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.     

N‐terminal fusion of a hyperthermophilic chitin‐binding domain to xylose isomerase from Thermotoga neapolitana enhances kinetics and thermostability of both free and immobilized enzymes

Immobilization of a thermostable D ‐xylose isomerase (EC 5.3.1.5) from Thermotoga neapolitana 5068 (TNXI) on chitin beads was accomplished via a N‐terminal fusion with a chitin‐binding domain (CBD) from a hyperthermophilic chitinase produced by Pyrococcus furiosus (PF1233) to create a fusion protein (CBD‐TNXI). The turnover numbers for glucose to fructose conversion for both unbound and immobilized CBD‐TNXI were greater than the wild‐type enzyme: k_cat (min^?1) was ～1,000, 3,800, and 5,800 at 80°C compared to 1,140, 10,350, and 7,000 at 90°C, for the wild‐type, unbound, and immobilized enzymes, respectively. These k_cat values for the glucose to fructose isomerization measured are the highest reported to date for any XI at any temperature. Enzyme kinetic inactivation at 100°C, as determined from a bi‐phasic inactivation model, showed that the CBD‐TNXI bound to chitin had a half‐life approximately three times longer than the soluble wild‐type TNXI (19.9 hours vs. 6.8 hours, respectively). Surprisingly, the unbound soluble CBD‐TNXI had a significantly longer half‐life (56.5 hours) than the immobilized enzyme. Molecular modeling results suggest that the N‐terminal fusion impacted subunit interactions, thereby contributing to the enhanced thermostability of both the unbound and immobilized CBD‐TNXI. These interactions likely also played a role in modifying active site structure, thereby diminishing substrate‐binding affinities and generating higher turnover rates in the unbound fusion protein. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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.     

Interactions between Clostridium beijerinckii and Geobacter metallireducens in co‐culture fermentation with anthrahydroquinone‐2, 6‐disulfonate (AH2QDS) for enhanced biohydrogen production from xylose

To enhance biohydrogen production, Clostridium beijerinckii was co‐cultured with Geobacter metallireducens in the presence of the reduced extracellular electron shuttle anthrahydroquinone‐2, 6‐disulfonate (AH₂QDS). In the co‐culture system, increases of up to 52.3% for maximum cumulative hydrogen production, 38.4% for specific hydrogen production rate, 15.4% for substrate utilization rate, 39.0% for substrate utilization extent, and 34.8% for hydrogen molar yield in co‐culture fermentation were observed compared to a pure culture of C. beijerinckii without AH₂QDS. G. metallireducens grew in the co‐culture system, resulting in a decrease in acetate concentration under co‐culture conditions and a presumed regeneration of AH₂QDS from AQDS. These co‐culture results demonstrate metabolic crosstalk between the fermentative bacterium C. beijerinckii and the respiratory bacterium G. metallireducens and suggest a strategy for industrial biohydrogen production. Biotechnol. Bioeng. 2013; 110: 164–172. © 2012 Wiley Periodicals, Inc.     

木糖异构酶在酿酒酵母细胞表面的展示

将来源于嗜热细菌Thermus thermophilus的木糖异构酶基因xylA,与酿酒酵母(Sac-charomyces cerevisiae)a-凝集素表面展示载体pYD1的Aga2p亚基C端序列融合。编码融合蛋白的基因序列前接上半乳糖诱导型启动子。用LiAc完整细胞法转化酿酒酵母EBY100。含重组质粒的菌株EBY100/pYD-xylA经半乳糖诱导表达外源融合蛋白,免疫荧光显微镜结果显示外源蛋白被锚定在细胞壁上,木糖异构酶活性测定结果表明,细胞壁上酶活测定值为1.52U,木糖异构酶在酿酒酵母细胞壁上得到活性表达。     

A Bacillus subtilis 168 mutant with increased xylose uptake can utilize xylose as sole carbon source

Abstract Bacillus subtilis 168 is unable to effectively utilize xylose as sole carbon source. We demonstrate here that this strain cannot actively transport xylose into the cell. After leaving B. subtilis 168 for a few days on minimal plates with xylose as sole carbon source large colonies arise with a frequency of 1 × 10⁻⁶/cell. These mutants grow well on xylose and efficiently take up that sugar. This new property is not inducible by xylose, indicating that the mutation is neither in the xyl nor in the xyn operon.     

