Kusol Pootanakit

In vivo identification of Bacillus thuringiensis Cry4Ba toxin receptors by RNA interference knockdown of glycosylphosphatidylinositol-linked aminopeptidase N transcripts in Aedes aegypti larvae.

Bacillus thuringiensis Cry4Ba toxin selectively kills Aedes aegypti mosquito larvae as it is in part due to the presence of specific membrane-bound protein receptors. In this study, using data mining approach, we initially identified three potential glycosylphosphatidylinositol-linked aminopeptidase N (GPI-APN) isoforms, APN2778, APN2783 and APN5808, which are believed to act as Cry4Ba toxin receptors. These three isoforms that are functionally expressed in the larval midgut can be sequence-specific knocked down (ranging from ∼80 % to 95 %) by soaking the Aedes aegypti larvae in buffer of long double-stranded GPI-APN RNAs (∼300-680 bp). Finally, to see the physiological effect of APN knockdowns, the larvae were fed with Escherichia coli expressing Cry4Ba toxin. The results revealed that all the three identified GPI-APN isoforms may possibly function as a Cry4Ba receptor, particularly for APN2783 as those larvae with this transcript knockdown showed a dramatic increase in resistance to Cry4Ba toxicity.

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.

Aedes aegypti Membrane-Bound Alkaline Phosphatase Expressed in Escherichia coli Retains High-Affinity Binding for Bacillus thuringiensis Cry4Ba Toxin

ABSTRACT Glycosylphosphatidylinositol-linked alkaline phosphatase (GPI-ALP) from the epithelial membrane of the larval midgut of Aedes aegypti was previously identified as a functional receptor of the Bacillus thuringiensis Cry4Ba toxin. Here, heterologous expression in Escherichia coli of the cloned ALP, lacking the secretion signal and GPI attachment sequences, and assessment of its binding characteristics were further investigated. The 54-kDa His tag-fused ALP overexpressed as an inclusion body was soluble when phosphate buffer (pH 7.5) was supplemented with 8 M urea. After renaturation in a nickel-nitrilotriacetic acid (Ni-NTA) affinity column, the refolded ALP protein was able to retain its phosphatase activity. This refolded ALP also showed binding to the 65-kDa activated Cry4Ba toxin under nondenaturing (dot blot) conditions. Quantitative binding analysis using a quartz crystal microbalance revealed that the purified ALP immobilized on a gold electrode was bound by the Cry4Ba toxin in a stoichiometry of approximately 1:2 and with high affinity (dissociation constant [K d ] of ∼14 nM) which is comparable to that calculated from kinetic parameters (dissociation rate constant [k off]/binding constant [k on]). Altogether, the data presented here of the E. coli-expressed ALP from A. aegypti retaining high-affinity toxin binding support our notion that glycosylation of this receptor is not required for binding to its counterpart toxin, Cry4Ba.

Engineered Escherichia coli for Short-Chain-Length Medium-Chain-Length Polyhydroxyalkanoate Copolymer Biosynthesis from Glycerol and Dodecanoate

Short-chain-length medium-chain-length polyhydroxyalkanoate (SCL-MCL PHA) copolymers are promising as bio-plastics with properties ranging from thermoplastics to elastomers. In this study, the hybrid pathway for the biosynthesis of SCL-MCL PHA copolymers was established in recombinant Escherichia coli by co-expression of β-ketothiolase (PhaA Re ) and NADPH-dependent acetoacetyl-CoA reductase (PhaB Re ) from Ralstonia eutropha together with PHA synthases from R. eutropha (PhaC Re ), Aeromonas hydrophila (PhaC Ah ), and Pseudomonas putida (PhaC2 Pp ) and with (R)-specific enoyl-CoA hydratases from P. putida (PhaJ1 Pp and PhaJ4 Pp ), and A. hydrophila (PhaJ Ah ). When glycerol supplemented with dodecanoate was used as primary carbon source, E. coli harboring various combinations of PhaABCJ produced SCL-MCL PHA copolymers of various monomer compositions varying from C4 to C10. In addition, polymer property analysis suggested that the copolymers produced from this recombinant source have thermal properties (lower glass transition and melting temperatures) superior to polyhydroxybutyrate homopolymer.

