1-2) was isolated from a PD strain from Florida. Fragment PD1-1-2 was cloned, sequenced, and found to be 1005 bp in length. PCR primers were designed to utilize these sequence data for PD strain detection. One primer set (XF176f–XF954r) amplified a 779-bp DNA fragment from 34 PD strains including seven pathotypes of X. fastidiosa, but not from strains of Xanthomonas campestris pv. campestris, Xan. vesicatoria or Escherichia coli. A second primer set (XF176f and XF686r) amplified a 511-bp fragment specific to 98 PD strains, but not from strains of citrus variegated chlorosis, mulberry leaf scorch, oak leaf scorch, periwinkle wilt, phony peach, or plum leaf scald. Sequence analysis indicated that RAPD fragment PD1-1-2 contains a Ser-tRNA gene. The PD-specific region includes a TaqI restriction site (TCGA) and is 150 bp downstream of the Ser-tRNA gene. Received: 1 March 1999 / Accepted: 5 April 1999     

Ethanol productions from D-xylose and cellobiose by Kluyveromyces cellobiovorus

Yeasts capable of fermenting both D-xylose and cellobiose to ethanol were screened. Of 213 species of yeasts surveyed, Kluyveromyces cellobiovorus sp. nov., a new species belonging to genus of Kluyveromyces, was selected as the sole strain. This strain accumulated 32, 22, and 19 g/L of ethanol from 8% glucose, D-xylose, and cellobiose, respectively. It was also shown that this strain produced ethanol from the enzymatic bagasse hydrolysate containing hexoses and pentoses more efficiently than Saccharomyces cerevisiae.     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

Genome-scale consequences of cofactor balancing in engineered pentose utilization pathways in Saccharomyces cerevisiae

Biofuels derived from lignocellulosic biomass offer promising alternative renewable energy sources for transportation fuels. Significant effort has been made to engineer Saccharomyces cerevisiae to efficiently ferment pentose sugars such as D-xylose and L-arabinose into biofuels such as ethanol through heterologous expression of the fungal D-xylose and L-arabinose pathways. However, one of the major bottlenecks in these fungal pathways is that the cofactors are not balanced, which contributes to inefficient utilization of pentose sugars. We utilized a genome-scale model of S. cerevisiae to predict the maximal achievable growth rate for cofactor balanced and imbalanced D-xylose and L-arabinose utilization pathways. Dynamic flux balance analysis (DFBA) was used to simulate batch fermentation of glucose, D-xylose, and L-arabinose. The dynamic models and experimental results are in good agreement for the wild type and for the engineered D-xylose utilization pathway. Cofactor balancing the engineered D-xylose and L-arabinose utilization pathways simulated an increase in ethanol batch production of 24.7% while simultaneously reducing the predicted substrate utilization time by 70%. Furthermore, the effects of cofactor balancing the engineered pentose utilization pathways were evaluated throughout the genome-scale metabolic network. This work not only provides new insights to the global network effects of cofactor balancing but also provides useful guidelines for engineering a recombinant yeast strain with cofactor balanced engineered pathways that efficiently co-utilizes pentose and hexose sugars for biofuels production. Experimental switching of cofactor usage in enzymes has been demonstrated, but is a time-consuming effort. Therefore, systems biology models that can predict the likely outcome of such strain engineering efforts are highly useful for motivating which efforts are likely to be worth the significant time investment.     

Effect of hydrogen acceptors on D-xylose fermentation by anaerobic culture of immobilized Pachysolen tannophilus cells

The effect of hydrogen acceptors on the kinetic parameters of D-xylose fermentation under anaerobic conditions was studied in a transient culture of immobilized Pachysolen tannophilus cells. Addition of oxygen to a steady-state culture resulted in a rapid increase (up to fivefold) in the rates of ethanol production and D-xylose uptake, but the rate of xylitol production was unaffected. Furthermore, the molar ethanol yield increased from 0.97 to 1.43 in the presence of oxygen. The moles of ethanol produced per moles of oxygen utilized were considerably greater than would be predicted from the stoichiometry of D-xylose fermentation, which suggests that the organism required oxygen for other functions in addition to its role as a hydrogen acceptor in D-xylose metabolism. When the artificial hydrogen acceptors acetone, acetaldehyde, and acetoin were added to the culture, the rate of ethanol production increased while the xylitol production rate decreased but the rate of xylose uptake was unaffected. The molar ethanol yields increased from 1.03 to 1.63, 1.43, and 1.24 upon addition of acetaldehyde, acetone, and acetoin, respectively, at the expense of the molar xylitol yields. The hydrogen acceptors sodium acetate, methylene blue, benzyl viologen, phenazine methosulfate, indigo carmine, and tetrazolium chloride had no effect on ethanol production.     

