Zhi-Wei Ye

Bioproduction of baccatin III, an advanced precursor of paclitaxol, with transgenic Flammulina velutipes expressing the 10‐deacetylbaccatin III‐10‐O‐acetyl transferase gene

BACKGROUND 10-Deacetylbaccatin III (10-DAB) and baccatin III are intermediates in the biosynthesis of Taxol (an anti-cancer drug) and useful precursors for semi-synthesis of the drug. In this study, a bioconversion system was established for the production of baccatin III, an advanced precursor of paclitaxel, in the transgenic mushroom Flammulina velutipes expressing the 10-deacetylbaccatin III-10β-O-acetyltransferase gene. The expression vector pgFvs-TcDBAT containing the 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT) gene was constructed and transformed into the cells of F. velutipes by polyethylene glycol-mediated protoplast transformation. RESULTS Polymerase chain reaction and Southern blotting analysis verified the successful integration of the exogenous DBAT gene into the genome of F. velutipes. Reverse transcription polymerase chain reaction and enzyme activity analyses confirmed that the DBAT gene was expressed in F. velutipes, and DBAT is able to convert substrate into baccatin III. CONCLUSION The DBAT gene from the plant Taxus chinensis can be functionally expressed in F. velutipes. Transgenic F. velutipes expressing the DBAT gene is able to produce the target product, baccatin III. This is the first report about the transformation and expression of paclitaxel biosynthetic gene in the edible mushroom F. velutipes. This represents a significant step towards bio-production of paclitaxel and its advanced precursor baccatin III in an edible fungus.

Compositional analysis of the fruiting body of transgenic Flammulina velutipes producing resveratrol.

Two strains of transgenic Flammulina velutipes TF71 and TF7H with 4cl and rs genes with the capability to produce resveratrol were obtained. In the nutrition assessment and analysis of transgenic strain and original strain (F7), the amino acid composition and proximate compositions of fruiting bodies were determined by amino acid automatic instrument and standard methods of the Association of Official Analytical Chemists (AOAC), while 4-coumaric acid, total flavonoids, and resveratrol were extracted by ethyl acetate and quantified by HPLC. Results indicated that significant differences were observed in proximate composition and amino acid between transgenic and original strains, but these detected components were within the normal ranges reported for F. velutipes. Total flavonoids and 4-coumaric acid contents of transgenic strains are lower than F7. Most important of all, resveratrol has been detected from TF71 and TF7H fruiting body but not found in F7, which was firstly produced by a transgenic mushroom.

Increased resveratrol production in wines using engineered wine strains Saccharomyces cerevisiae EC1118 and relaxed antibiotic or auxotrophic selection

Resveratrol is a polyphenolic compound with diverse beneficial effects on human health. Red wine is the major dietary source of resveratrol but the amount that people can obtain from wines is limited. To increase the resveratrol production in wines, two expression vectors carrying 4‐coumarate: coenzyme A ligase gene (4CL) from Arabidopsis thaliana and resveratrol synthase gene (RS) from Vitis vinifera were transformed into industrial wine strain Saccharomyces cerevisiae EC1118. When cultured with 1 mM p‐coumaric acid, the engineered strains grown with and without the addition of antibiotics produced 8.249 and 3.317 mg/L of trans‐resveratrol in the culture broth, respectively. Resveratrol content of the wine fermented with engineered strains was twice higher than that of the control, indicating that our engineered strains could increase the production of resveratrol during wine fermentation.

Use of autochthonous lactic acid bacteria starters to ferment mango juice for promoting its probiotic roles

ABSTRACT Strains of Leuconostoc mesenteroides, Pediococcus pentosaceus, and Lactobacillus brevis were identified from mango fruits by partial 16S rDNA gene sequence. Based on the ability of producing mannitol and diacetyl, Leuconostoc mesenteroides MPL18 and MPL39 were selected within the lactic acid bacteria isolates, and used as mixed starters to ferment mango juice (MJ). Both the autochthonous strains grew well in fermented mango juice (FMJ) and remained viable at 9.81 log cfu mL−1 during 30 days of storage at 4°C. The content of total sugar of FMJ was lower than that of MJ, while the concentration of mannitol was higher than that of MJ, and the concentration of diacetyl was 3.29 ± 0.12 mg L−1. Among detected organic acids including citric acid, gallic acid, lactic acid, and acetic acid, only citric acid and gallic acid were found in MJ, while all detected organic acids were found in FMJ. The concentration of lactic acid of FMJ was the highest (78.62 ± 13.66 mM) among all detected organic acids. The DPPH radical scavenging capacity of FMJ was higher than that of MJ. Total phenolic compounds were better preserved in FMJ. The acidity and sweetness had a noticeable impact on the overall acceptance of the treated sample.

