Tao Wei

Genomics-guided discovery and structure identification of cyclic lipopeptides from the Bacillus siamensis JFL15

In this research, a strain with broad-spectrum antimicrobial activities was isolated from the gastrointestinal tract of hairtail (Trichiurus haumela) and identified as Bacillus siamensis JFL15 through morphological, 16S rRNA, and average nucleotide identity analyses. The genome of B. siamensis JFL15 was sequenced, and three gene clusters involved in the biosynthesis of surfactin (srf), bacillibactin (dhb), and fengycin (fen) were predicted through antiSMASH analysis. The combined genomics-metabolics profiling of the strain revealed 20 active compounds, which belong to four main types of cyclic lipopeptides produced by Bacillus species: bacillibactin, iturin, fengycin, and surfactin. Among these lipopeptides, two high-purity antifungal components, namely, components b and c, were successfully identified as iturin A and bacillomycin F. The minimum inhibitory concentrations (MICs) of iturin A for Magnapothe grisea, Rhizoctorzia solani, and Colletotrichum gloeosporioides were 125.00, 62.50, and 125.00 μg/ml, respectively, whereas the MICs of bacillomycin F for these three organisms were 62.50, 31.25, and 62.50 μg/ml, respectively. The mechanism of bacillomycin F and iturin A against M. grisea was also investigated. Scanning electron microscopy (SEM) indicated that the surface of the hypha treated with iturin A or bacillomycin F became sunk, lumpy, and wrinkled. The diversity of the identified and predicted compounds from B. siamensis JFL15 suggested that this strain might be a promising biocontrol agent for an effective and environmentally friendly control of pathogenic microorganisms. To the best of our knowledge, this study is the first to describe cyclic lipopeptides purified and identified from B. siamensis.

Bio-production of Baccatin III, an Important Precursor of Paclitaxel by a Cost-Effective Approach

Natural production of anti-cancer drug taxol from Taxus has proved to be environmentally unsustainable and economically unfeasible. Currently, bioengineering the biosynthetic pathway of taxol is an attractive alternative production approach. 10-deacetylbaccatin III-10-O-acetyl transferase (DBAT) was previously characterized as an acyltransferase, using 10-deacetylbaccatin III (10-DAB) and acetyl CoA as natural substrates, to form baccatin III in the taxol biosynthesis. Here, we report that other than the natural acetyl CoA (Ac-CoA) substrate, DBAT can also utilize vinyl acetate (VA), which is commercially available at very low cost, acylate quickly and irreversibly, as acetyl donor in the acyl transfer reaction to produce baccatin III. Furthermore, mutants were prepared via a semi-rational design in this work. A double mutant, I43S/D390R was constructed to combine the positive effects of the different single mutations on catalytic activity, and its catalytic efficiency towards 10-DAB and VA was successfully improved by 3.30-fold, compared to that of wild-type DBAT, while 2.99-fold higher than the catalytic efficiency of WT DBAT towards 10-DAB and Ac-CoA. These findings can provide a promising economically and environmentally friendly method for exploring novel acyl donors to engineer natural product pathways.

Improving the thermal stability of anisyl alcohol by β-galactosidase enzymatic glycosylation

1 Institute of Food Biotechnology, South China Agriculture University, 482 Wu-Shan Street, Tian-He, Guangzhou 510640, China 2 Department of Bioengineering, College of Food Science, South China Agricultural University, 482 Wu-Shan Street, Tian-He, Guangzhou 510640, China 3 James Clark School of Engineering, University of Maryland, College Park, MD 20742, USA 4 Alchemy Biotechnology Co. Ltd. of Guangzhou City, Guangzhou 510760, China

Efficient CRISPR-Cas9 Gene Disruption System in Edible-Medicinal Mushroom Cordyceps militaris

Cordyceps militaris is a well-known edible medicinal mushroom in East Asia that contains abundant and diverse bioactive compounds. Since traditional genome editing systems in C. militaris were inefficient and complicated, here, we show that the codon-optimized cas9, which was used with the newly reported promoter Pcmlsm3 and terminator Tcmura3, was expressed. Furthermore, with the help of the negative selection marker ura3, a CRISPR-Cas9 system that included the Cas9 DNA endonuclease, RNA presynthesized in vitro and a single-strand DNA template efficiently generated site-specific deletion and insertion. This is the first report of a CRISPR-Cas9 system in C. militaris, and it could accelerate the genome reconstruction of C. militaris to meet the need for rapid development in the fungi industry.

