CRISPR-Cas9-mediated seb1 disruption in Talaromyces pinophilus EMU for its enhanced cellulase production.

Filamentous fungi are working horses for industrial enzyme production. Combinatory approaches, such as random mutagenesis and rational genetic engineering, were adopted to improve their enzyme productivity. The filamentous fungus Talaromyces pinophilus EMU is a hyper cellulase-producing filamentous fungus obtained through random mutagenesis.

High-copy genome integration of 2,3-butanediol biosynthesis pathway in Saccharomyces cerevisiae via in vivo DNA assembly and replicative CRISPR-Cas9 mediated delta integration.

CRISPR Cas9 system is becoming an emerging genome-editing platform and has been widely used for multiplex genome engineering of Saccharomyces cerevisiae. In this study, we developed a novel replicative and integrative CRISPR Cas9 genome-editing platform for large DNA construct in vivo assembly, replication, and high-copy genome integration in Saccharomyces cerevisiae. It harnessed advantages of autonomous replicative sequence in S.

Enhanced cellulase and reducing sugar production by a new mutant strain Trichoderma harzianum EUA20.

Trichoderma harzianum EU2-77 was a mutant strain of the wild-type strain T. harzianum NP13a isolated in Singapore. A multi-mutagenesis one-screening (MMOS) method was developed to further improve strain EU2-77 and a new mutant EUA20 was obtained.

Enzyme production and oil palm empty fruit bunch bioconversion to ethanol using a hybrid yeast strain.

Oil palm empty fruit bunch (OPEFB) is a lignocellulosic biomass generated in palm oil mills. It is a sustainable resource for fuels and chemicals. In this study, OPEFB was converted to ethanol by an integrative OPEFB conversion process including dilute alkaline pretreatment, cellulolytic enzyme production, separate OPEFB hydrolysis, and cofermentation using a hybrid xylose-fermenting yeast.

Enhanced ethanol production from industrial lignocellulose hydrolysates by a hydrolysate-cofermenting Saccharomyces cerevisiae strain.

Industrial production of lignocellulosic ethanol requires a microorganism utilizing both hexose and pentose, and tolerating inhibitors. In this study, a hydrolysate-cofermenting Saccharomyces cerevisiae strain was obtained through one step in vivo DNA assembly of pentose-metabolizing pathway genes, followed by consecutive adaptive evolution in pentose media containing acetic acid, and direct screening in biomass hydrolysate media. The strain was able to coferment glucose and xylose in synthetic media with the respective maximal specific rates of glucose and xylose consumption, and ethanol production of 3.

Directed Evolution of a Homodimeric Laccase from BBP6 by Random Mutagenesis and In Vivo Assembly.

Laccases have great potential for industrial applications due to their green catalytic properties and broad substrate specificities, and various studies have attempted to improve the catalytic performance of these enzymes. Here, to the best of our knowledge, we firstly report the directed evolution of a homodimeric laccase from BBP6 fused with α-factor prepro-leader that was engineered through random mutagenesis followed by in vivo assembly in . Three evolved fusion variants selected from ~3500 clones presented 31- to 37-fold increases in total laccase activity, with better thermostability and broader pH profiles.

A novel homodimer laccase from Cerrena unicolor BBP6: Purification, characterization, and potential in dye decolorization and denim bleaching.

The white-rot fungus Cerrena unicolor BBP6 produced up to 243.4 U mL-1 laccase. A novel laccase isoform LacA was purified; LacA is a homodimer with an apparent molecular mass of 55 kDa and an isoelectric point of 4.

Bacterial xylose isomerases from the mammal gut Bacteroidetes cluster function in Saccharomyces cerevisiae for effective xylose fermentation.

Background: Xylose isomerase (XI) catalyzes the conversion of xylose to xylulose, which is the key step for anaerobic ethanolic fermentation of xylose. Very few bacterial XIs can function actively in Saccharomyces cerevisiae. Here, we illustrate a group of XIs that would function for xylose fermentation in S.

Polyhydroxybutyrate production from oil palm empty fruit bunch using Bacillus megaterium R11.

Oil palm empty fruit bunch (OPEFB), contains abundant cellulose and hemicelluloses and can be used as a renewable resource for fuel and chemical production. This study, as the first attempt, aims to convert OPEFB derived sugars to polyhydroxybutyrate (PHB). OPEFB collected from a Malaysia palm oil refinery plant was chemically pretreated and enzymatically hydrolyzed by an in-house prepared cellulase cocktail.

Effects of lignin-derived phenolic compounds on xylitol production and key enzyme activities by a xylose utilizing yeast Candida athensensis SB18.

Candida athensensis SB18 is potential xylitol producing yeast isolated in Singapore. It has excellent xylose tolerance and is able to produce xylitol in high titer and yield. However, by-products, such as phenolic compounds, derived in lignocellulosic biomass hydrolysate might negatively influence the performance of this strain for xylitol production.

