De novo production of bioactive sesterterpenoid ophiobolins in Saccharomyces cerevisiae cell factories.

BACKGROUND: Sesterterpenoids are rare species among the terpenoids family. Ophiobolins are sesterterpenes with a 5-8-5 tricyclic skeleton. The oxidized ophiobolins exhibit significant cytotoxic activity and potential medicinal value. There is an urgent need for large amounts of ophiobolins supplication for drug development. The synthetic biology approach has been successfully employed in lots of terpene compound production and inspired us to develop a cell factory for ophiobolin biosynthesis. RESULTS: We developed a systematic metabolic engineering strategy to construct an ophiobolin biosynthesis chassis based on Saccharomyces cerevisiae. The whole-cell biotransformation methods were further combined with metabolic engineering to enhance the expression of key ophiobolin biosynthetic genes and improve the supply of precursors and cofactors. A high yield of 5.1 g/L of ophiobolin F was reached using ethanol and fatty acids as substrates. To accumulate oxidized ophiobolins, we optimized the sources and expression conditions for P450-CPR and alleviated the toxicity of bioactive compounds to cells through PDR engineering. We unexpectedly obtained a novel ophiobolin intermediate with potent cytotoxicity, 5-hydroxy-21-formyl-ophiobolin F, and the known bioactive compound ophiobolin U. Finally, we achieved the ophiobolin U titer of 128.9 mg/L. CONCLUSIONS: We established efficient cell factories based on S. cerevisiae, enabling de novo biosynthesis of the ophiobolin skeleton ophiobolin F and oxidized ophiobolins derivatives. This work has filled the gap in the heterologous biosynthesis of sesterterpenoids in S. cerevisiae and provided valuable solutions for new drug development based on sesterterpenoids.

Structural Insights Into the Terpene Cyclization Domains of Two Fungal Sesterterpene Synthases and Enzymatic Engineering for Sesterterpene Diversification.

Little is known about the structures and catalytic mechanisms of sesterterpene synthases (StTSs), which greatly hinders the structure-based engineering of StTSs for structural diversity expansion of sesterterpenes. We here report on the crystal structures of the terpene cyclization (TC) domains of two fungal StTSs: sesterfisherol synthase (NfSS) and sesterbrasiliatriene synthase (PbSS). Both TC structures contain benzyltriethylammonium chloride (BTAC), pyrophosphate (PPi), and magnesium ions (Mg2+), clearly defining the catalytic active sites. A combination of theory and experiments including carbocationic intermediates modeling, site-directed mutagenesis, and isotope labeling provided detailed insights into the structural basis for their catalytic mechanisms. Structure-based engineering of NfSS and PbSS resulted in the formation of 20 sesterterpenes including 13 new compounds and four pairs of epimers with different configurations at C18. These results expand the structural diversity of sesterterpenes and provide important insights for future synthetic biology research.

Two Sesterterpene Synthases from Lentzea atacamensis Demonstrate the Role of Conformational Variability in Terpene Biosynthesis.

Mining of two multiproduct sesterterpene synthases from Lentzea atacamensis resulted in the identification of the synthases for lentzeadiene (LaLDS) and atacamatriene (LaATS). The main product of LaLDS (lentzeadiene) is a new compound, while one of the side products (lentzeatetraene) is the enantiomer of brassitetraene B and the other side product (sestermobaraene F) is known from a surprisingly distantly related sesterterpene synthase. LaATS produces six new compounds, one of which is the enantiomer of the known sesterterpene Bm1. Notably, for both enzymes the products cannot all be explained from one and the same starting conformation of geranylfarnesyl diphosphate, demonstrating the requirement of conformational flexibility of the substrate in the enzymes' active sites. For lentzeadiene an intriguing thermal [1,5]-sigmatropic rearrangement was discovered, reminiscent of the biosynthesis of vitamin D3. All enzyme reactions and the [1,5]-sigmatropic rearrangement were investigated through isotopic labeling experiments and DFT calculations. The results also emphasize the importance of conformational changes during terpene cyclizations.