Continuous conversion of D-xylose to ethanol by immobilized pachysolen tannophilus

The yeast Pachysolen tannophilus was entrapped in calcium alginate beads to ferment D-xylose on a continous basis in the presence of high cell densities. Experimental operating variables included the feed D-xylose concentration, the dilution rate, and the fermentor biomass concentration. Under favorable operating conditions, cultures retained at least 50% of their initial productivity after 26 days of operation. The specific ehanol production rate was dependent on the substrate level in the fermentor, passing through an optimum when the D-xylose concentration was between 28 and 35 g/L. Consequently, reactor productivity increased with dilution rate and feed D-xylose concentration until a maximum was reached. The ethanol content of the effluent always decreased with increasing dilution rate, but excessive dilution rates diminished the ethanol content without increasing productivity. Unlike production rate, ethanol yield declined monotonically from 0.35 g/g as the fermentor substrate concentration increased. The yield was 69% of that theoretically possible when the D-xylose concentration was near zero, as opposed to 42% when it was in the range supporting the optimum specific rate of ethanol production. As long as D-xylose was supplied to cells faster than they could consume it, productivity increased with the mass of cells immobilized. The effectiveness factor associated with the calcium alginte beads used in this system was 0.4, indicating that only 40% of the entrapped biomass was effective in converting D-xylose to ethanol because of diffusion limitations.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Effect of nitrogen sources on xylitol production from D-xylose by Candida Sp. L-102

Summary The production of extracellular xylitol from D-xylose by an efficient xylitol-producing yeast, Candida sp. L-102, was studied in shake flask cultures with different nitrogen sources in the basic salt medium. Maximum xylitol production was obtained with urea as the nitrogen source. A final concentration of 100 g/L of xylitol from 114 g/L D-xylose was obtained from the yeast with an indicated yield of 87.7% (based on D-xylose consumed). The average specific xylitol production rate of 0.46 g/g.h was achieved within 65 hours of incubation using 0.3% urea.     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.     

Bioethanol fermentation by recombinant E. coli FBR5 and its robust mutant FBHW using hot-water wood extract hydrolyzate as substrate

Hemicellulose is a potential by-product currently under-utilized in the papermaking industry. It is a hetero-carbohydrate polymer. For hardwood hemicelluloses, D-xylose is the major component upon depolymerization. At SUNY-ESF, wood extracts were obtained by extracting sugar maple wood chips with hot water at an elevated temperature. The wood extracts were then concentrated and acid hydrolyzed. Ethanologenic bacteria, E. coli FBR5, had a good performance in pure xylose medium for ethanol production. However, FBR5 was strongly inhibited in dilute sulfuric acid hydrolyzate of hot-water wood extract. FBR5 was challenged by hot-water wood extract hydrolyzate in this study. After repeated strain adaptation, an improved strain: E. coli FBHW was obtained. Fermentation experiments indicated that FBHW was resistant to the toxicity of hydrolyzate in the fermentation media of concentrated hydrolyzate, and xylose was completely utilized by the strain to produce ethanol. FBHW was grown in the concentrated hydrolyzate without any detoxification treatment and has yielded 36.8 g/L ethanol.     

D-Xylose fermentation to ethanol bySchizosaccharomyces pombe cloned with xylose isomerase gene

Summary Schizosaccharomyces pombe cloned with the xylose isomerase gene from E. coli is able to grow on YNB and YMP broths containing xylose as the sole carbon source. This yeast can ferment D-xylose to ethanol directly; however, the ethanol production rate and the yield were dependent on the nitrogen source. With the YMP broth as a nitrogen source, the final ethanol concentration can reach 3.7% (w/v), and the ethanol yield was 80% of the theoretical value based on the amount of xylose that was metabolized. The ethanol production is slow, and the xylitol production is still very active; apparently, the limiting step is the isomerization of xylose to xylulose.     