Improved ethanol production by a xylose-fermenting recombinant yeast strain constructed through a modified genome shuffling method.

Background: Xylose is the second most abundant carbohydrate in the lignocellulosic biomass hydrolysate. The fermentation of xylose is essential for the bioconversion of lignocelluloses to fuels and chemicals. However the wild-type strains of Saccharomyces cerevisiae are unable to utilize xylose.

Xylitol production from D-xylose and horticultural waste hemicellulosic hydrolysate by a new isolate of Candida athensensis SB18.

This paper describes the production of xylitol from d-xylose and horticultural waste hemicellulosic hydrolysate by a new strain of Candida athensensis SB18. Strain SB18 completely consumed 250 and 300 g L(-1) D-xylose and successful converted it to xylitol in the respective yield of 0.83 and 0.

Ethanol production from horticultural waste treated by a modified organosolv method.

In this study, we investigated the use of horticultural waste (HW) collected in Singapore as a renewable raw material for bioethanol production. A modified organosolv method using ethanol cooking under mild conditions followed by H(2)O(2) post-treatment was investigated for HW pretreatment. It was found that the addition of acid catalysts in the pretreatment process was not critical and post-treatment using H(2)O(2) was essential for the enhancement of HW digestibility.

New isolate of Trichoderma viride strain for enhanced cellulolytic enzyme complex production.

A new Trichoderma viride stain was isolated from Singapore soil samples. Its mutants were developed by using ethyl methyl sulfonate (EMS) treatment and UV-irradiation followed by a semi-quantitative plate clearing assay on phosphoric-acid-swollen cellulose plates. Mutant EU2-77 proved to be the most promising extracellular cellulase producer among 20 mutants in a screening program performed in shake flask fermentation after plate screening.

Utilization of horticultural waste for laccase production by Trametes versicolor under solid-state fermentation.

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.

Enzymatic hydrolysis of sodium dodecyl sulphate (SDS)-pretreated newspaper for cellulosic ethanol production by Saccharomyces cerevisiae and Pichia stipitis.

Fermentation of enzymatic hydrolysate of waste newspaper was investigated for cellulosic ethanol production in this study. Various nonionic and ionic surfactants were applied for waste newspaper pretreatment to increase the enzymatic digestibility. The surfactant-pretreated newspaper was enzymatically digested in 0.

Horticultural waste as the substrate for cellulase and hemicellulase production by Trichoderma reesei under solid-state fermentation.

Horticultural waste in wood chips form collected from a landscape company in Singapore was utilized as the substrate for the production of cellulase and hemicellulase under solid-state fermentation by Trichoderma reesei RUT-C30. The effects of substrate pretreatment methods, substrate particle size, incubation temperature and time, initial medium pH value, and moisture content on cellulase and hemicellulase production were investigated. Enzyme complex was obtained at the optimal conditions.

Induction of ortho- and meta-cleavage pathways in Pseudomonas in biodegradation of high benzoate concentration: MS identification of catabolic enzymes.

The degradation pathways of benzoate at high concentration in Pseudomonas putida P8 were directly elucidated through mass spectrometric identification of some key catabolic enzymes. Proteins from P. putida P8 grown on benzoate or succinate were separated using two-dimensional gel electrophoresis.

Isolation and characterization of a phenol-degrading bacterium from an industrial activated sludge.

This paper reports the successful isolation and characterization of a new phenol-degrading bacterium, strain EDP3, from activated sludge. Strain EDP3 is a nonmotile, strictly aerobic, Gram-negative, and short-rod or coccobacillary bacterium, which occurs singly, in pairs, or in clusters. 16S rRNA gene sequence analysis revealed that strain EDP3 belonged to the gamma group of Proteobacteria, with a 97.

Proteome analysis of gentisate-induced response in Pseudomonas alcaligenes NCIB 9867.

Pseudomonas alcaligenes NCIB 9867 (P25X wild-type) is capable of degrading aromatic hydrocarbons via the gentisate pathway. Biochemical characterization of P25X mutants indicated that it has isofunctional enzymes for the mono- and dioxygenase-catalyzed reactions. One set of the enzymes is constitutive whereas the other is strictly inducible.

Heterogeneity of Surface Energies in Reversed-Phase Perfusive Packings.

The surface energetic heterogeneity of the packing media used in perfusion chromatography was investigated based on the liquid-solid adsorption information of phenol. The adsorption isotherms on two perfusive packings, POROS R1 and POROS R2, were measured by stepwise frontal experiments at varying mobile-phase concentrations and temperatures. The isosteric heat of adsorption was calculated from the isotherm data and the adsorption energy distributions (AEDs) were obtained numerically by the expectation maximization (EM) method.