Improved ethanol productivity from lignocellulosic hydrolysates by Escherichia coli with regulated glucose utilization.

BACKGROUND: Lignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge. RESULTS: An engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed. In E. coli B0013-2021HPA, pta-ackA, ldhA and pflB were deleted to block the formation of acetate, lactate and formate and additional three mutations at glk, ptsG and manZ generated to block the glucose uptake and catabolism, followed by the replacement of the wild-type frdA locus with the ptsG expression cassette under the control of the temperature-inducible λ pR and pL promoters, and the final introduction of pEtac-PA carrying Zymomonas mobilis pdc and adhB for the ethanol pathway. B0013-2021HPA was able to utilize almost all xylose, galactose and arabinose but not glucose for cell propagation at 34 °C and converted all sugars to ethanol at 42 °C under oxygen-limited fermentation conditions. CONCLUSIONS: Engineered E. coli strain with regulated glucose utilization showed efficient metabolism of mixed sugars in lignocellulosic hydrolysates and thus higher productivity of ethanol production.

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate.

OBJECTIVES: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery. RESULTS: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate. CONCLUSIONS: By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.

Optimization of cultivation medium and cyclic fed-batch fermentation strategy for enhanced polyhydroxyalkanoate production by Bacillus thuringiensis using a glucose-rich hydrolyzate.

The accumulation of petrochemical plastic waste is detrimental to the environment. Polyhydroxyalkanoates (PHAs) are bacterial-derived polymers utilized for the production of bioplastics. PHA-plastics exhibit mechanical and thermal properties similar to conventional plastics. However, high production cost and obtaining high PHA yield and productivity impedes the widespread use of bioplastics. This study demonstrates the concept of cyclic fed-batch fermentation (CFBF) for enhanced PHA productivity by Bacillus thuringiensis using a glucose-rich hydrolyzate as the sole carbon source. The statistically optimized fermentation conditions used to obtain high cell density biomass (OD600 of 2.4175) were: 8.77 g L-1 yeast extract; 66.63% hydrolyzate (v/v); a fermentation pH of 7.18; and an incubation time of 27.22 h. The CFBF comprised three cycles of 29 h, 52 h, and 65 h, respectively. After the third cyclic event, cell biomass of 20.99 g L-1, PHA concentration of 14.28 g L-1, PHA yield of 68.03%, and PHA productivity of 0.219 g L-1 h-1 was achieved. This cyclic strategy yielded an almost threefold increase in biomass concentration and a fourfold increase in PHA concentration compared with batch fermentation. FTIR spectra of the extracted PHAs display prominent peaks at the wavelengths unique to PHAs. A copolymer was elucidated after the first cyclic event, whereas, after cycles CFBF 2-4, a terpolymer was noted. The PHAs obtained after CFBF cycle 3 have a slightly higher thermal stability compared with commercial PHB. The cyclic events decreased the melting temperature and degree of crystallinity of the PHAs. The approach used in this study demonstrates the possibility of coupling fermentation strategies with hydrolyzate derived from lignocellulosic waste as an alternative feedstock to obtain high cell density biomass and enhanced PHA productivity.

Crystal structure of a thermophilic fungal cyanase and its implications on the catalytic mechanism for bioremediation.

Cyanase catalyzes the bicarbonate-dependent degradation of cyanate to produce ammonia and carbon dioxide, and ammonia is a considerable alternative nitrogen source. Strikingly, the cyanase from the thermophilic fungus Thermomyces lanuginosus (Tl-Cyn) has the highest catalytic efficiency reported among these enzymes. However, its molecular mechanism of action is not clearly understood, because currently there is no structural information available on fungal cyanases. Here we report the crystal structure of Tl-Cyn in complex with inhibitors malonate and formate at 2.2 Å resolution. The structure reveals extensive interactions at the subunit interfaces in a dimer, and a decamer is formed by a pentamer of these dimers. Our biochemical, kinetic and mutagenesis studies confirm the structural observations on the complex and provide further insights into its catalytic mechanism and inhibition. The structure has also aided the creation of a mutant enzyme with enhanced catalytic activity, and such enzymes may have the potential for biotechnological applications, including biotransformation and bioremediation. Moreover, other fungal cyanases with potentially high catalytic activity could also be predicted based on the Tl-Cyn structure, as the active site region among fungal cyanases are highly conserved.