Enhanced direct ethanol production by cofactor optimization of cell surface‐displayed xylose isomerase in yeast

Xylose isomerase (XylC) from Clostridium cellulovorans can simultaneously perform isomerization and fermentation of d ‐xylose, the main component of lignocellulosic biomass, and is an attractive candidate enzyme. In this study, we optimized a specified metal cation in a previously established Saccharomyces cerevisiae strain displaying XylC. We investigated the effect of each metal cation on the catalytic function of the XylC‐displaying S. cerevisiae. Results showed that the divalent cobalt cations (Co²⁺) especially enhanced the activity by 46‐fold. Co²⁺ also contributed to d ‐xylose fermentation, which resulted in improving ethanol yields and xylose consumption rates by 6.0‐ and 2.7‐fold, respectively. Utility of the extracellular xylose isomerization system was exhibited in the presence of mixed sugar. XylC‐displaying yeast showed the faster d ‐xylose uptake than the yeast producing XI intracellularly. Furthermore, direct xylan saccharification and fermentation was performed by unique yeast co‐culture system. A xylan‐degrading yeast strain was established by displaying two kinds of xylanases; endo‐1,4‐β‐xylanase (Xyn11B) from Saccharophagus degradans, and β‐xylosidase (XlnD) from Aspergillus niger. The yeast co‐culture system enabled fine‐tuning of the initial ratios of the displayed enzymes (Xyn11B:XlnD:XylC) by adjusting the inoculation ratios of Xylanases (Xyn11B and XlnD)‐displaying yeast and XylC‐displaying yeast. When the enzymes were inoculated at the ratio of 1:1:2 (1.39 × 10¹³: 1.39 × 10¹³: 2.78 × 10¹³ molecules), 6.0 g/L ethanol was produced from xylan. Thus, the cofactor optimization and the yeast co‐culture system developed in this study could expand the prospect of biofuels production from lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1068–1076, 2017     

Overexpression of bacterial xylose isomerase and yeast host xylulokinase improves xylose alcoholic fermentation in the thermotolerant yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha is able to ferment xylose to ethanol. To improve characteristics of xylose fermentation, the recombinant strain Delta xyl1 Delta xyl2-ADelta xyl2-B, with deletions of genes encoding first enzymes of xylose utilization (NAD(P)H-dependent xylose reductase and NAD-dependent xylitol dehydrogenases, respectively), was constructed and used as a recipient for co-overexpression of the Escherichia coli xylA gene coding for xylose isomerase and endogenous XYL3 gene coding for xylulokinase. The expression of both genes was driven by the H. polymorpha glyceraldehyde-3-phosphate dehydrogenase promoter. Xylose isomerase activities of obtained transformants amounted to approximately 80% of that of the bacterial host strain. Xylulokinase activities of the transformants increased twofold when compared with the parental strain. The recombinant strains displayed improved ethanol production during the fermentation of xylose.     

Kinetic studies of the enzymatic isomerization of xylose

d-Xylose isomerase catalyses the conversion of the common pentose, d-xylose, to its keto-isomer, d-xylose. This reaction is of interest because many microorganisms that are unable to metabolize d-xylose can utilize d-xylulose. The kinetics of a commonly used immobilized whole-cell isomerase, Sweetzyme Q, have been determined from initial rate studies on the forward and reverse reactions. The effect of pH, temperature, and substrate and product concentration on enzyme activity have all been examined. Reaction rates were modelled with the Michaelis-Menten equation. Using constants determined from Lineweaver-Burk plots, the rate equation accurately simulated experimental conversion data.     

Formation mechanism of cross‐linking Maillard compounds in peptide–xylose systems

The formation mechanism of Maillard peptides was explored in Maillard reaction through diglycine/glutathione(GSH)/(Cys‐Glu‐Lys‐His‐Ile‐Met)–xlyose systems by heating at 120 °C for 30–120 min. Maximum fluorescence intensity of Maillard reaction products (MRPs) with an emission wavelength of 420~430 nm in all systems was observed, and the intensity values were proportional to the heating time. Taken diglycine/GSH–[¹³C₅]xylose systems as a control, it was proposed that the compounds with high m/z values of 379 and 616 have the high molecular weight (HMW) products formed by cross‐linking of peptides and sugar. In (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the m/z value of HMW MRPs was not observed, which might be due to the weak signals of these products. According to the results of gel permeation chromatography, HMW MRPs were formed by Maillard reaction, especially in (Cys‐Glu‐Lys‐His‐Ile‐Met)–xylose system, the percentage of Maillard peptides reached 52.90%. It was concluded that Maillard peptides can be prepared through the cross‐linking of sugar and small peptides with a certain MW range. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

木糖发酵生产乙醇的研究

选育出一株优良的木糖发酵菌株树干毕赤酵母菌7124,并利用纯木糖优化了木糖发酵条件,利用海藻酸钠固定化树干毕赤酵母菌增殖细胞,不仅能较好满足限氧发酵条件,而且能耐较高糖浓度,使乙醇发酵浓度提高到20g/L。利用半纤维素水解液进行了乙醇发酵的初步研究,基本达到了纯木糖发酵的效果。