Enhanced catalytic activities and modified substrate preferences for taxoid 10β- O -acetyl transferase mutants by engineering catalytic histidine residues

ObjectivesTaxoid 10β-O-acetyl transferase (DBAT) was redesigned to enhance its catalytic activity and substrate preference for baccatin III and taxol biosynthesis.ResultsResidues H162, D166 and R363 were determined as potential sites within the catalytic pocket of DBAT for molecular docking and site-directed mutagenesis to modify the activity of DBAT. Enzymatic activity assays revealed that the kcat/KM values of mutant H162A/R363H, D166H, R363H, D166H/R363H acting on 10-deacetylbaccatin III were about 3, 15, 26 and 60 times higher than that of the wild type of DBAT, respectively. Substrate preference assays indicated that these mutants (H162A/R363H, D166H, R363H, D166H/R363H) could transfer acetyl group from unnatural acetyl donor (e.g. vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-deacetylbaccatin III.ConclusionTaxoid 10β-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.

Activity Essential Residue Analysis of Taxoid 10β-O-Acetyl Transferase for Enzymatic Synthesis of Baccatin

Taxoid 10β-O-acetyl transferase (DBAT) is a key enzyme in the biosynthesis of the famous anticancer drug paclitaxel, which catalyses the formation of baccatin III from 10-deacetylbaccatin III (10-DAB). However, the activity essential residues of the enzyme are still unknown, and the acylation mechanism from its natural substrate 10-deacetylbaccatin III and acetyl CoA to baccatin III remains unclear. In this study, the homology modelling, molecular docking, site-directed mutagenesis, and kinetic parameter determination of the enzyme were carried out. The results showed that the enzyme mutant DBATH162A resulted in complete loss of enzymatic activity, suggesting that the residue histidine at 162 was essential to DBAT activity. Residues D166 and R363 which were located in the pocket of the enzyme by homology modelling and molecular docking were also important for DBAT activity through the site-directed mutations. Furthermore, four amino acid residues including S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG also played important roles on acylation. This was the first report of the elucidation of the activity essential residues of DBAT, making it possible for the further structural-based re-design of the enzyme for efficient biotransformation of baccatin III and paclitaxel.

Isolation and characterization of cyclic lipopeptides with broad-spectrum antimicrobial activity from Bacillus siamensis JFL15

In this research, the antimicrobial substance anti-JFL15 was partially purified using a simple two-step extraction process from the cell-free supernatants of Bacillus siamensis JFL15. Anti-JFL15 exhibited a strong antibacterial activity against various multidrug-resistant aquatic bacterial pathogens, including Escherichia coli, Edwardsiella tarda, Pseudomonas aeruginosa, Aeromonas hydrophila, and Vibrio. Liquid chromatography–mass spectrometry revealed that anti-JFL15 contained eight cyclic lipopeptides belonging to two families: bacillomycin F (m/z 1056.56–1084.59) and surfactin (m/z 1007.65–1049.70) analogs. PCR analysis showed the presence of genes (i.e., sfp gene, surfactin synthetase D, fengycin synthetase B, iturin synthetase A, iturin synthetase C and bacillomycin synthetase D) involved in the biosynthesis of cyclic lipopeptides. This study is the first to identify cyclic lipopeptides from B. siamensis and use them to suppress the growth of various multidrug-resistant aquatic bacterial pathogens. Results indicated that B. siamensis JFL15 is a promising biocontrol agent for the effective and environmentally friendly control of various multidrug-resistant aquatic bacterial pathogens.