Heteromerization of short-chain trans-prenyltransferase controls precursor allocation within a plastidial terpenoid network.

Terpenes are the largest and most diverse class of plant specialized metabolites. Sesterterpenes (C25), which are derived from the plastid methylerythritol phosphate pathway, were recently characterized in plants. In Arabidopsis thaliana, four genes encoding geranylfarnesyl diphosphate synthase (GFPPS) (AtGFPPS1 to 4) are responsible for the production of GFPP, which is the common precursor for sesterterpene biosynthesis. However, the interplay between sesterterpenes and other known terpenes remain elusive. Here, we first provide genetic evidence to demonstrate that GFPPSs are responsible for sesterterpene production in Arabidopsis. Blockage of the sesterterpene pathway at the GFPPS step increased the production of geranylgeranyl diphosphate (GGPP)-derived terpenes. Interestingly, co-expression of sesterTPSs in GFPPS-OE (overexpression) plants rescued the phenotypic changes of GFPPS-OE plants by restoring the endogenous GGPP. We further demonstrated that, in addition to precursor (DMAPP/IPP) competition by GFPPS and GGPP synthase (GGPPS) in plastids, GFPPS directly decreased the activity of GGPPS through protein-protein interaction, ultimately leading to GGPP deficiency in planta. Our study provides a new regulatory mechanism of the plastidial terpenoid network in plant cells.

Chemical control over the conversion between bicyclic and polycyclic terpenes by fungal bifunctional terpene synthases.

Fungal bifunctional terpene synthases (BFTSs) reportedly associate with a series of new skeletons of di/sesterterpenes. However, the molecular mechanisms underlying the variabilities in the ring system of BFTS-catalyzed products are not well understood. In this study, we identified a key site, S89/L89, that controls the conversion between bicyclic and polycyclic terpene skeletons catalyzed by two BFTSs, BsPS and FoFS. Our analysis revealed that a mutation on site 89 in the BFTSs alters the carbocation transportation pathway and redirects the competing reactions for previously unreported terpenes.

Schultriene and nigtetraene: two sesterterpenes characterized from pathogenetic fungi via genome mining approach.

Fungal bifunctional terpene synthases (BFTSs) have been reported to contribute to the biosynthesis of a variety of di/sesterterpenes via different carbocation transportation pathways. Genome mining of new BFTSs from unique fungal resources will, theoretically, allow for the identification of new terpenes. In this study, we surveyed the distribution of BFTSs in our in-house collection of 430 pathogenetic fungi and preferred two BFTSs (CsSS and NnNS), long distance from previously characterized BFTSs and located in relatively independent branches, based on the established phylogenetic tree. The heterologous expression of the two BFTSs in Aspergillus oryzae and Saccharomyces cerevisiae led to the identification of two new sesterterpenes separately, 5/12/5 tricyclic type-A sesterterpene (schultriene, 1) for CsSS and 5/11 bicyclic type-B sesterterpene (nigtetraene, 2) for NnNS. In addition, to the best of our knowledge, 2 is the first 5/11 bicyclic type-B characterized sesterterpene to date. On the basis of this, the plausible cyclization mechanisms of 1 and 2 were proposed based on density functional theory calculations. These new enzymes and their corresponding terpenes suggest that the chemical spaces produced by BFTSs remain large and also provide important evidences for further protein engineering for new terpenes and for understanding of cyclization mechanism catalyzed by BFTSs. KEY POINTS: • Genome mining of two BFTSs yields two new sesterterpenoids correspondingly. • Identification of the first 5/11 ring system type-B product. • Parse out the rational cyclization mechanism of isolated sesterterpenoids.

Occurrence and biosynthesis of plant sesterterpenes (C25), a new addition to terpene diversity.

