Jin-Ling Yang

Improving 10-deacetylbaccatin III-10-β- O -acetyltransferase catalytic fitness for Taxol production

The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-β-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-β-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a β-xylosidase to obtain an in vitro one-pot conversion of 7-β-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml−1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.

Production of a bioactive unnatural ginsenoside by metabolically engineered yeasts based on a new UDP-glycosyltransferase from Bacillus subtilis

Ginsenosides are the main bioactive constituents of Panax species, which are biosynthesized by glycosylation at C3-OH and/or C20-OH of protopanaxadiol (PPD), C6-OH and/or C20-OH of protopanaxatriol (PPT). The C12-glycosylated ginsenosides have scarcely been identified from Panax species. The C12-glycosylated ginsenosides produced from PPD by chemical semi-synthesis have been reported to exhibit higher cytotoxicity than the natural ginsenosides. However, the chemical semi-synthesis approach is not practical due to its complexity and high cost. In our study, a new UDP-glycosyltransferase UGT109A1 was identified from Bacillus subtilis. This enzyme transferred a glucose moiety to C3-OH and C20-OH of dammarenediol-II (DM), C3-OH and C12-OH of PPD and PPT respectively to produce the unnatural ginsenosides 3β-O-Glc-DM, 3β,20S-Di-O-Glc-DM, 3β,12β-Di-O-Glc-PPD and 3β,12β-Di-O-Glc-PPT. Among these unnatural ginsenosides, 3β,12β-Di-O-Glc-PPT is a new compound which has never been reported before. The anti-cancer activities of these unnatural ginsenosides were evaluated in vitro and in vivo. 3β,12β-Di-O-Glc-PPD exhibited higher anti-lung cancer activity than Rg3, which is the most active natural ginsenoside against lung cancer. Finally, we constructed metabolically engineered yeasts to produce 3β,12β-Di-O-Glc-PPD by introducing the genes encoding B. subtilis UGT109A1, Panax ginseng dammarenediol-II synthase (DS), P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS) together with Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1) into Saccharomyces cerevisiae INVSc1. The yield of 3β,12β-Di-O-Glc-PPD was increased from 6.17mg/L to 9.05mg/L by overexpressing tHMG1. Thus, this study has established an alternative route to produce the unnatural ginsenoside 3β,12β-Di-O-Glc-PPD by synthetic biology strategies, which provides a promising candidate for anti-cancer drug discovery.

Tetrocarcin Q, a New Spirotetronate with a Unique Glycosyl Group from a Marine-Derived Actinomycete Micromonospora carbonacea LS276

A new spirotetronate glycoside tetrocarcin Q (1) and six known analogues tetrocarcin A (2), AC6H (3), tetrocarcin N (4), tetrocarcin H (5), arisostatin A (6), and tetrocarcin F1 (7) were isolated from the fermentation broth of the marine-derived actinomycete Micromonospora carbonacea LS276. Their chemical structures were established on the basis of 1D- and 2D-NMR spectroscopy, as well as HR-ESI-MS analysis. The absolute configurations of their stereogenic carbons were determined by circular dichroism (CD) analysis. Compound 1 possesses 2-deoxy-allose, which is a unique sugar type at the C-9 position. This type has not been found in the previously reported spirotetronate glycosides. Compound 1 displayed moderate antibacterial activity against Bacillus subitlis ATCC 63501 with minimum inhibitory concentration (MIC) value of 12.5 μM.

Enhancement of recombinant BmK AngM1 production in Pichia pastoris by regulating gene dosage, co-expressing with chaperones and fermenting in fed-batch mode

Abstract The scorpion peptide BmK AngM1 was reported to exhibit evident analgesic effect, but its yield by extraction from scorpion venom limits the research and application. The heterologous expression of BmK AngM1 was achieved in Pichia pastoris in our previous study. In order to realize high-level expression of recombinant BmK AngM1 (rBmK AngM1), the gene dosage of BmK AngM1 was optimized in engineered strains. The yield of rBmK AngM1 in the four-copy strain reached up to 100 mg/L, which was further enhanced to 190 mg/L by co-expressing with chaperones of PDI, BiP, and HAC1. Moreover, the yield of rBmK AngM1 was up to 1200 mg/L by high-density fermentation in 10 L fermenter. Finally, 360 mg rBmK AngM1 was purified from 1 L cultures by a two-step purification method. The efficient and convenient techniques presented in this study could facilitate further scale-up for industrial production of rBmK AngM1.