Metabolic engineering of Saccharomyces cerevisiae for bioconversion of D-xylose to D-xylonate

An NAD(+)-dependent D-xylose dehydrogenase, XylB, from Caulobacter crescentus was expressed in Saccharomyces cerevisiae, resulting in production of 17 ± 2 g D-xylonate l(-1) at 0.23 gl(-1)h(-1) from 23 g D-xylose l(-1) (with glucose and ethanol as co-substrates). D-Xylonate titre and production rate were increased and xylitol production decreased, compared to strains expressing genes encoding T. reesei or pig liver NADP(+)-dependent D-xylose dehydrogenases. D-Xylonate accumulated intracellularly to ～70 mgg(-1); xylitol to ～18 mgg(-1). The aldose reductase encoding gene GRE3 was deleted to reduce xylitol production. Cells expressing D-xylonolactone lactonase xylC from C. crescentus with xylB initially produced more extracellular D-xylonate than cells lacking xylC at both pH 5.5 and pH 3, and sustained higher production at pH 3. Cell vitality and viability decreased during D-xylonate production at pH 3.0. An industrial S. cerevisiae strain expressing xylB efficiently produced 43 g D-xylonate l(-1) from 49 g D-xylose l(-1).     

Engineering of ethanolic E. coli with the Vitreoscilla hemoglobin gene enhances ethanol production from both glucose and xylose

Escherichia coli strain FBR5, which has been engineered to direct fermentation of sugars to ethanol, was further engineered, using three different constructs, to contain and express the Vitreoscilla hemoglobin gene (vgb). The three resulting strains expressed Vitreoscilla hemoglobin (VHb) at various levels, and the production of ethanol was inversely proportional to the VHb level. High levels of VHb were correlated with an inhibition of ethanol production; however, the strain (TS3) with the lowest VHb expression (approximately the normal induced level in Vitreoscilla) produced, under microaerobic conditions in shake flasks, more ethanol than the parental strain (FBR5) with glucose, xylose, or corn stover hydrolysate as the predominant carbon source. Ethanol production was dependent on growth conditions, but increases were as high as 30%, 119%, and 59% for glucose, xylose, and corn stover hydrolysate, respectively. Only in the case of glucose, however, was the theoretical yield of ethanol by TS3 greater than that achieved by others with FBR5 grown under more closely controlled conditions. TS3 had no advantage over FBR5 regarding ethanol production from arabinose. In 2 L fermentors, TS3 produced about 10% and 15% more ethanol than FBR5 for growth on glucose and xylose, respectively. The results suggest that engineering of microorganisms with vgb/VHb could be of significant use in enhancing biological production of ethanol.     

不同信号肽对酿酒酵母蔗糖转化酶Suc2分泌表达水平的影响

目前，绝大多数酿酒酵母（Saccharomyces cerevisiae）菌株利用菊糖生产乙醇的能力有限，而蔗糖转化酶Suc2是酿酒酵母水解菊糖的关键酶，其分泌水平直接影响酿酒酵母转化菊糖为乙醇的性能。为提高酿酒酵母中蔗糖转化酶Suc2的分泌表达水平，利用生物信息学的分析方法选择出11种不同的分泌信号肽，包括酿酒酵母内源性、其他菌株来源以及已报道序列优化改造的信号肽，将它们融合至Suc2并构建了相应的酿酒酵母BY4741重组菌。其中，酿酒酵母内源分泌信号肽AGA2能使蔗糖转化酶Suc2更有效的分泌，含有信号肽AGA2的重组菌BY-AG的蔗糖酶酶活和菊糖酶酶活相对于含有天然信号肽的原始菌BY-S分别提高42%和26%，其利用菊糖产乙醇能力较原始菌提高了32%，乙醇产量达到78.11 g/L。在使用毕赤酵母（Pichia pastoris）分泌信号肽MSB2时，蔗糖转化酶Suc2的分泌水平也有提高，含有信号肽MSB2的重组菌BY-MS较原始菌BY-S的蔗糖酶酶活和菊糖酶酶活分别提高了80%和74%，同时，利用菊糖产乙醇能力也提高了56%，产量达到86.31 g/L。最后，对重组菌BY-MS摇瓶发酵过程中的生物量、蔗糖酶酶活、残糖总量和乙醇产量进行了监测，结果表明，重组菌BY-MS的发酵性能较原始菌BY-S有显著提高。本研究为提高蔗糖转化酶Suc2的分泌水平、构建高效菊糖基乙醇生产菌株提供参考。     

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

The yeast Pachysolen tannophilus was found to be capable of converting D-xylose to ethanol. Batch cultures initially containing 50 g/L D-xylose yielded 0.34 g of ethanol per gram of pentose consumed. Aerobic conditions were required for cell growth but not for ethanol production. Both alcohol formation and growth were optimum when incubation temperature was 32 degrees C, when pH was near 2.5, and when D-xylose and ethanol concentrations did not exceed 50 and 20 g/L, respectively.