NAD+-dependent xylitol dehydrogenase (xdhA) and l-arabitol-4-dehydrogenase (ladA) deletion mutants of Aspergillus oryzae for improved xylitol production

Xylitol dehydrogenase (XDHA) and l-arabitol dehydrogenase (LADA) are two key enzymes in xylan metabolism catalyzing the oxidation of xylitol to d-xylulose and arabitol to l-xylulose, respectively. In Aspergillus oryzae, XDHA and LADA are encoded by xdhA and ladA. We deleted xdhA and ladA and xdhA–ladA to generate mutants with decreased dehydrogenase activities and increased xylitol production. The mutants were constructed by homologous transformation into A. oryzae P4 (?pyrG) using pyrG as a selectable marker. The xylitol productivity of the mutants was measured using d-xylose as the sole carbohydrate source. xdhA, ladA, and the double-deletion mutant produced, respectively, 12.4 g xylitol/l with a yield of 0.24 g/g d-xylose, 12.4 g/l with a yield of 0.33 g/g d-xylose, and 8.6 g/l at a yield of 0.26 g/g d-xylose.     

Genetically engineered Pichia pastoris yeast for conversion of glucose to xylitol by a single-fermentation process

Xylitol is industrially synthesized by chemical reduction of d-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce d-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of d-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the d-xylulose-forming d-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a single-fermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078 g xylitol/g glucose and productivity of 0.29 g xylitol/L h. This was the first report to convert xylitol from glucose by the pathway of glucose–d-arabitol–d-xylulose–xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.     

Cloning, Heterologous Expression, and Characterization of the Xylitol and l-Arabitol Dehydrogenase Genes, Texdh and Telad, from the Thermophilic Fungus Talaromyces emersonii

The genes encoding xylitol dehydrogenase (Texdh) and l-arabitol dehydrogenase (Telad) are involved in the fungal pentose pathway and were isolated from the thermophilic fungus Talaromyces emersonii, expressed in Escherichia coli, and the products purified to homogeneity. TeXDH showed activity toward xylitol and d-sorbitol. TeLAD was active with l-arabitol, xylitol, and d-sorbitol. Phylogenetic analysis showed TeLAD has evolved from d-sorbitol dehydrogenase as a result of environmental adaptation. Substrate specificity studies indicate that TeXDH is likely to have evolved from the more broadly acting TeLAD. Texdh and Telad expression was inducible by the same carbon sources responsible for induction of genes involved in biomass degradation, suggesting for the first time a coordinated regulatory control mechanism for expression of genes encoding extracellular hydrolases and intracellular metabolic genes in the pentose utilization pathways of T. emersonii. These data also suggest that TeXDH and TeLAD may be valuable in the production of xylitol, l-arabitol, and ethanol from renewable resources rich in pentose sugars.     

Biosynthesis of ethylene glycol in Escherichia coli

Ethylene glycol (EG) is an important platform chemical with steadily expanding global demand. Its commercial production is currently limited to fossil resources; no biosynthesis route has been delineated. Herein, a biosynthesis route for EG production from d-xylose is reported. This route consists of four steps: d-xylose?→?d-xylonate?→?2-dehydro-3-deoxy-d-pentonate?→?glycoaldehyde?→?EG. Respective enzymes, d-xylose dehydrogenase, d-xylonate dehydratase, 2-dehydro-3-deoxy-d-pentonate aldolase, and glycoaldehyde reductase, were assembled. The route was implemented in a metabolically engineered Escherichia coli, in which the d-xylose?→?d-xylulose reaction was prevented by disrupting the d-xylose isomerase gene. The most efficient construct produced 11.7 g?L^?1 of EG from 40.0 g?L^?1 of d-xylose. Glycolate is a carbon-competing by-product during EG production in E. coli; blockage of glycoaldehyde?→?glycolate reaction was also performed by disrupting the gene encoding aldehyde dehydrogenase, but from this approach, EG productivity was not improved but rather led to d-xylonate accumulation. To channel more carbon flux towards EG than the glycolate pathway, further systematic metabolic engineering and fermentation optimization studies are still required to improve EG productivity.     

