Ergot alkaloids are a class of indole derivatives produced by the genera of Ascomycota including Claviceps, Aspergillus, Penicillium, and Epichloë. Many natural and semi-synthetic ergot alkaloids exhibit valuable pharmacological activities and have been widely used in the therapy of human CNS disorders. Owing to the development of genome sequencing technology, the gene clusters involved in the biosynthesis of ergot alkaloids have been identified from these fungi. In this review, we briefly introduce the pharmacological activities and possible mechanisms of action of some ergot alkaloids. Then we summarize the recent progress in the functional characterization of the key genes and gene clusters involved in the biosynthetic pathways of ergot alkaloids from different genera. Particularly, we summarize and discuss the constructions of ergot alkaloid biosynthetic pathways in different heterologous hosts and the optimization strategies performed on the recombinant strains, which provide references for producing ergot alkaloids and the derivatives in cell factories by synthetic biology in the future.

The protection and sustainable utilization of natural resources are among the most pressing global problems of the 21st century. Taxol is a well-known anticancer agent with a unique active mechanism, mainly isolated from the slow-growing yew tree, which has achieved the status of “blockbuster drug” due to its popularity in the treatment of cancers. However, the source of Taxol has always been a primary concern, because its content in the plant is extremely low. In this review, we introduce the advances in exploring alternative Taxol sources aside from total synthesis, including Taxol from nursery cultivated Taxus, semi-synthesis of Taxol, Taxol from Taxus cell culture, production of Taxol by synthetic biology, Taxol from the endophytic fungi and Taxol from other non-Taxus plants. The future perspective of Taxol production is also discussed.

Paclitaxel content in yew tree is extremely low, causing a worldwide shortage of this important anticancer drug. Yew tree can also produce abundant 7-β-xylosyl-10-deacetyltaxol that can be bio-converted into 10-deacetyltaxol for semi-synthesis of paclitaxel. However, the bio-conversion by the screened natural microorganisms was inefficient. We have constructed the recombinant yeast with a glycoside hydrolase gene from Lentinula edodes and explored the bioconversion. Based on previously established reaction conditions, the bioconversion of 7-β-xylosyl-10-deacetyltaxol or its extract was further optimized and scaled up with the engineered yeast harvested from 200-L scale high-cell-density fermentation. The optimization included the freeze-dried cell amount, dimethyl sulfoxide concentration, addition of 0.5 % antifoam supplement, and substrate concentration. A 93–95 % bioconversion and 83 % bioconversion of 10 and 15 g/L 7-β-xylosyltaxanes in 10 L reaction volume were achieved, respectively. The yield of 10-deacetyltaxol reached 10.58 g/L in 1 L volume with 15 g/L 7-β-xylosyl-10-deacetyltaxol. The conversion efficiencies were not only much higher than those of other reports and our previous work, but also realized in 10 L reaction volume. A pilot-scale product purification was also established. Our study bridges the gap between the basic research and commercial utilization of 7-β-xylosyl-10-deacetyltaxol for the industrial production of semi-synthetic paclitaxel.

•Two new cadinane-type sesquiterpenes were isolated from Hypocreales sp.•Five other known compounds were also isolated.•The absolute configurations of the new compounds were deduced.•Two compounds showed moderate anti-inflammatory activity.Two new cadinane-type sesquiterpenes, hypocreaterpenes A (1) and B (2), along with five known compounds (3–7) were isolated from a marine-derived fungus Hypocreales sp. strain HLS-104 isolated from a sponge Gelliodes carnosa. Their structures were determined by a combination of spectroscopic methods. All compounds were tested for the inhibitory effects on the nitric oxide (NO) production in lipopolysaccharide (LPS)-treated RAW264.7 cells. Among them, compounds 3 and 6 showed moderate anti-inflammatory activity with average maximum inhibition (Emax) values of 10.22% and 26.46% at 1 μM, respectively.

The glycoside hydrolase of 7-β-xylosyltaxanes (designated as LXYL-P1-2) is encoded by Lxyl-p1-2 isolated from Lentinula edodes. This hydrolase specifically removes C-7 xylose from 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes, which can be used for the semi-synthesis of paclitaxel or its analogues. In our present study, we established a high-cell-density fermentation of the recombinant Pichia pastoris harboring the Lxyl-p1-2 gene. Moreover, we further optimized the fermentation conditions, including the initial cell density and the dissolved oxygen level in the induction phase. Under optimized conditions, the biomass of 312.3 g/l (wet cell weight, WCW) was obtained, and the biomass activity of the recombinant enzyme reached 6.55 × 104 U/g (WCW). The freeze-dried cells (32 g/l) were used to convert 7-β-xylosyltaxanes (10 g/l, 7-β-xylosyl-10-deacetyltaxol = 62.12 %) in a 5-l reaction volume, and a bioconversion rate about 80 % was achieved. The product purification was performed by ethyl acetate, silica gel chromatography, and preparative HPLC (prep-HPLC), yielding 15.13 g of 10-deacetyltaxol, 3.07 g of 10-deacetylcephalomanine, and 3.47 g of 10-deacetyltaxol C, respectively. In addition, the average recovery rate was around 70 %. Our work provided a foundation for the industrial utilization of the recombinant enzyme on the semi-synthesis of paclitaxel using 7-β-xylosyltaxanes.