Terpenes, the largest group of plant-specialized metabolites, have received considerable attention for their highly diverse biological activities. Monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), and triterpenes (C30) have been extensively investigated at both the biochemical and molecular levels over the past two decades. Sesterterpenes (C25), an understudied terpenoid group, were recently described by plant scientists at the molecular level. This review summarizes the plant species that produce sesterterpenes and describes recent developments in the field of sesterterpene biosynthesis, placing a special focus on the catalytic mechanism and evolution of geranylfarnesyl diphosphate synthase and sesterterpene synthase. Finally, we propose several questions to be addressed in future studies, which may help to elucidate sesterterpene metabolism in plants.

Systematic mining of fungal chimeric terpene synthases using an efficient precursor-providing yeast chassis.

Chimeric terpene synthases, which consist of C-terminal prenyltransferase (PT) and N-terminal class I terpene synthase (TS) domains (termed PTTSs here), is unique to fungi and produces structurally diverse di- and sesterterpenes. Prior to this study, 20 PTTSs had been functionally characterized. Our understanding of the origin and functional evolution of PTTS genes is limited. Our systematic search of sequenced fungal genomes among diverse taxa revealed that PTTS genes were restricted to Dikarya. Phylogenetic findings indicated different potential models of the origin and evolution of PTTS genes. One was that PTTS genes originated in the common Dikarya ancestor and then underwent frequent gene loss among various subsequent lineages. To understand their functional evolution, we selected 74 PTTS genes for biochemical characterization in an efficient precursor-providing yeast system employing chassis-based, robot-assisted, high-throughput automatic assembly. We found 34 PTTS genes that encoded active enzymes and collectively produced 24 di- and sesterterpenes. About half of these di- and sesterterpenes were also the products of the 20 known PTTSs, indicating functional conservation, whereas the PTTS products included the previously unknown sesterterpenes, sesterevisene (1), and sesterorbiculene (2), suggesting that a diversity of PTTS products awaits discovery. Separating functional PTTSs into two monophyletic groups implied that an early gene duplication event occurred during the evolution of the PTTS family followed by functional divergence with the characteristics of distinct cyclization mechanisms.

The untapped potential of plant sesterterpenoids: chemistry, biological activities and biosynthesis.

Covering: 1969 up to 2021Sesterterpenoids, biosynthetically derived from the precursor, namely geranylfarnesyl diphosphate (GFDP) are amongst the rarest of all isoprenoids with approximately 1300 compounds known. Most sesterterpenoids originate from marine organisms (especially sponges), while only about 15% of these compounds are isolated from several families of plants such as Lamiaceae, Gentianaceae, and Nartheciaceae. Many plant sesterterpenoids possess highly oxygenated and complex cyclic skeletons and exhibit remarkable biological activities involving cytotoxic, anti-inflammatory, antimicrobial, and antifeedant properties. Thus, due to their intrinsic chemical complexity and intriguing biological profiles, plant sesterterpenoids have attracted continuing interest from both chemists and biologists. However, the biosynthesis and distribution of sesterterpenoids in the plant kingdom still remain elusive, although substantial progress has been achieved in recent years. This review provides an overall coverage of sesterterpenoids originating from plant sources, followed by a classification of their chemical skeletons, which summarizes the distribution, chemistry, biological activities, biosynthesis and evolution of plant sesterterpenoids, aiming at strengthening the research efforts toward the untapped great potential of these unique natural product resources.

Genome-Based Discovery of Enantiomeric Pentacyclic Sesterterpenes Catalyzed by Fungal Bifunctional Terpene Synthases.