Characterization of the sugar alcohol-producing yeast Pichia anomala

Sugar alcohols have been widely applied in the field of food and medicine for their unique properties. Compared to chemical production, microbial production of sugar alcohol has become attractive for its environmental and sustainable pattern. In this study, a potential yeast isolated from soil of Beijing suburbs was identified as Pichia anomala TIB-x229, and its key enzyme of d-arabitol dehydrogenase for microbial production of sugar alcohols was functionally characterized. This yeast could simultaneously produce d-arabitol, xylitol, and/or ribitol from a different ratio of sugar substrates at a high efficiency by bioconversion, and no glucose repression happened when mixed sugars of xylose and glucose were used as the substrates during the bioconversion. This yeast could also efficiently convert complicated feedstock such as xylose mother liquor to d-arabitol, xylitol, and ribitol with 55 % yields. To elucidate the conversion relationship of the sugar alcohols, especially d-arabitol and xylitol, the key d-arabitol dehydrogenase gene from P. anomala was cloned, expressed and purified for further in vitro characterization. The results showed that this d-arabitol dehydrogenase could catalyze arabitol to xylulose further, which is significant for xylitol production from glucose. Our study laid the foundation for improving the production of sugar alcohols by metabolic and fermentation engineering strategies.     

Properties of the sugar carrier in Baker's yeast

Incubation ofSaccharomyces cerevisiae cells withd-galactose induced the formation of galactose-utilizing enzymes, among them a monosaccharide carrier, apparently synthesized as a proteinde novo. The synthesis of the carrier preceded that of galactokinase by as much as 2 h. The inducible carrier shows a preference for monosaccharides with an axial hydroxyl group at carbon 4 of theC1 chair conformation or at carbon 2 of the1C chair conformation. Through its mediation, some sugars normally poorly transported (d-galactose,d-fucose,l-xylose,l-arabinose) can enter into the entire cell water, occupying then one more kinetic (and morphological ?) compartment than before induction. Some other monosaccharides, readily transported even by a constitutive carrier system (e.g.l-sorbose,d-xylose,d-arabinose) share the newly synthesized carrier.     

Taxonomic characteristic of the strain ETH 7437 producing granaticin

The streptomycete strain ETH 7437, producing granaticin, was characterized according to present taxonomic standards and compared with the strainsStreptomyces olivaceus (Waksman) Waksman and Henrici,Streptomyces coelicolor (Müller) andStreptomyces violaceruber (Waksman & Curtis). The strain ETH 7437 formed gray aerial mycelium, straight and flexuos sporophores, even single hooks and open spirals. Normally the amount of spores in chains was over 50, the surface of spores being smooth. The strain utilizedd-glucose,d-arabinose,d-xylose andd-fructose. The strain was capable of forming antibiotic pigment stably. For the strain ETH 7437 the termStreptomyces granaticolor was proposed.     

Effect of sugars on the morphogenesis ofAureobasidium pullulans

The dominance of individual elements of the vegetative fructification of five selected strains of the polymorphic organismAureobasidium pullulans (de Baby)Arnaud was studied in media with basic assimilable sugars,d-glucose,d-galactose,d-xylose, maltose, sucrose, lactose and a mixture ofl -arabinose andd-mannitol. Pronounced differences between cultures grown in the presence of monosaccharides and those cultivated in the presence of disaccharides were detected.     

Engineering lower inhibitor affinities in β-d-xylosidase

β-d-Xylosidase catalyzes hydrolysis of xylooligosaccharides to d-xylose residues. The enzyme, SXA from Selenomonas ruminantium, is the most active catalyst known for the reaction; however, its activity is inhibited by d-xylose and d-glucose (K _i values of ～10^?2?M). Higher K _i’s could enhance enzyme performance in lignocellulose saccharification processes for bioethanol production. We report here the development of a two-tier high-throughput screen where the 1° screen selects for activity (active/inactive screen) and the 2° screen selects for a higher K _i(d-xylose) and its subsequent use in screening ～5,900 members of an SXA enzyme library prepared using error-prone PCR. In one variant, termed SXA-C3, K _i(d-xylose) is threefold and K _i(d-glucose) is twofold that of wild-type SXA. C3 contains four amino acid mutations, and one of these, W145G, is responsible for most of the lost affinity for the monosaccharides. Experiments that probe the active site with ligands that bind only to subsite ?1 or subsite +1 indicate that the changed affinity stems from changed affinity for d-xylose in subsite +1 and not in subsite ?1 of the two-subsite active site. Trp145 is 6 Å from the active site, and its side chain contacts three active-site residues, two in subsite +1 and one in subsite ?1.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Biochemical and kinetic characterization of GH43 β-d-xylosidase/α-l-arabinofuranosidase and GH30 α-l-arabinofuranosidase/β-d-xylosidase from rumen metagenome