Several molecular techniques were employed to document the bacterial diversity associated with the marine sponge Gelliodes carnosa. Cultivation-dependent and cultivation-independent methods were used to obtain the 16S rRNA gene sequences of the bacteria. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the bacterial community structure was highly diverse with representatives of the high G + C Gram-positive bacteria, cyanobacteria, low G + C Gram-positive bacteria, and proteobacteria (α-, β-, and γ-), most of which were also found in other marine environments, including in association with other sponges. Overall, 300 bacterial isolates were cultivated, and a total of 62 operational taxonomic units (OTUs) were identified from these isolates by restriction fragment length polymorphism (RFLP) analysis and DNA sequencing of the 16S rRNA genes. Approximately 1,000 16S rRNA gene clones were obtained by the cultivation-independent method. A total of 310 clones were randomly selected for RFLP analysis, from which 33 OTUs were acquired by further DNA sequencing and chimera checking. A total of 12 cultured OTUs (19.4% of the total cultured OTUs) and 13 uncultured OTUs (39.4% of the total uncultured OTUs) had low sequence identity (≤97%) with their closest matches in GenBank and were probably new species. Our data provide strong evidence for the presence of a diverse variety of unidentified bacteria in the marine sponge G. carnosa. A relatively high proportion of the isolates exhibited antimicrobial activity, and the deferred antagonism assay showed that over half of the active isolates exhibited a much stronger bioactivity when grown on medium containing seawater. In addition to demonstrating that the sponge-associated bacteria could be a rich source of new biologically active natural products, the results may have ecological implications. This study expands our knowledge of the diversity of sponge-associated bacteria and contributes to the growing database of the bacterial communities within sponges.

The diversity and biological activities of fungi associated with the two sponges, Haliclona simulans and Gelliodes carnosa, were investigated using a culture-dependent method followed by analysis of the fungal rDNA-ITS sequences. The two sponges were collected from the coastal waters of Lingshui Bay of Hainan Island in the South China Sea. A total of 37 independent fungal isolates corresponding to 30 different species were obtained from the two sponges. Nearly two thirds of the strains (n = 24, 64.9%) had close affiliations (identity (ID) or similarity ≥ 98%) with their best matches in GenBank. Another one third of the isolates (n = 13, 35.1%) were distantly related to their closest relatives (ID < 98%), implying that these species are possibly different from those previously reported. The two sponges possessed similar fungal diversities. Haliclona simulans harbored a mainly different fungal consortium as compared with that of the same sponge species collected from Irish coastal waters, suggesting that the fungal diversity associated with the sponges is more dependent on the surrounding environment than on the sponge species. Biological activities of the fungal culture extracts were tested against the human tumor cell lines, mainly, a human lung carcinoma cell line (A-549), a human liver carcinoma cell line (Bel-7402), and a human colon carcinoma cell line (HCT-8), and against the Gram positive bacterium Bacillus subtilis. A relatively high proportion of positive results were obtained in this study, demonstrating that fungi isolated from sponges could be a rich source of new biologically active natural products.

The gene encoding squalene synthase (GfSQS) was cloned from Fusarium fujikuroi (Gibberella fujikuroi MP-C) and characterized. The cloned genomic DNA is 3,267 bp in length, including the 5′-untranslated region (UTR), 3′-UTR, four exons, and three introns. A noncanonical splice-site (CA-GG, or GC-AG) was found at the first intron. The open reading frame of the gene is 1,389 bp in length, corresponding to a predicted polypeptide of 462 amino acid residues with a MW 53.4 kDa. The predicted GfSQS shares at least four conserved regions involved in the enzymatic activity with the SQSs of varied species. The recombinant protein was expressed in E. coli and detected by SDS–PAGE and western blot. GC–MS analysis showed that the wild-type GfSQS could catalyze the reaction from farnesyl diphosphate (FPP) to squalene, while the mutant mGfSQS (D82G) lost total activity, supporting the prediction that the aspartate-rich motif (DTXED) in the region I of SQS is essential for binding of the diphosphate substrate.

BmK AngM1 is an analgesic peptide from the venom of Buthus martensii Karsch (BmK). The synthetic gene encoding BmK AngM1 was optimized on the basis of its cDNA sequence and the codon usage preference of Pichia pastoris. The codon-optimized gene was cloned into pPIC9K and then transformed into P. pastoris. SDS-PAGE and Western blot analysis showed that the recombinant BmK AngM1 (rBmK AngM1) was expressed by the addition of methanol to the medium, and its maximum production reached above 500 mg/l. The purified rBmK AngM1 could be obtained efficiently by Nickel affinity chromatography. Analgesic bioassay, by the mouse-twisting model, showed that rBmK AngM1 had evident analgesic effect with an ED50 of 0.5 mg/kg.