Genome-based discovery of two previously unreported fungal bifunctional terpene synthases (BFTSs) from phytopathogenic fungi are reported: FoFS catalyzing the formation of fusoxypenes A-C (1-3) and (-)-astellatene (4) and AtAS capable of synthesizing preaspterpenacid I (6). Interestingly, FoFS and AtAS catalyzed the formation of enantiomeric sesterterpenes with a 5-6-7-3-5 ring system. C22-oxidative modification of preaspterpenacid I by AtP450 was characterized as well. Plausible cyclization pathways of the fusoxypenes were illustrated by DFT calculations.

Genome Mining Reveals a Multiproduct Sesterterpenoid Biosynthetic Gene Cluster in Aspergillus ustus.

Genome mining of Aspergillus ustus 094102 enabled the discovery of a multiproduct bifunctional terpene synthase (BTS), AuAS. Heterologous expression of AuAS led to the discovery of five new sesterterpenes, and coexpression of the upstream CYP450 monooxygenase (AuAP450) generated four new sesterterpene alcohols. Additionally, aspergilol A showed cytotoxic activities against MCF-7, MDA-MB231, and HepG2 cancer cells (IC50 21.20-48.76 µM), and aspergilol B exhibited a cytotoxic effect on MCF-7 cells (IC50 27.41 µM).

Molecular Basis for Sesterterpene Diversity Produced by Plant Terpene Synthases.

Class I terpene synthase (TPS) generates bioactive terpenoids with diverse backbones. Sesterterpene synthase (sester-TPS, C25), a branch of class I TPSs, was recently identified in Brassicaceae. However, the catalytic mechanisms of sester-TPSs are not fully understood. Here, we first identified three nonclustered functional sester-TPSs (AtTPS06, AtTPS22, and AtTPS29) in Arabidopsis thaliana. AtTPS06 utilizes a type-B cyclization mechanism, whereas most other sester-TPSs produce various sesterterpene backbones via a type-A cyclization mechanism. We then determined the crystal structure of the AtTPS18-FSPP complex to explore the cyclization mechanism of plant sester-TPSs. We used structural comparisons and site-directed mutagenesis to further elucidate the mechanism: (1) mainly due to the outward shift of helix G, plant sester-TPSs have a larger catalytic pocket than do mono-, sesqui-, and di-TPSs to accommodate GFPP; (2) type-A sester-TPSs have more aromatic residues (five or six) in their catalytic pocket than classic TPSs (two or three), which also determines whether the type-A or type-B cyclization mechanism is active; and (3) the other residues responsible for product fidelity are determined by interconversion of AtTPS18 and its close homologs. Altogether, this study improves our understanding of the catalytic mechanism of plant sester-TPS, which ultimately enables the rational engineering of sesterterpenoids for future applications.

Biosynthetic Gene Cluster for Asperterpenols A and B and the Cyclization Mechanism of Asperterpenol A Synthase.

The biosynthetic gene cluster for the sesterterpenoids asperterpenols A and B from Aspergillus calidoustus CBS121601 is described. The cluster contains two genes, acldAS for a chimeric sesterterpene synthase and acldA-P450 for a cytochrome P450 monooxygenase. The cyclization mechanism of the asperterpenol A synthase, catalyzing the formation of a unique sesterterpene with a 6/6/8/5 tetracyclic ring system, was studied using isotopically labeled terpene precursors.

The Biosynthetic Gene Cluster for Sestermobaraenes-Discovery of a Geranylfarnesyl Diphosphate Synthase and a Multiproduct Sesterterpene Synthase from Streptomyces mobaraensis.

A biosynthetic gene cluster from Streptomyces mobaraensis encoding the first cases of a bacterial geranylfarnesyl diphosphate synthase and a type I sesterterpene synthase was identified. The structures of seven sesterterpenes produced by these enzymes were elucidated, including their absolute configurations. The enzyme mechanism of the sesterterpene synthase was investigated by extensive isotope labeling experiments.

On the mechanism of ophiobolin F synthase and the absolute configuration of its product by isotopic labelling experiments.