The present study focuses on characterization of two hemicellulases, RuXyn1 and RuXyn2, from rumen bacterial metagenome and their capabilities for degradation of xylans. Glycosyl hydrolase (GH) family?43 ??-d-xylosidase/??-l-arabinofuranosidase RuXyn1 can hydrolyze p-nitrophenyl-??-d-xylopyranoside (pNPX), p-nitrophenyl-??-l-arabinofuranoside (pNPA), and xylo-oligosaccharide substrates, while GH30 1,5-??-l-arabinofuranosidase/??-d-xylosidase RuXyn2, the first ??-l-arabinofuranosidase assigned to this GH family, shows activities towards 1,5-??-l-arabinobiose and pNPX substrates but no activity for pNPA. Kinetic analysis for aryl-glycosides revealed that RuXyn2 had higher catalytic efficiency than RuXyn1 toward pNPX substrate. RuXyn1 shows high synergism with endoxylanase, elevating by 73% the reducing sugars released from brichwood xylans, and converted most intermediate xylo-oligosaccharide hydrolysate into xylose. The high xylose conversion capability of RuXyn1 suggests it has potential applications in enzymatic production of xylose and improvement of hemicellulose saccharification for production of biofuels. RuXyn2 shows no obviously synergistic effect in the endoxylanase-coupled assay for enzymatic saccharification of xylan. Further cosmid DNA sequencing revealed a neighboring putative GH43 ??-l-arabinofuranosidase RuAra1 and two putative GH3 ??-xylosidase/arabinosidases, RuXyn3 and RuXyn5, downstream of RuXyn2, indicating that this hemicellulase gene cluster may be responsible for production of end-product, xylose and arabinose, from hemicellulose biomass.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

Molecular analysis of NAD+-dependent xylitol dehydrogenase from the zygomycetous fungus Rhizomucor pusillus and reversal of the coenzyme preference

The zygomycetous fungus Rhizomucor pusillus NBRC 4578 is able to ferment not only d-glucose but also d-xylose into ethanol. Xylitol dehydrogenase from R. pusillus NBRC 4578 (RpXDH), which catalyzes the second step of d-xylose metabolism, was purified, and its enzymatic properties were characterized. The purified RpXDH preferred NAD⁺ as its coenzyme and showed substrate specificity for xylitol, d-sorbitol, and ribitol. cDNA cloning of xyl2 gene encoding RpXDH revealed that the gene included a coding sequence of 1,092?bp with a molecular mass of 39,185?kDa. Expression of the xyl2 in R. pusillus NBRC 4578 was induced by d-xylose, and the expression levels were increased with accumulation of xylitol. The xyl2 gene was expressed in Escherichia coli, and coenzyme preference of the recombinant RpXDH was reversed from NAD⁺ to NADP⁺ in the double mutant D205A/I206R by site-directed mutagenesis.     

Improved Protease Stability of the Antimicrobial Peptide Pin2 Substituted with d-Amino Acids

Cationic antimicrobial peptides (AMPs) have attracted a great interest as novel class of antibiotics that might help in the treatment of infectious diseases caused by pathogenic bacteria. However, some AMPs with high antimicrobial activities are also highly hemolytic and subject to proteolytic degradation from human and bacterial proteases that limit their pharmaceutical uses. In this work a d-diastereomer of Pandinin 2, d-Pin2, was constructed to observe if it maintained antimicrobial activity in the same range as the parental one, but with the purpose of reducing its hemolytic activity to human erythrocytes and improving its ability to resist proteolytic cleavage. Although, the hydrophobic and secondary structure characteristics of l- and d-Pin2 were to some extent similar, an important reduction in d-Pin2 hemolytic activity (30–40 %) was achieved compared to that of l-Pin2 over human erythrocytes. Furthermore, d-Pin2 had an antimicrobial activity with a MIC value of 12.5 μM towards Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae and two strains of Pseudomonas aeruginosa in agar diffusion assays, but it was half less potent than that of l-Pin2. Nevertheless, the antimicrobial activity of d-Pin2 was equally effective as that of l-Pin2 in microdilution assays. Yet, when d- and l-Pin2 were incubated with trypsin, elastase and whole human serum, only d-Pin2 kept its antimicrobial activity towards all bacteria, but in diluted human serum, l- and d-Pin2 maintained similar peptide stability. Finally, when l- and d-Pin2 were incubated with proteases from P. aeruginosa DFU3 culture, a clinical isolated strain, d-Pin2 kept its antibiotic activity while l-Pin2 was not effective.     

Enhanced saccharification of rice straw using hypochlorite-hydrogen peroxide

Rice straw is a lignocellulosic biomass, and has been recognized as a renewable organic substance and alternative energy source. In this study, rice straw was pretreated with hypochlorite-hydrogen peroxide (Ox-B) solution. The optimal pretreatment conditions were determined via response surface methodology, and the pretreated rice straw was hydrolyzed with exo-glucanase, endoglucanase, hemicellulase, and β-glucosidase Accellerase 1000? (endo-glucanase equivalent activity of 1,250 carboxy methyl cellulose (CMC) U/g of rice straw pretreated for 24 h). The optimal conditions were as follows: 60 min pretreatment using Ox-B solution containing 0.6% hypochlorite and 25% hydrogen peroxide for 1 g of rice straw in a total reaction volume of 240 mL. Under these conditions, 406.8 mg of d-glucose and 224.0 mg of d-xylose were obtained from 1 g of rice straw. The fermentation of enzymatic hydrolysates containing 8.14 g/L d-glucose and 4.49 g/L d-xylose with Pichia stipitis generated 3.65 g/L of ethanol with a corresponding yield of 0.37 g/g. The maximum possible ethanol conversion rate is 72.54%.     

