Complete pathway elucidation and heterologous reconstitution of (+)-nootkatone biosynthesis from Alpinia oxyphylla.

(+)-Nootkatone is a natural sesquiterpene ketone widely used in food, cosmetics, pharmaceuticals, and agriculture. It is also regarded as one of the most valuable terpenes used commercially. However, plants contain trace amounts of (+)-nootkatone, and extraction from plants is insufficient to meet market demand. Alpinia oxyphylla is a well-known medicinal plant in China, and (+)-nootkatone is one of the main components within the fruits. By transcriptome mining and functional screening using a precursor-providing yeast chassis, the complete (+)-nootkatone biosynthetic pathway in Alpinia oxyphylla was identified. A (+)-valencene synthase (AoVS) was identified as a novel monocot-derived valencene synthase; three (+)-valencene oxidases AoCYP6 (CYP71BB2), AoCYP9 (CYP71CX8), and AoCYP18 (CYP701A170) were identified by constructing a valencene-providing yeast strain. With further characterisation of a cytochrome P450 reductase (AoCPR1) and three dehydrogenases (AoSDR1/2/3), we successfully reconstructed the (+)-nootkatone biosynthetic pathway in Saccharomyces cerevisiae, representing a basis for its biotechnological production. Identifying the biosynthetic pathway of (+)-nootkatone in A. oxyphylla unravelled the molecular mechanism underlying its formation in planta and also supported the bioengineering production of (+)-nootkatone. The highly efficient yeast chassis screening method could be used to elucidate the complete biosynthetic pathway of other valuable plant natural products in future.

Redox signaling-driven modulation of microbial biosynthesis and biocatalysis.

Microbial communication can drive coordinated functions through sensing, analyzing and processing signal information, playing critical roles in biomanufacturing and life evolution. However, it is still a great challenge to develop effective methods to construct a microbial communication system with coordinated behaviors. Here, we report an electron transfer triggered redox communication network consisting of three building blocks including signal router, optical verifier and bio-actuator for microbial metabolism regulation and coordination. In the redox communication network, the Fe3+/Fe2+ redox signal can be dynamically and reversibly transduced, channeling electrons directly and specifically into bio-actuator cells through iron oxidation pathway. The redox communication network drives gene expression of electron transfer proteins and simultaneously facilitates the critical reducing power regeneration in the bio-actuator, thus enabling regulation of microbial metabolism. In this way, the redox communication system efficiently promotes the biomanufacturing yield and CO2 fixation rate of bio-actuator. Furthermore, the results demonstrate that this redox communication strategy is applicable both in co-culture and microbial consortia. The proposed electron transfer triggered redox communication strategy in this work could provide an approach for reducing power regeneration and metabolic optimization and could offer insights into improving biomanufacturing efficiency.

Exploring the Application and Prospects of Synthetic Biology in Engineered Living Materials.

At the intersection of synthetic biology and materials science, engineered living materials (ELMs) exhibit unprecedented potential. Possessing unique "living" attributes, ELMs represent a significant paradigm shift in material design, showcasing self-organization, self-repair, adaptability, and evolvability, surpassing conventional synthetic materials. This review focuses on reviewing the applications of ELMs derived from bacteria, fungi, and plants in environmental remediation, eco-friendly architecture, and sustainable energy. The review provides a comprehensive overview of the latest research progress and emerging design strategies for ELMs in various application fields from the perspectives of synthetic biology and materials science. In addition, the review provides valuable references for the design of novel ELMs, extending the potential applications of future ELMs. The investigation into the synergistic application possibilities amongst different species of ELMs offers beneficial reference information for researchers and practitioners in this field. Finally, future trends and development challenges of synthetic biology for ELMs in the coming years are discussed in detail.

Characterization of Tremulane Sesquiterpene Synthase from the Basidiomycete Irpex lacteus.

Tremulane sesquiterpenoids are key secondary metabolites of the basidiomycete Irpex lacteus, which displays structural diversity and various bioactivities. However, tremulane sesquiterpene synthases have not been reported to date. The tremulane sesquiterpene synthase of I. lacteus was characterized by genome mining, heterologous expression, an in vitro assay, and substrate feeding. Moreover, the structures of the corresponding products were elucidated by NMR spectroscopy and X-ray diffraction analysis.

Discovery of non-squalene triterpenes.