An ophiobolin F synthase homolog was discovered from Aspergillus calidoustus CBS121601. The cyclisation mechanism of this terpene synthase was investigated by extensive isotopic labelling experiments and the absolute configuration of its product ophiobolin F was elucidated by enantioselective deuteration.

New secondary metabolites with immunosuppressive activity from the phytopathogenic fungus Bipolaris maydis.

Three previously undescribed compounds, including a meroterpenoid, guignardone T (1), and two ophiobolin-type sesterterpenoids, maydispenoids A and B (2 and 3), along with four known compounds (4-7), were isolated from the phytopathogenic fungus Bipolaris maydis collected from Anoectochilus roxburghii (Wall.) Lindl leaves. The structures of all undescribed compounds were elucidated by spectroscopic analysis, electronic circular dichroism (ECD) calculations and single-crystal X-ray diffraction. Structurally, maydispenoids A was characterized by a fascinating decahydro-3-oxacycloocta[cd]pentalene fragment. It is notable that the compounds 2 and 3 exhibited potential inhibitory activity in anti-CD3/anti-CD28 monoclonal antibodies (mAbs) stimulated murine splenocytes proliferation, with IC50 values of 5.28 and 9.38 µM, respectively, and also suppress the murine splenocytes proliferation activated by lipopolysaccharide (LPS), with IC50 values of 7.25 and 16.82 µM, respectively. This is the first report of ophiobolin-type sesterterpenoids as immunosuppressor, and may provide new chemical templates for the development of new immunosuppressive drugs for autoimmune disease treatment.

High ophiobolin A production in endophytic fungus Bipolaris sp. associated with Datura metel.

Ophiobolin A, a metabolite of fungi, is known to induce cell death and have anticancer activity. Therefore, obtaining ophiobolin A has become an important aspect in studying activity with medicinal properties that are affected by it. Ophiobolin A-producing filamentous fungi are endophytic or infectious microbes that attack annual and short-cycle plants. Here we isolated the endophyte of Datura metel, which is an annual plant that produces ophiobolin A. Results of this study have led to the identification of an endophytic filamentous fungus Bipolaris sp. with high ophiobolin A production (235 mg/L) in liquid culture after 21 days. Our findings further indicate that ophiobolin A-producing fungi live in short-cycle plants, and a method of finding the fungus is described.

Production of sesterterpene ophiobolin by a bifunctional terpene synthase in Escherichia coli.

Ophiobolins (ophs) are characteristic 5-8-5 tricyclic sesterterpenes with potential pharmaceutical activities. Ophiobolin synthase is a bifunctional terpene synthase (BTS) that catalyzes both chain elongation and cyclization. In Aspergillus ustus 094102, ophiobolin accumulation was involved with not only ophiobolin synthase C25 (Au8003) but also other four gene clusters containing C15 (Au6298), C20 (Au13192 and Au11565), and C30 (Au3446) terpene synthases. In this report, overexpression of codon-optimized gene Au8003 resulted in a detectable production of oph F in E. coli. In subsequent modulation of culture conditions, pentose arabinose allowed a more than 10-fold improvement of production than that of glycerol. To achieve a higher titer, the whole mevalonate pathway and an additional copy of isopentenyl diphosphate isomerase gene were assembled, leading to approximately 24-fold and 60-fold yield increases, respectively. The above four terpene synthase genes related to ophiobolin production in strain 094102 were individually or combinatorially overexpressed with Au8003 to mimic the original fungal biosynthesis. The biosynthesis of oph scaffold was increased by short-chain terpene synthases (C15 and C20), among which the C15 synthase gene contributed the highest yield of 82.76 mg/L at 96 h; the multi-gene combinatorial results suggested that cyclization might be a rate-limiting step. Further protein engineering including fusion tags and phylogenetically based mutations on the rate-limiting cyclization part of Au8003 enabled a further yield improvement (> 150 mg/L at 96 h) in shake flasks. These multiple approaches for sesterterpene skeleton production using engineered E. coli may be applicable for cost-effective, high-yield productions of ophiobolins and other compounds synthesized by BTSs.

