Enhancement of catalytic activity and alkaline stability of cellobiohydrolase by structure-based protein engineering.

Alkaline cellobiohydrolases have the potential for application in various industries, including pulp processing and laundry where operation under high pH conditions is preferred. In this study, variants of CtCel6A cellobiohydrolase from Chaetomium thermophilum were generated by structural-based protein engineering with the rationale of increasing catalytic activity and alkaline stability. The variants included removal of the carbohydrate-binding module (CBM) and substitution of residues 173 and 200. The CBM-deleted enzyme with Y200F mutation predicted to mediate conformational change at the N-terminal loop demonstrated increased alkaline stability at 60 °C, pH 8.0 for 24 h up to 2.25-fold compared with the wild-type enzyme. Another CBM-deleted enzyme with L173E mutation predicted to induce a new hydrogen bond in the substrate-binding cleft showed enhanced hydrolysis yield of pretreated sugarcane trash up to 4.65-fold greater than that of the wild-type enzyme at the pH 8.0. The variant enzymes could thus be developed for applications on cellulose hydrolysis and plant fiber modification operated under alkaline conditions. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-022-03339-4.

Engineered Production of Isobutanol from Sugarcane Trash Hydrolysates in Pichia pastoris.

Concerns over climate change have led to increased interest in renewable fuels in recent years. Microbial production of advanced fuels from renewable and readily available carbon sources has emerged as an attractive alternative to the traditional production of transportation fuels. Here, we engineered the yeast Pichia pastoris, an industrial powerhouse in heterologous enzyme production, to produce the advanced biofuel isobutanol from sugarcane trash hydrolysates. Our strategy involved overexpressing a heterologous xylose isomerase and the endogenous xylulokinase to enable the yeast to consume both C5 and C6 sugars in biomass. To enable the yeast to produce isobutanol, we then overexpressed the endogenous amino acid biosynthetic pathway and the 2-keto acid degradation pathway. The engineered strains produced isobutanol at a titer of up to 48.2 ± 1.7 mg/L directly from a minimal medium containing sugarcane trash hydrolysates as the sole carbon source. To our knowledge, this is the first demonstration of advanced biofuel production using agricultural waste-derived hydrolysates in the yeast P. pastoris. We envision that our work will pave the way for a scalable route to this advanced biofuel and further establish P. pastoris as a versatile production platform for fuels and high-value chemicals.

Enhancement of catalytic performance of a metagenome-derived thermophilic oligosaccharide-specific xylanase by binding module removal and random mutagenesis.

Xylo-oligosaccharide (XO) is a promising pre-biotic with applications in food, feed and healthcare products. XO can be produced by enzymatic digestion of xylan with xylanase. In this study, we aimed to improve the biochemical properties relevant to catalysis and kinetics of X11, a thermophilic glycosyl hydrolase (GH) family 11 endo-ß-1,4-xylanase derived from a metagenomic library isolated from sugarcane bagasse, under high-temperature conditions preferred for XO synthesis. Removal of a carbohydrate-binding module (X11C) resulted in 6.5 fold greater catalytic efficiency. X11C was further improved by a Pro71Thr mutation in the X11P variant obtained from a random mutagenesis library, which exhibited 15.9 fold greater catalytic efficiency compared with wild-type X11 under the enzyme's optimal conditions of 80°C and pH 6.0. Homology modeling suggested that the improved performance of X11P could be attributed to formation of an extra H-bond between Thr71 and Ser75, which stabilizes the key catalytic residue Glu180 at the active pocket and ß-sheet layers and agrees with the respective increase in melting temperature (Tm) where X11P >X11C >X11 as determined by differential scanning fluorimetry. The X11P variant was tested for hydrolysis of beechwood xylan, which showed X6 as the major product followed by X3 and X4 XOs. The highest yield of 5.5 g total XOs product/mg enzyme was observed for X11P, equivalent to 3.7 fold higher than that of wild-type with XO production of >800 mg/g xylan. The X11P enzyme could be developed as a thermophilic biocatalyst for XO synthesis in biorefineries.