All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene1. This approach is fundamentally different from the biosynthesis of short-chain (C10-C25) terpenes that are formed from polyisoprenyl diphosphates2-4. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.

Solar-Driven Overproduction of Biofuels in Microorganisms.

Microbial cell factories reinvigorate current industries by producing complex fine chemicals at low costs. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) is the main reducing power to drive the biosynthetic pathways in microorganisms. However, insufficient intrinsic NADPH limits the productivity of microorganisms. Here, we report that supplying microorganisms with long-lived electrons from persistent phosphor mesoporous Al2 O3 (meso-Al2 O3 ) can elevate the NADPH level to facilitate efficient fine chemical production. The defects in meso-Al2 O3 were demonstrated to be highly efficient in prolonging electrons' lifetime. The long-lived electrons in meso-Al2 O3 can pass the material-microorganism interface and power the biosynthetic pathways of E. coli to produce jet fuel farnesene. This work represents a reliable strategy to design photo-biosynthesis systems to improve the productivity of microorganisms with solar energy.

Systematic identification of Ocimum sanctum sesquiterpenoid synthases and (-)-eremophilene overproduction in engineered yeast.

Plant-derived natural active products have attracted increasing attention for use in flavors and perfumes. These compounds also have applications in insect pest control because of their environment-friendly properties. Holy basil (Ocimum sanctum), a famous herb used in Ayurveda in India, is a natural source of medical healing agents and insecticidal repellents. Despite the available genomic sequences and genome-wide bioinformatic analysis of terpene synthase genes, the functionality of the sesquiterpene genes involved in the unique fragrance and insecticidal activities of Holy basil are largely unknown. In this study, we systematically screened the sesquiterpenoid biosynthesis genes in this plant using a precursor-providing yeast system. The enzymes that synthesize ß-caryophyllene and its close isomer α-humulene were successfully identified. The enzymatic product of OsaTPS07 was characterized by in vivo mining, in vitro reaction, and NMR detection. This product was revealed as (-)-eremophilene. We created a mutant yeast strain that can achieve a high-yield titer by adjusting the gene copy number and FPP precursor enhancement. An optimized two-stage fed-batch fermentation method achieved high biosynthetic capacity, with a titer of 34.6 g/L cyclic sesquiterpene bioproduction in a 15-L bioreactor. Further insect-repelling assays demonstrated that (-)-eremophilene repelled the insect pest, fall leafworm, suggesting the potential of (-)-eremophilene as an alternative to synthetic chemicals for agricultural pest control. This study highlights the potential of our microbial platform for the bulk mining of plant-derived ingredients and provides an impressive cornerstone for their industrial utilization.

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.

Discovery of the cryptic function of terpene cyclases as aromatic prenyltransferases.

Catalytic versatility is an inherent property of many enzymes. In nature, terpene cyclases comprise the foundation of molecular biodiversity as they generate diverse hydrocarbon scaffolds found in thousands of terpenoid natural products. Here, we report that the catalytic activity of the terpene cyclases AaTPS and FgGS can be switched from cyclase to aromatic prenyltransferase at basic pH to generate prenylindoles. The crystal structures of AaTPS and FgGS provide insights into the catalytic mechanism of this cryptic function. Moreover, aromatic prenyltransferase activity discovered in other terpene cyclases indicates that this cryptic function is broadly conserved among the greater family of terpene cyclases. We suggest that this cryptic function is chemoprotective for the cell by regulating isoprenoid diphosphate concentrations so that they are maintained below toxic thresholds.

Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.

Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.

A Family of Related Fungal and Bacterial Di- and Sesterterpenes: Studies on Fusaterpenol and Variediene.

The absolute configuration of fusaterpenol (GJ1012E) has been revised by an enantioselective deuteration strategy. A bifunctional enzyme with a terpene synthase and a prenyltransferase domain from Aspergillus brasiliensis was characterised as variediene synthase, and the absolute configuration of its product was elucidated. The uniform absolute configurations of these and structurally related di- and sesterterpenes together with a common stereochemical course for the geminal methyl groups of GGPP unravel a similar conformational fold of the substrate in the active sites of the terpene synthases. For variediene, a thermal reaction observed during GC/MS analysis was studied in detail for which a surprising mechanism was uncovered.

Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products.