Simultaneous removal of heavy metals and cyanate in a wastewater sample using immobilized cyanate hydratase on magnetic-multiwall carbon nanotubes.

Global environmental problems allied with waste management require novel approaches for the simultaneous removal of heavy metals and other associated compounds including cyanate. In this study, iron-oxide filled multi-walled carbon nanotubes (m-MWCNTs) were successfully synthesized and characterized by field emission gun scanning electron microscopy (FEGSEM), high-resolution transmission electron microscopy (HRTEM) and X-ray diffraction (XRD). The m-MWCNTs were amino-functionalized for the covalent immobilization of a recombinant cyanate hydratase (rTl-Cyn), and were characterized by fourier transform infrared (FTIR) spectroscopy. The immobilized rTl-Cyn on the m-MWCNTs (m-MWCNT-rTl-Cyn) had long term storage stability and showed great potential towards cyanate biodegradability. We found that m-MWCNT-rTl-Cyn retained >94% of the initial activity even after 10 repeated cycles of bio-catalysis. Strikingly, the m-MWCNT-rTl-Cyn simultaneously reduced the concentration of chromium (Cr), iron (Fe), lead (Pb) and copper (Cu) by 39.31, 35.53, 34.48 and 29.63%, respectively as well as the concentration of cyanate by ≥84%, in a synthetic wastewater sample.

A novel strategy for the efficient removal of toxic cyanate by the combinatorial use of recombinant enzymes immobilized on aminosilane modified magnetic nanoparticles.

Cyanase detoxifies cyanate by transforming it to ammonia and carbon dioxide in a bicarbonate-dependent reaction, however, dependence on bicarbonate limits its utilization in large-scale applications. A novel strategy was therefore developed for overcoming this bottleneck by the combined application of cyanase (rTl-Cyn) and carbonic anhydrase (rTl-CA). The synergistic effect of rTl-Cyn and rTl-CA could reduce the dependence of bicarbonate by 80%, compared to using rTl-Cyn alone. Complete degradation of cyanate (4 mM) was achieved with buffered conditions and 85 ±â€¯5% degradation with industrial wastewater sample, when 20 U of rTl-Cyn was applied. Furthermore, a similar percentage of degradation was achieved using 80% less bicarbonate, when rTl-Cyn and rTl-CA were used together under identical conditions. In addition, rTl-Cyn and rTl-CA were immobilized onto the magnetic nanoparticles and their catalytic activity, stability and reusability were also evaluated. This is the first report on the synergistic biocatalysis by rTl-Cyn and rTl-CA, for cyanate detoxification.

Production of gellan gum, an exopolysaccharide, from biodiesel-derived waste glycerol by Sphingomonas spp.

In the present study, biodiesel-derived waste glycerol (WG) was used for the isolation and production of gellan, an exopolysaccharide, on media containing WG as the main carbon source. Two bacterial isolates showed gellan producing potential which were identified as Sphingomonas pseudosanguinis (Accession No. GI:724472387) and Sphingomonas yabuuchiae (GI:724472388) by 16S rRNA gene sequencing. To maximize gellan production by S. pseudosanguinis and S. yabuuchiae, media optimization was performed at different pHs and glycerol concentrations. Morphological observations through microscopic images showed the production of gellan from these isolates. Simple linear regression showed better utilization of WG by S. pseudosanguinis than S. yabuuchiae at pH 6 and pH 7. Though, both the strains showed reverse trend at pH 8. Both the strains were able to produce high amounts of gellan gum (51.6 and 52.6 g/l, respectively) using WG (80 g/l) as the sole carbon source, in a minimal medium. This is the first report on the efficient degradation of WG and low-cost production of gellan. Owing to these characteristics, S. pseudosanguinis and S. yabuuchiae demonstrate great potential for use in the commercial production of gellan and in the bioremediation of WG.

Exploring the Metabolomic Responses of Bacillus licheniformis to Temperature Stress by Gas Chromatography/Mass Spectrometry.

