Gene_locus Report for: thite-g2rae6

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 degrees C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 degrees C. Among tested p-nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p-nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4U/mg and 1152.5U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.

Cutinases are esterases that release fatty acids from the apoplastic layer in plants. As they accept bulky and hydrophobic substrates, cutinases could be used in many applications, ranging from valorization of bark-rich side streams to plastic recycling. Advancement of these applications with cutinases as biocatalysts, however, requires deeper knowledge of the enzymes' biodiversity and structure-function relationships. Here, we mined over 3000 members from Carbohydrate Esterase family 5 (CE5) for putative cutinases and condensed it to 151 genes from known or putative lignocellulose-targeting organisms. The 151 genes were subjected to a phylogenetic analysis. While cutinases with available crystal structures were phylogenetically closely related, we selected nine phylogenic diverse cutinases for characterization. The nine selected cutinases were recombinantly produced and their kinetic activity was characterized against para-nitrophenol substrates esterified with consecutively longer alkyl chains (pNP-C(2) to C(16)). The investigated cutinases each had a unique activity fingerprint against tested pNP-substrates. The five enzymes with the highest activity on pNP-C(12) and C(16), indicative of activity on bulky hydrophobic compounds, were selected for in-depth kinetic and structure-function analysis. All five enzymes showed a decrease in k(cat) values with increasing substrate chain length, while K(M) values and binding energies (calculated from in silico docking analysis) improved. Two cutinases from Fusarium solani and Cryptococcus sp. exhibited outstandingly low K(M) values, resulting in high catalytic efficiencies towards pNP-C(16). Docking analysis suggested that different clades of the phylogenetic tree may harbor enzymes with different modes of substrate interaction, involving a solvent-exposed catalytic triad, a lipase-like lid, or a clamshell-like active site possibly formed by flexible loops.

A codon-optimized cutinase gene (TtCutopt) from Thielavia terrestris was over-expressed in Pichia pastoris. An extracellular activity reached 10,200U/mL using high cell density fermentation. The optimal pH and temperature of TtCutopt were 7.0 and 50 degrees C, respectively. It displayed high stability over a wide range of pH from 3.0 to 11.0 and up to 85 degrees C. Among tested p-nitrophenyl esters and triglycerides, TtCutopt showed the highest activity towards p-nitrophenyl butyrate and tributyrin, with specificity activity of 2322.4U/mg and 1152.5U/mg, respectively. It was extremely stable in organic solvents and surfactants. TtCutopt efficiently catalyzed the synthesis of butyl butyrate, hexyl butyrate, butyl hexanoate and hexyl hexanoate with esterification efficiency of >95%. Furthermore, it catalyzed the degradation of >90% of dimethyl phthalate, diethyl phthalate, dipropyl phthalate and dibutyl phthalate to release their corresponding monoalkyl phthalates within 24h. Thus, high yield, high stability, and esterification efficiency of TtCutopt make it an attractive candidate for ester biosynthesis and biodegradation.

An acidic cutinase (TtcutB) from Thielavia terrestris CAU709 was purified to apparent homogeneity with 983Umg(-1) specific activity. The molecular mass of the enzyme was estimated to be 27.3 and 27.9kDa by SDS-PAGE and gel filtration, respectively. A peptide sequence homology search revealed no homologous cutinases from T. terrestris, except for one putative cutinase gene (XP003656017.1), indicating that TtcutB is a novel enzyme. TtcutB exhibited an acidic pH optimum of 4.0, and stability at pH 2.5-10.5. Optimal activity was at 55 degrees C, it was stable up to 65 degrees C, and retained over 30% activity at 0 degrees C. Km values toward p-nitrophenyl (pNP) acetate, pNP-butyrate and pNP-caproate were 8.3, 1.1 and 0.88mM, respectively. The cutinase exhibited strong synthetic activity on flavor ester butyl butyrate under non-aqueous environment, and the highest esterification efficiency of 95% was observed under the optimized reaction conditions. The enzyme's unique biochemical properties suggest great potential in flavor esters-producing industries.

Thermostable enzymes and thermophilic cell factories may afford economic advantages in the production of many chemicals and biomass-based fuels. Here we describe and compare the genomes of two thermophilic fungi, Myceliophthora thermophila and Thielavia terrestris. To our knowledge, these genomes are the first described for thermophilic eukaryotes and the first complete telomere-to-telomere genomes for filamentous fungi. Genome analyses and experimental data suggest that both thermophiles are capable of hydrolyzing all major polysaccharides found in biomass. Examination of transcriptome data and secreted proteins suggests that the two fungi use shared approaches in the hydrolysis of cellulose and xylan but distinct mechanisms in pectin degradation. Characterization of the biomass-hydrolyzing activity of recombinant enzymes suggests that these organisms are highly efficient in biomass decomposition at both moderate and high temperatures. Furthermore, we present evidence suggesting that aside from representing a potential reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using classical and molecular genetics.