Sesquiterpene synthases in Trichoderma viride have been seldom studied, despite the efficiency of filamentous fungi for terpenoid production. Using the farnesyl diphosphate-overexpressing Saccharomyces cerevisiae platform to produce diverse terpenoids, we herein identified an unknown sesquiterpene synthase from T. viride by genome mining and determined the structure of its corresponding products. One new 5/6 bicyclic sesquiterpene and its esterified derivative were characterised by GC-MS and 1D and 2D NMR spectroscopy. To the best of our knowledge, this is the first well-identified sesquiterpene synthase from T. viride to date.

Sesquiterpenoids Produced by Combining Two Sesquiterpene Cyclases with Promiscuous Myxobacterial CYP260B1.

Sesquiterpenes represent a class of important terpenoids with high structural diversity and a wide range of applications. The cyclized core skeletons are generated by sesquiterpene cyclases, and the structural diversity is further increased by a series of modification steps. Cytochromes P450 (P450s) are a class of monooxygenases and one of the main contributors to the structural diversity of natural products. Some of these P450s show a broad substrate range and might be promising candidates for the implementation of cascade reactions. In this study, a combinatorial biosynthesis approach was utilized by the combination of a promiscuous myxobacterial P450 (CYP260B1) with two sesquiterpene cyclases (FgJ01056, FgJ09920) of filamentous fungi. Two oxygenated products, culmorin and culmorone, and a new compound, koraidiol, were successfully generated and characterized. This approach suggests the potential use of noncognate P450s to produce novel oxygenated terpenoids, or to generate a novel biosynthetic route for known terpenoids by a combinatorial biosynthesis strategy.

A Clade II-D Fungal Chimeric Diterpene Synthase from Colletotrichum gloeosporioides Produces Dolasta-1(15),8-diene.

Based on a terpenoid overproduction platform in yeast for genome mining, a chimeric diterpene synthase from the endophytic fungus Colletotrichum gloeosporioides ES026 was characterized as the (5R,12R,14S)-dolasta-1(15),8-diene synthase. The absolute configuration was independently verified through the use of enantioselectively deuterated terpene precursors, which unequivocally established the predicted C1-III-IV cyclization mode for this first characterized clade II-D enzyme. Extensive isotopic labeling experiments and isolation of the intermediate (1R)-δ-araneosene supported the proposed cyclization mechanism.

In Vivo Platforms for Terpenoid Overproduction and the Generation of Chemical Diversity.

Terpenoids represent a highly diverse group of natural products with wide applications. Engineering approaches have been used to increase titers of many value-added terpenoids, such as farnesene, taxadiene, lycopene, and astaxanthin. In this chapter, we review the in vitro reconstitution-based targeted engineering of terpenoids, as well as approaches for the mining of terpene cyclases and for increasing the chemical diversity. Information gained from in vitro reconstitution extends our understanding of the mechanisms underlying terpenoid biosynthesis, the contributions of enzymes and cofactors, and key enzymes and rate-limiting steps for the development of an ideal biosynthetic production system. The in vitro reconstitution-based targeted engineering strategy provides a rational and accurate engineering approach for terpenoid overproduction with high efficiency. Furthermore, an efficient terpenoid overproduction platform can accelerate the entire process for the mining of terpene cyclases and the discovery of novel terpenoids and can substantially increase the chemical diversity of these kinds of terpenoids.

Metabolic Engineering-Based Rapid Characterization of a Sesquiterpene Cyclase and the Skeletons of Fusariumdiene and Fusagramineol from Fusarium graminearum.

The potential power of sesquiterpene synthase FgJ03939 from Fusarium graminearum was fully exploited in a farnesyl diphosphate-overexpressing Saccharomyces cerevisiae chassis to produce the novel sesquiterpenes fusariumdiene (1), epi-fusagramineol (2), and fusagramineol (3) with 5/7 bicyclic and 5/6/3 tricyclic ring systems, respectively, as well as five known sesquiterpenes (4-8). The structure of the unusual skeletons was characterized, and an absolute configuration was proposed. A mechanism for the biosynthesis of 1-8 was also proposed.

Strategies for terpenoid overproduction and new terpenoid discovery.

Terpenoids comprise the largest family of natural products and have widespread applications. The overproduction of value-added terpenoids and the efficient mining of new terpenoids represent major challenges in the fields of metabolic engineering and natural product discovery. For terpenoid overproduction in microbial systems, in vitro reconstitution strategy guided rational engineering was emphasized, and -omics studies provide further information for targeted engineering. In addition, systematic engineering, including host and pathway engineering for terpenoid overproduction was reviewed. Furthermore, robust precursor supplies with optimized high-production hosts can also be used as an efficient platform to rapidly screen terpene cyclases and accelerate the process of mining new terpenoids.