Phylogenetic Analysis and Metabolic Potential of Microbial Communities in an Industrial Bagasse Collection Site

Industrial bagasse collection sites at sugar mills are an important resource for biomass-based industries and represent a unique ecological niche in lignocellulose degradation. In this study, microbial community structures at regions with varying microenvironmental conditions contained within a bagasse collection site were explored using tagged 16S rRNA gene pyrosequencing. Overall, remarkable differences in microbial community structures were found in aerobic surface and oxygen-limited interior regions of the pile. A variety of Alphaproteobacteria and Gammaproteobacteria represented the majority of bacteria in the aerobic upper-pile regions with the predominance of acetic acid bacteria towards the outer surface. Diverse Proteobacteria, Bacteroidetes, and Acidobacteria represented the predominant phyla at the exterior soil-contact pile base with an increasing abundance of anaerobic Spirochaetes with the increasing depth, where it shared similar community structures to that in the open-field soil from decomposed bagasse. Using complementary shotgun pyrosequencing, a variety of genes encoding various glycosyl hydrolases targeting cellulose and hemicellulose degradation were identified in the oxygen-limited interior pile base. Most were relevant to orders Clostridiales, Bacteroidales, Sphingobacteriales, and Cytophagales, suggesting their role in lignocellulose degradation in this region, as evidenced by the decrease in cellulose and respective increase in lignin fractions of the biomass. Partial carbon flux in the anoxic region was metabolized through mixed methanogenesis pathways as suggested by the annotated functional genes in methane synthesis. This study gives insights into native microbial community structures and functions in this unique lignocellulose degrading environment and provides the basis for controlling microbial processes important for utilization of bagasse in bio-industries.

Bacillus thuringiensis Cry4Ba toxin employs two receptor-binding loops for synergistic interactions with Cyt2Aa2

We previously demonstrated that co-expression in Escherichia coli of Bacillus thuringiensis (Bt) subsp. israelensis Cry4Ba and Bt subsp. darmstadiensis Cyt2Aa2 shows high synergistic toxicity against target mosquito larvae. Here, further insights into synergistic interactions between these two toxins were revealed through bioactivity restoration of particular inactive Cry4Ba-mutant toxins altered within the receptor-binding domain. Specific mutations at β2-β3 (Y332A) or β4-β5 (F364A) loops, but neither at three other β-hairpin loops (β6-β7, β8-β9 and β10-β11) of Cry4Ba, adversely affect toxicity restoration by Cyt2Aa2. Binding analysis using quartz crystal microbalance verified a decrease in binding of these two bioinactive-mutant toxins (Y332A and F364A) to the immobilized Cyt2Aa2. This suggests that Cry4Ba utilizes these two critical aromatic loop-residues, Tyr(332) and Phe(364), for synergistic toxicity with its alternative receptor-Cyt2Aa2.

Functional expression in Beauveria bassiana of a chitinase gene from Ophiocordyceps unilateralis, an ant-pathogenic fungus

Abstract A gene encoding chitinase was cloned from Ophiocordyceps unilateralis, a Formamidae-specific fungus, collected from Sirindhorn Peat Swamp Forest, Thailand. The O. unilateralis chitinase (OuChi) full-length gene (1311 bp) encodes 436 amino acids with the first 20 amino acids as a putative signal peptide. The gene showed highest identity (78%) to Isaria farinose endochitinase. To investigate if cross-species chitinase expression also enhances fungal toxicity, the mature OuChi gene was subcloned into an Agrobacterium binary vector pPZP-bar and then transformed into Beauveria bassiana strain BCC2659. Chitinase activity was detected using 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside. The fungal transformant expressing O. unilateralis chitinase showed higher toxicity against Spodoptera exigua. These results support the hypothesis that chitinolytic enzymes are one of several ‘virulence’ factors produced by entomopathogenic fungi during host encounter.

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 ∼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.

Bacterial Diversity and Phylogenetic Analysis of Type II Polyketide Synthase Gene from Manao-Pee Cave, Thailand

ABSTRACT A subterranean limestone cave, Manao-Pee, was investigated for bacterial diversity and potential secondary metabolites production. Comparative 16 S rRNA analysis revealed that cave soil was highly dominated by Actinobacteria; whereas, Proteobacteria was highly abundant outside the cave. As Actinobacteria are biotechnologically valuable for their secondary metabolites, the diversity of the β-ketoacyl synthase (KSβ) was investigated. The results showed that the identified KSβ has 61–80% amino acid sequence identity to known sequences. Phylogenetic analysis placed some of the sequences in novel clades, suggesting the presence of novel KSβ domains. Thus, Manao-Pee cave is a promising habitat to discover potential novel bioactive compounds.