Identification and evaluation of novel anchoring proteins for cell surface display on Saccharomyces cerevisiae.

The development of arming yeast strains as whole-cell biocatalysts involves a selection of effective anchoring proteins to display enzymes and proteins on yeast cell surface. To screen for novel anchoring proteins with improved efficiency, a bioinformatics pipeline for the identification of glycosylphosphatidylinositol-anchored cell wall proteins (GPI-CWPs) suitable for attaching passenger proteins to the cell surface of Saccharomyces cerevisiae has been developed. Here, the C-terminal sequences (CTSs) of putative GPI-CWPs were selected based on the criteria that the sequence must contain a serine/threonine-rich (S/T) region of at least 30% S/T content, a total threonine content of at least 10%, a continuous S/T stretch of at least 130 amino acids in length, and a continuous T-rich region of at least 10 amino acids in length. Of the predicted 790 proteins, 37 putative GPI-CWPs were selected from different yeast and fungal species to be evaluated for their performance in displaying yeast-enhanced green fluorescent protein and ß-glucosidase enzyme. This led to the identification of five novel anchoring proteins with higher performance compared to α-agglutinin used as benchmark. In particular, the CTS of uncharacterized protein in Kluyveromyces lactis, namely 6_Kl, is the most efficient anchoring protein of the group. The CTS of 6_Kl protein provided a ß-glucosidase activity of up to 23.5 U/g cell dry weight, which is 2.8 times higher than that of the CTS of α-agglutinin. These identified CTSs could be potential novel anchoring protein candidates for construction of efficient arming yeasts for biotechnology applications in the future.

Structure-based protein engineering for thermostable and alkaliphilic enhancement of endo-ß-1,4-xylanase for applications in pulp bleaching.

In the pulp bleaching industry, enzymes with robust activity at high pH and temperatures are desirable for facilitating the pre-bleaching process with simplified processing and minimal use of chlorinated compounds. To engineer an enzyme for this purpose, we determined the crystal structure of the Xyn12.2 xylanase, a xylan-hydrolyzing enzyme derived from the termite gut symbiont metagenome, as the basis for structure-based protein engineering to improve Xyn12.2 stability in high heat and alkaline conditions. Engineered cysteine pairs that generated exterior disulfide bonds increased the kcat of Xyn12.2 variants and melting temperature at all tested conditions. These improvements led to up to 4.2-fold increases in catalytic efficiency at pH 9.0, 50°C for 1h and up to 3-fold increases at 60°C. The most effective variants, XynTT and XynTTTE, exhibited 2-3-fold increases in bagasse hydrolysis at pH 9.0 and 60°C compared to the wild-type enzyme. Overall, engineering arginines and phenylalanines for increased pKa and hydrogen bonding improved enzyme catalytic efficiency at high stringency conditions. These modifications were the keys to enhancing thermostability and alkaliphilicity in our enzyme variants, with XynTT and XynTTTE being especially promising for their application to the pulp and paper industry.

Alkaliphilic endoxylanase from lignocellulolytic microbial consortium metagenome for biobleaching of eucalyptus pulp.

Enzymatic pre-bleaching by modification of pulp fibers with xylanases is an attractive approach to reduce the consumption of toxic bleaching chemicals in the paper industry. In this study, an alkaliphilic endoxylanase gene was isolated from metagenomic DNA of a structurally stable thermophilic lignocellulose-degrading microbial consortium using amplification with conserved glycosyl hydrolase family 10 primers and subsequent genome walking. The full-length xylanase showed 78% sequence identity to an endo-beta-1,4-xylanase of Clostridium phytofermentans and was expressed in a mature form with an N-terminal His6 tag fusion in Escherichia coli. The recombinant xylanase Xyn3F was thermotolerant and alkaliphilic, working optimally at 65-70 degrees C with an optimal pH at 9- 10 and retaining >80% activity at pH 9, 60 degrees C for 1 h. Xyn3F showed a Vmax of 2,327 IU/mg and Km of 3.5 mg/ml on birchwood xylan. Pre-bleaching of industrial eucalyptus pulp with no prior pH adjustment (pH 9) using Xyn3F at 50 IU/g dried pulp led to 4.5-5.1% increase in final pulp brightness and 90.4-102.4% increase in whiteness after a single-step hypochlorite bleaching over the untreated pulp, which allowed at least 20% decrease in hypochlorite consumption to achieve the same final bleaching indices. The alkaliphilic xylanase is promising for application in an environmentally friendly bleaching step of kraft and soda pulps with no requirement for pH adjustment, leading to improved economic feasibility of the process.