Unusual and Highly Bioactive Sesterterpenes Synthesized by Pleurotus ostreatus during Coculture with Trametes robiniophila Murr.

Candida albicans and Cryptococcus neoformans, human-pathogenic fungi found worldwide, are receiving increasing attention due to high morbidity and mortality in immunocompromised patients. In the present work, 110 fungus pairs were constructed by coculturing 16 wood-decaying basidiomycetes, among which coculture of Trametes robiniophila Murr and Pleurotus ostreatus was found to strongly inhibit pathogenic fungi through bioactivity-guided assays. A combination of metabolomics and molecular network analysis revealed that 44 features were either newly synthesized or produced at high levels in this coculture system and that 6 of the features that belonged to a family of novel and unusual linear sesterterpenes contributed to high activity with MICs of 1 to 32 µg/ml against pathogenic fungi. Furthermore, dynamic 13C-labeling analysis revealed an association between induced features and the corresponding fungi. Unusual sesterterpenes were 13C labeled only in P. ostreatus in a time course after stimulation by the coculture, suggesting that these sesterterpenes were synthesized by P. ostreatus instead of T. robiniophila Murr. Sesterterpene compounds 1 to 3 were renamed postrediene A to C. Real-time reverse transcription-quantitative PCR (RT-qPCR) analysis revealed that transcriptional levels of three genes encoding terpene synthase, farnesyl-diphosphate farnesyltransferase, and oxidase were found to be 8.2-fold, 88.7-fold, and 21.6-fold higher, respectively, in the coculture than in the monoculture, indicating that biosynthetic gene cluster 10 was most likely responsible for the synthesis of these sesterterpenes. A putative biosynthetic pathway of postrediene A to postrediene C was then proposed based on structures of sesterterpenes and molecular network analysis.IMPORTANCE A number of gene clusters involved in biosynthesis of secondary metabolites are presumably silent or expressed at low levels under conditions of standard laboratory cultivation, resulting in a large gap between the pool of discovered metabolites and genome capability. This work mimicked naturally occurring competition by construction of an artificial coculture of basidiomycete fungi for the identification of secondary metabolites with novel scaffolds and excellent bioactivity. Unusual linear sesterterpenes of postrediene A to C synthesized by P. ostreatus not only were promising lead drugs against human-pathogenic fungi but also highlighted a distinct pathway for sesterterpene biosynthesis in basidiomycetes. The current work provides an important basis for uncovering novel gene functions involved in sesterterpene synthesis and for gaining insights into the mechanism of silent gene activation in fungal defense.

Recently duplicated sesterterpene (C25) gene clusters in Arabidopsis thaliana modulate root microbiota.

Land plants co-speciate with a diversity of continually expanding plant specialized metabolites (PSMs) and root microbial communities (microbiota). Homeostatic interactions between plants and root microbiota are essential for plant survival in natural environments. A growing appreciation of microbiota for plant health is fuelling rapid advances in genetic mechanisms of controlling microbiota by host plants. PSMs have long been proposed to mediate plant and single microbe interactions. However, the effects of PSMs, especially those evolutionarily new PSMs, on root microbiota at community level remain to be elucidated. Here, we discovered sesterterpenes in Arabidopsis thaliana, produced by recently duplicated prenyltransferase-terpene synthase (PT-TPS) gene clusters, with neo-functionalization. A single-residue substitution played a critical role in the acquisition of sesterterpene synthase (sesterTPS) activity in Brassicaceae plants. Moreover, we found that the absence of two root-specific sesterterpenoids, with similar chemical structure, significantly affected root microbiota assembly in similar patterns. Our results not only demonstrate the sensitivity of plant microbiota to PSMs but also establish a complete framework of host plants to control root microbiota composition through evolutionarily dynamic PSMs.