Jun-Fang Lin

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.

Composition of transgenic soybean seeds with higher γ-linolenic acid content is equivalent to that of conventional control.

γ-Linolenic acid (GLA) has been used as a general nutraceutical for pharmacologic applications, particularly in the treatment of skin conditions such as eczema. Four transgenic soybean lines that produce GLA at high yields (4.21% of total fatty acids, up to 1002-fold) were generated through the stable insertion of the Delta-6-fatty acid desaturase gene isolated from Borago officinalis into the genome of a conventional soybean cultivar. As part of the safety assessment of genetically engineered crops, the transgenic soybean seeds were compared with their parental soybean seeds (nontransgenic) by applying the principle of substantial equivalence. Compositional analyses were conducted by measuring the fatty acids, proximate analysis (moisture, crude protein, crude fat, carbohydrates, TDF, and ash contents), amino acids, lectins, and trypsin inhibitor activity. The present results showed that the specific transgenic cultivar studied was similar to the conventional control.

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.

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.

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.

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