Targeted Gene Deletion in Cordyceps militaris Using the Split-Marker Approach

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.

Heterologous expression of the multi-functional cellulase gene (mfc) from the mollusc Ampullaria crossean, in Volvariella volvacea

ABSTRACT Volvariella volvacea, or straw mushroom, is a widely cultivated and important edible fungus. In this paper, we used straw mushroom as a host to express an exogenous mfc gene encoding a multi-functional cellulase (MFC) cloned from the mollusc Ampullaria crossean. The purpose was to increase expression of MFC in the new host, as well as to improve cellulose degradation and increase future yields of V. volvacea. MFC is an enzyme with exo-β-1,4-glucanase, endo-β-1,4-glucanase, and endo-β-xylanase activities. The A. crossean mfc gene was expressed in V. volvacea under the control of an endogenous promoter (gpd-Vvs) using the expression vector pgVvs-mfc, constructed by ligating the gpd-Vvs promoter with the mfc gene. The expression plasmid, pgVvs-afp, containing an afp gene encoding an anti-freeze protein (AFP) cloned from spruce budworm as the selective marker gene, was co-transformed into protoplasts of V. volvacea along with the vector pgVvs-mfc using polyethylene glycol (PEG)-mediated transformation. Putative transformants of V. volvacea were obtained by exposing the mycelia to regeneration medium (200 g l−1 potato extract, 20 g l−1 dextrose, 20 g l−1 agar, and 0.8 M D-mannitol) at 0°C. PCR and Southern blotting were used to identify positive transformants. The results indicated that the mfc gene had been integrated into the genome of V. volvacea. The total cellulose activity, based on filter paper degradation, and carboxymethyl cellulase and xylanase activities in the transformants were 3.92, 4.75, and 58.99 Units ml−1, respectively. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showed an approx. 46 kDa band, which was equivalent to the expected size of the MFC protein of A. crossean. Thus, the mfc gene appeared to have been expressed heterologously in V. volvacea.

Complete Genome Sequence of Probiotic Lactobacillus plantarum Strain FMNP01, Isolated from Mango Fruit

ABSTRACT Lactobacillus plantarum strain FMNP01 is a new strain with probiotic properties that was isolated from fresh mango from Guangzhou, China. Here, we report the complete genome of this organism.

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.

De novo transcriptome sequencing of Flammulina velutipes uncover candidate genes associated with cold-induced fruiting

To understand molecular mechanism of cold‐induced fruiting in Flammulina velutipes, which is one of most popular edible fungi in east Asia, de novo assembly of the F. velutipes transcriptome was carried out. There were 26,888,494 and 26,275,146 clean reads obtained from mycelium and primordia of F. velutipes, respectively. A total of 20,157 unigenes were de novo assembled and 15,058 of them were annotated. Moreover, 7935 unigenes were differentially expressed between mycelium and primordia, 4025 of them were up‐regulated and 3910 were down‐regulated. GO and KEGG pathway analysis of the differentially expressed unigenes indicated that functional groups associated with two‐component signaling pathway, calcium signaling, mitogen‐actived protein kinase pathway, molecular chaperones, cell wall and membrane system, play an important role in cold‐induced fruiting of F. velutipes. In this work 643 EST‐SSRs were identified in 20,157 unigenes and 1560 EST‐SSRs primers pairs were designed. Moreover, 5548 and 5955 SNPs were detected in mycelium and primordia, respectively. Consequently, results of this work can serve as a valuable resource for functional genomics study of cold‐induced fruiting in F. velutipes.

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.