Two new monoterpenoid α-pyrones from a fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa

Two new monoterpenoid α-pyrones, named nectriapyrones C and D (1 and 2), along with a known α-pyrone (nectriapyrone, 3) were isolated from a marine-derived fungus Nectria sp. HLS206 associated with the marine sponge Gelliodes carnosa collected from the South China Sea. Their structures were determined on the basis of 1D NMR, 2D NMR, HR-ESI-MS methods.

Author Correction: Improving 10-deacetylbaccatin III-10-β- O -acetyltransferase catalytic fitness for Taxol production

This corrects the article DOI: 10.1038/ncomms15544.

Enzymatic Synthesis of Unnatural Ginsenosides Using a Promiscuous UDP-Glucosyltransferase from Bacillus subtilis

Glycosylation, which is catalyzed by UDP-glycosyltransferases (UGTs), is an important biological modification for the structural and functional diversity of ginsenosides. In this study, the promiscuous UGT109A1 from Bacillus subtilis was used to synthesize unnatural ginsenosides from natural ginsenosides. UGT109A1 was heterologously expressed in Escherichia coli and then purified by Ni-NTA affinity chromatography. Ginsenosides Re, Rf, Rh1, and R1 were selected as the substrates to produce the corresponding derivatives by the recombinant UGT109A1. The results showed that UGT109A1 could transfer a glucosyl moiety to C3-OH of ginsenosides Re and R1, and C3-OH and C12-OH of ginsenosides Rf and Rh1, respectively, to produce unnatural ginsenosides 3,20-di-O-β-d-glucopyranosyl-6-O-[α-l-rhamnopyrano-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (1), 3,20-di-O-β-d-glucopyranosyl-6-O-[β-d-xylopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (6), 3-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (3), 3,12-di-O-β-d-glucopyranosyl-6-O-[β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl]-dammar-24-ene-3β,6α,12β,20S-tetraol (2), 3,6-di-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (5), and 3,6,12-tri-O-β-d-glucopyranosyl-dammar-24-ene-3β,6α,12β,20S-tetraol (4). Among the above products, 1, 2, 3, and 6 are new compounds. The maximal activity of UGT109A1 was achieved at the temperature of 40 °C, in the pH range of 8.0–10.0. The activity of UGT109A1 was considerably enhanced by Mg2+, Mn2+, and Ca2+, but was obviously reduced by Cu2+, Co2+, and Zn2+. The study demonstrated that UGT109A1 was effective in producing a series of unnatural ginsenosides through enzymatic reactions, which could pave a way to generate promising leads for new drug discovery.

Progress on the Studies of the Key Enzymes of Ginsenoside Biosynthesis

As the main bioactive constituents of Panax species, ginsenosides possess a wide range of notable medicinal effects such as anti-cancer, anti-oxidative, antiaging, anti-inflammatory, anti-apoptotic and neuroprotective activities. However, the increasing medical demand for ginsenosides cannot be met due to the limited resource of Panax species and the low contents of ginsenosides. In recent years, biotechnological approaches have been utilized to increase the production of ginsenosides by regulating the key enzymes of ginsenoside biosynthesis, while synthetic biology strategies have been adopted to produce ginsenosides by introducing these genes into yeast. This review summarizes the latest research progress on cloning and functional characterization of key genes dedicated to the production of ginsenosides, which not only lays the foundation for their application in plant engineering, but also provides the building blocks for the production of ginsenosides by synthetic biology.

Recent progress in ergot alkaloid research

Ergot alkaloids are a class of indole derivatives produced by the genera of Ascomycota including Claviceps, Aspergillus, Penicillium, and Epichloe. 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.