Biochemical characterization and in vitro digestibility assay of Eupenicillium parvum (BCC17694) phytase expressed in Pichia pastoris.

A mature phytase cDNA, encoding 441 amino acids, from Eupenicillium parvum (BCC17694) was cloned into a Pichia pastoris expression vector, pPICZ alpha A, and was successfully expressed as active extracellular glycosylated protein. The recombinant phytase contained the active site RHGXRXP and HD sequence motifs, a large alpha/beta domain and a small alpha-domain that are typical of histidine acid phosphatase. Glycosylation was found to be important for enzyme activity which is most active at 50 degrees C and pH 5.5. The recombinant phytase displayed broad substrate specificity toward p-nitrophenyl phosphate, sodium-, calcium-, and potassium-phytate. The enzyme lost its activity after incubating at 50 degrees C for 5 min and is 50% inhibited by 5mM Cu(2+). However, the enzyme exhibits broad pH stability from 2.5 to 8.0 and is resistant to pepsin. In vitro digestibility test suggested that BCC17694 phytase is at least as effective as another recombinant phytase (r-A170) which is comparable to Natuphos, a commercial phytase, in releasing phosphate from corn-based animal feed, suggesting that BCC17694 phytase is suitable for use as phytase supplement in the animal diet.

Cloning, expression and characterization of a thermotolerant endoglucanase from Syncephalastrum racemosum (BCC18080) in Pichia pastoris.

Endoglucanase is a major cellulolytic enzyme produced by Syncephalastrum racemosum (BCC18080). Preliminary results showed that this endoglucanase is thermotolerant as it retained more than 50% of its activity after incubation at 80 degrees C for an hour. As this property may be of industrial use, we have cloned the full-length BCC18080 endoglucanase gene of 1020 nucleotides. Sequence analysis suggested that it belonged to the glycosyl hydrolase family 45. N-terminal sequencing and analysis by SignalP program suggested that the first 32 amino acid residues encoded the signal peptide. Expression of the recombinant clones with and without its own signal peptide in Pichia pastoris demonstrated that P. pastoris produced active 55 and 30 kDa secreted proteins. N-terminal sequencing suggested that the 55 kDa band was the mature protein while the 30 kDa band was the truncated protein. Glycoprotein analysis showed that the 55 kDa protein was glycosylated; while the smaller protein was not. All recombinant endoglucanases showed optimal temperature of 70 degrees C and optimal pH of 5-6. They retained more than 50% activity for 4h at 70 degrees C. In addition, high k(cat) and low apparent K(m) of these recombinant proteins indicated good properties of this enzyme against carboxylmethylcellulose.

Cloning and expression of xylanase 10 from Cryptovalsa mangrovei (BCC7197) in Pichia pastoris.

Xylanases are one of the industrially valuable enzymes. Using RT-PCR and 5'- and 3'-RACE procedures, we have cloned a full-length xylanase encoding gene from a filamentous fungus, Cryptovalsa mangrovei (BCC7197) from Phuket, Thailand. The results showed that BCC7197 xylanase cDNA has an open reading frame of 978 bp encoding 325 amino acid residues. Further sequence analysis revealed that this xylanase gene is belonged to the glycosyl hydrolase family 10 and has approximately 50-60% amino acid sequence similarity to other fungal xylanases. Furthermore, expression of BCC7197 xylanase in the Pichia pastoris was also performed. The results demonstrated that the active BCC7197 xylanase protein was successfully produced and secreted from P. pastoris.