d-Amino Acid-Induced Expression of d-Amino Acid Oxidase in the Yeast Schizosaccharomyces pombe

We investigated d-amino acid oxidase (DAO) induction in the popular model yeast Schizosaccharomyces pombe. The product of the putative DAO gene of the yeast expressed in E.?coli displayed oxidase activity to neutral and basic d-amino acids, but not to an l-amino acid or acidic d-amino acids, showing that the putative DAO gene encodes catalytically active DAO. DAO activity was weakly detected in yeast cells grown on a culture medium without d-amino acid, and was approximately doubled by adding d-alanine. The elimination of ammonium chloride from culture medium induced activity by up to eight-fold. l-Alanine also induced the activity, but only by about half of that induced by d-alanine. The induction by d-alanine reached a maximum level at 2?h cultivation; it remained roughly constant until cell growth reached a stationary phase. The best inducer was d-alanine, followed by d-proline and then d-serine. Not effective were N-carbamoyl-d,l-alanine (a better inducer of DAO than d-alanine in the yeast Trigonopsis variabilis), and both basic and acidic d-amino acids. These results showed that S. pombe DAO could be a suitable model for analyzing the regulation of DAO expression in eukaryotic organisms.     

Deletion of FPS1, Encoding Aquaglyceroporin Fps1p,Improves Xylose Fermentation by Engineered Saccharomyces cerevisiae

Accumulation of xylitol in xylose fermentation with engineered Saccharomyces cerevisiae presents a major problem that hampers economically feasible production of biofuels from cellulosic plant biomass. In particular, substantial production of xylitol due to unbalanced redox cofactor usage by xylose reductase (XR) and xylitol dehydrogenase (XDH) leads to low yields of ethanol. While previous research focused on manipulating intracellular enzymatic reactions to improve xylose metabolism, this study demonstrated a new strategy to reduce xylitol formation and increase carbon flux toward target products by controlling the process of xylitol secretion. Using xylitol-producing S. cerevisiae strains expressing XR only, we determined the role of aquaglyceroporin Fps1p in xylitol export by characterizing extracellular and intracellular xylitol. In addition, when FPS1 was deleted in a poorly xylose-fermenting strain with unbalanced XR and XDH activities, the xylitol yield was decreased by 71% and the ethanol yield was substantially increased by nearly four times. Experiments with our optimized xylose-fermenting strain also showed that FPS1 deletion reduced xylitol production by 21% to 30% and increased ethanol yields by 3% to 10% under various fermentation conditions. Deletion of FPS1 decreased the xylose consumption rate under anaerobic conditions, but the effect was not significant in fermentation at high cell density. Deletion of FPS1 resulted in higher intracellular xylitol concentrations but did not significantly change the intracellular NAD⁺/NADH ratio in xylose-fermenting strains. The results demonstrate that Fps1p is involved in xylitol export in S. cerevisiae and present a new gene deletion target, FPS1, and a mechanism different from those previously reported to engineer yeast for improved xylose fermentation.     

Isolation of proline-based cyclic dipeptides from Bacillus sp. N strain associated with rhabitid entomopathogenic nematode and its antimicrobial properties

Entomopathogenic nematodes (EPN) are well-known as biological control agents and are found to have associated bacteria which can produce a wide range of bioactive secondary metabolites. We report herewith isolation of six proline containing cyclic dipeptides cyclo(d-Pro-l-Leu), cyclo(l-Pro-l-Met), cyclo(d-Pro-l-Phe), cyclo(l-Pro-l-Phe), cyclo(l-Pro-l-Tyr) and cyclo(l-Pro-d-Tyr) from ethyl acetate extract of the Luria Broth (LB) cell free culture filtrate of Bacillus sp. strain N associated with a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute farm. Antimicrobial studies of these 2,5-diketopiperazines (DKPs) against both medicinally and agriculturally important bacterium and fungi showed potent inhibitory values in the range of μg/mL. Cyclic dipeptides showed significantly higher activity than the commercial fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani, and Pencillium expansum. The highest activity of 2 μg/mL by cyclo(l-Pro-l-Phe) was recorded against P. expansum, a plant pathogen responsible for causing post harvest decay of stored apples and oranges. To our knowledge, this is the first report on the isolation of these DKPs from Rhabditis EPN bacterial strain Bacillus sp.