Owing to its high protein secretion capacity, simple nutritional requirements, and GRAS (generally regarded as safe) status, Bacillus licheniformis is widely used as a host for the industrial production of enzymes, antibiotics, and peptides. However, as compared with its close relative Bacillus subtilis, little is known about the physiology and stress responses of B. licheniformis. To explore its temperature-stress metabolome, B. licheniformis strains ATCC 14580 and B186, with respective optimal growth temperatures of 42°C and 50°C, were cultured at 42°C, 50°C, and 60°C and their corresponding metabolic profiles were determined by gas chromatography/mass spectrometry and multivariate statistical analyses. It was found that with increased growth temperatures, the two B. licheniformis strains displayed elevated cellular levels of proline, glutamate, lysine, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, and octadecanoic acid, and decreased levels of glutamine and octadecenoic acid. Regulation of amino acid and fatty acid metabolism is likely to be associated with the evolution of protective biochemical mechanisms of B. licheniformis. Our results will help to optimize the industrial use of B. licheniformis and other important Bacillus species.

Expression of a novel recombinant cyanate hydratase (rTl-Cyn) in Pichia pastoris, characteristics and applicability in the detoxification of cyanate.

A recombinant Pichia pastoris harbouring the cyanate hydratase gene (rTl-Cyn) from the thermophilic fungus Thermomyces lanuginosus SSBP yielded a high titre of extracellular cyanate hydratase (100±13UmL-1) which was ∼10-fold higher than the native fungal strain. The purified rTl-Cyn had a molecular mass of ∼20kDa on SDS-PAGE, with Km, Vmax, kcat and kcat/Km values of 0.34mM, 2857.14µmolesmg-1min-1, 2.14×104s-1 and 6.3 ×107M-1s-1, respectively. Its properties of thermostability, pH stability, and heavy metals insensitivity, make it a suitable candidate for bioremediation in extreme environments. The rTl-Cyn was able to degrade toxic cyanate completely with the liberation of ammonia, which was confirmed by FTIR analysis. This is the first report of any known cyanate hydratase that has been expressed in P. pastoris, characterized and effectively evaluated for cyanate detoxification.

Cloning, expression, and molecular dynamics simulations of a xylosidase obtained from Thermomyces lanuginosus.

The aim of this study was to clone, express, and characterize a ß-xylosidase (Tlxyn1) from the thermophilic fungus Thermomyces lanuginosus SSBP in Pichia pastoris GS115 as well as analyze optimal activity and stability using computational and experimental methods. The enzyme was constitutively expressed using the GAP promoter and secreted into the medium due to the alpha-mating factor secretion signal present on the expression vector pBGPI. The 1276 bp gene consists of an open reading frame that does not contain introns. A 12% SDS-PAGE gel revealed a major protein band at an estimated molecular mass of 50 kDa which corresponded to zymogram analysis. The three-dimensional structure of ß-xylosidase was predicted, and molecular dynamics simulations at different ranges of temperature and pH were performed in order to predict optimal activity and folding energy. The results suggested a strong conformational temperature and pH dependence. The recombinant enzyme exhibited optimal activity at pH 7 and 50°C and retained 80% activity at 50°C, pH 7 for about 45 min. This is the first report of the cloning, functional expression, and simulations study of a ß-xylosidase from Thermomyces species in a fungal host.

Gene cloning, heterologous expression, and characterization of a high maltose-producing α-amylase of Rhizopus oryzae.

A putative α-amylase gene, designated as RoAmy, was cloned from Rhizopus oryzae. The deduced amino acid sequence showed the highest (42.8%) similarity to the α-amylase from Trichoderma viride. The RoAmy gene was successfully expressed in Pichia pastoris GS115 under the induction of methanol. The molecular weight of the purified RoAmy determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was approximately 48 kDa. The optimal pH and temperature were 4-6 and 60 °C, respectively. The enzyme was stable at pH ranges of 4.5-6.5 and temperatures below 50 °C. Purified RoAmy had a K(m) and V(max) of 0.27 mg/ml and 0.068 mg/min, respectively, with a specific activity of 1,123 U/mg on soluble starch. Amylase activity was strongly inhibited by 5 mM Cu(2+) and 5 mM Fe(2+), whereas 5 mM Ca(2+) showed no significant effect. The RoAmy hydrolytic activity was the highest on wheat starch but showed only 55% activity on amylopectin relative to soluble corn starch, while the pullulanase activity was negligible. The main end products of the polysaccharides tested were glucose and maltose. Maltose reached a concentration of 74% (w/w) with potato starch as the substrate. The enzyme had an extremely high affinity (K(m) = 0.22 mM) to maltotriose. A high ratio of glucose/maltose of 1:4 was obtained when maltotriose was used at an initial concentration of 40 mM.