Releasing the potential power of terpene synthases by a robust precursor supply platform.

Terpenoids represent the largest family of natural products. Their structural diversity is largely due to variable skeletons generated by terpene synthases. However, terpene skeletons found in nature are much more than those generated from known terpene synthases. Most promiscuous terpene synthases (i.e. those that can generate more than one product) have not been comprehensively characterised. Here, we first demonstrated that the promiscuous terpene synthases can produce more variable terpenoids in vivo by converting precursor polyisoprenoid diphosphates of different lengths (C10, C15, C20, C25). To release the synthetic potential of these enzymes, we integrated the engineered MVA pathway, combinatorial biosynthesis, and point mutagenesis to depict the comprehensive product profiles. In total, eight new terpenoids were characterised by NMR and three new skeletons were revealed. This work highlights the key role of metabolic engineering for natural product discovery.

Production of taxadiene by engineering of mevalonate pathway in Escherichia coli and endophytic fungus Alternaria alternata TPF6.

Taxol (paclitaxel) is a diterpenoid compound with significant and extensive applications in the treatment of cancer. The production of Taxol and relevant intermediates by engineered microbes is an attractive alternative to the semichemical synthesis of Taxol. In this study, based on a previously developed platform, the authors first established taxadiene production in mutant E. coli T2 and T4 by engineering of the mevalonate (MVA) pathway. The authors then developed an Agrobacterium tumefaciens-mediated transformation (ATMT) method and verified the strength of heterologous promoters in Alternaria alternata TPF6. The authors next transformed the taxadiene-producing platform into A. alternata TPF6, and the MVA pathway was engineered, with introduction of the plant taxadiene-forming gene. Notably, by co-overexpression of isopentenyl diphosphate isomerase (Idi), a truncated version of 3-hydroxy-3-methylglutaryl-CoA reductase (tHMG1), and taxadiene synthase (TS), the authors could detect 61.9 ± 6.3 µg/L taxadiene in the engineered strain GB127. This is the first demonstration of taxadiene production in filamentous fungi, and the approach presented in this study provides a new method for microbial production of Taxol. The well-established ATMT method and the known promoter strengths facilitated further engineering of taxaenes in this fungus.

Saccharopolyspora dendranthemae sp. nov. a halotolerant endophytic actinomycete isolated from a coastal salt marsh plant in Jiangsu, China.

A halotolerant actinomycete strain, designated strain KLBMP 1305(T), was isolated from a salt marsh plant Dendranthema indicum (Linn.) Des Moul collected from the coastal region of Nantong, Jiangsu Province, in east China and was studied in detail for its taxonomic position. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain KLBMP 1305(T) is a member of the genus Saccharopolyspora. The 16S rRNA gene sequence similarity indicated that strain KLBMP 1305(T) was most closely related to 'Saccharopolyspora pathumthaniensis' S582(T) (99.31 %), 'Saccharopolyspora endophytica' YIM 61095(T) (99.17 %) and Saccharopolyspora tripterygii YIM 65359(T) (99.15 %); similarity to other type strains of the genus Saccharopolyspora was <97.2 %. The organism had chemical and morphological features consistent with its classification in the genus Saccharopolyspora such as meso-diaminopimelic acid as the diagnostic diamino acid in the cell wall peptidoglycan and arabinose and galactose as the diagnostic sugars. The predominant menaquinone was MK-9(H4). The polar lipids detected were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylcholine, an unknown glycolipid and an unknown lipid. The major fatty acids were iso-C16:0, iso-C15:0, anteiso-C15:0, anteiso-C17:0 and sum in feature 8 (18:1ω7c/18:1ω6c). The G+C content of the genomic DNA of the type strain was 68.7 mol%. DNA-DNA relatedness data, together with phenotypic differences, clearly distinguished the isolate from its closest relatives. On the basis of these phenotypic and genotypic data, the isolate represents a novel species, for which the name Saccharopolyspora dendranthemae sp. nov. is proposed. The type strain is KLBMP 1305(T) (=KCTC 19889(T) = NBRC 108675(T)).