Identification of the cytochrome P450s responsible for the biosynthesis of two types of aporphine alkaloids and their de novo biosynthesis in yeast.

Aporphine alkaloids have diverse pharmacological activities; however, our understanding of their biosynthesis is relatively limited. Previous studies have classified aporphine alkaloids into two categories based on the configuration and number of substituents of the D-ring and have proposed preliminary biosynthetic pathways for each category. In this study, we identified two specific cytochrome P450 enzymes (CYP80G6 and CYP80Q5) with distinct activities toward (S)-configured and (R)-configured substrates from the herbaceous perennial vine Stephania tetrandra, shedding light on the biosynthetic mechanisms and stereochemical features of these two aporphine alkaloid categories. Additionally, we characterized two CYP719C enzymes (CYP719C3 and CYP719C4) that catalyzed the formation of the methylenedioxy bridge, an essential pharmacophoric group, on the A- and D-rings, respectively, of aporphine alkaloids. Leveraging the functional characterization of these crucial cytochrome P450 enzymes, we reconstructed the biosynthetic pathways for the two types of aporphine alkaloids in budding yeast (Saccharomyces cerevisiae) for the de novo production of compounds such as (R)-glaziovine, (S)-glaziovine, and magnoflorine. This study provides key insight into the biosynthesis of aporphine alkaloids and lays a foundation for producing these valuable compounds through synthetic biology.

Diterpene synthases from Leonurus japonicus elucidate epoxy-bridge formation of spiro-labdane diterpenoids.

Spiro-9,13-epoxy-labdane diterpenoids are commonly found in Leonurus species, particularly in Leonurus japonicus Houtt., which is a medicinal herb of long-standing use in Asia and in which such spiro-heterocycles are present in at least 38 diterpenoids. Here, through generation of a transcriptome and functional characterization of six diterpene synthases (diTPSs) from L. japonicus, including three class II diTPSs (LjTPS1, LjTPS3, and LjTPS4) and three class I diTPSs (LjTPS5, LjTPS6, and LjTPS7), formation of the spiro-9,13-epoxy-labdane backbone was elucidated, along with identification of the relevant diTPSs for production of other labdane-related diterpenes. Similar to what has been found with diTPSs from other plant species, while LjTPS3 specifically produces the carbon-9 (C9) hydroxylated bicycle peregrinol diphosphate (PPP), the subsequently acting LjTPS6 yields a mixture of four products, largely labda-13(16),14-dien-9-ol, but with substantial amounts of viteagnusin D and the C13-S/R epimers of 9,13-epoxy-labda-14-ene. Notably, structure-function analysis identified a critical residue in LjTPS6 (I420) in which single site mutations enable specific production of the 13S epimer. Indeed, extensive mutagenesis demonstrated that LjTPS6:I420G reacts with PPP to both specifically and efficiently produce 9,13S-epoxy-labda-14-ene, providing a specialized synthase for further investigation of derived diterpenoid biosynthesis. The results reported here provide a strong foundation for future studies of the intriguing spiro-9,13-epoxy-labdane diterpenoid metabolism found in L. japonicus.

Structure-function analysis of CYP719As involved in methylenedioxy bridge-formation in the biosynthesis of benzylisoquinoline alkaloids and its de novo production.

Benzylisoquinoline alkaloids (BIAs) are a type of secondary metabolite with clinical application value. (S)-stylopine is a special BIA which contains methylenedioxy bridge structures. CYP719As could catalyze the methylenedioxy bridge-formation on the A or D rings of protoberberine alkaloids, while displaying significant substrate regiospecificity. To explore the substrate preference of CYP719As, we cloned and identified five CyCYP719A candidates from Corydalis yanhusuo. Two CyCYP719As (CyCYP719A39 and CyCYP719A42) with high catalytic efficiency for the methylenedioxy bridge-formation on the D or A rings were characterized, respectively. The residues (Leu 294 for CyCYP719A42 and Asp 289 for CyCYP719A39) were identified as the key to controlling the regioselectivity of CYP719As affecting the methylenedioxy bridge-formation on the A or D rings by homology modeling and mutation analysis. Furthermore, for de novo production of BIAs, CyCYP719A39, CyCYP719A42, and their mutants were introduced into the (S)-scoulerine-producing yeast to produce 32 mg/L (S)-stylopine. These results lay a foundation for understanding the structure-function relationship of CYP719A-mediated methylenedioxy bridge-formation and provide yeast strains for the BIAs production by synthetic biology.

[Systematic identification of chemical forms of key terpene synthase in Cinnamomum camphora].

Cinnamomum camphora is an important economic tree species in China. According to the type and content of main components in the volatile oil of leaf, C. camphora were divided into five chemotypes, including borneol-type, camphor-type, linalool-type, cineole-type, and nerolidol-type. Terpene synthase(TPS) is the key enzyme for the formation of these compounds. Although several key enzyme genes have been identified, the biosynthetic pathway of(+)-borneol, which has the most economic value, has not been reported. In this study, nine terpenoid synthase genes CcTPS1-CcTPS9 were cloned through transcriptome analysis of four chemical-type leaves. After the recombinant protein was induced by Escherichia coli, geranyl pyrophosphate(GPP) and farnesyl pyrophosphate(FPP) were used as substrates for enzymatic reaction, respectively. Both CcTPS1 and CcTPS9 could catalyze GPP to produce bornyl pyrophosphate, which could be hydrolyzed by phosphohydrolase to obtain(+)-borneol, and the product of(+)-borneol accounted for 0.4% and 89.3%, respectively. Both CcTPS3 and CcTPS6 could catalyze GPP to generate a single product linalool, and CcTPS6 could also react with FPP to generate nerolidol. CcTPS8 reacted with GPP to produce 1,8-cineol(30.71%). Nine terpene synthases produced 9 monoterpene and 6 sesquiterpenes. The study has identified the key enzyme genes responsible for borneol biosynthesis in C. camphora for the first time, laying a foundation for further elucidating the molecular mechanism of chemical type formation and cultivating new varieties of borneol with high yield by using bioengineering technology.

Integrating Metabolomics and Transcriptomics to Unveil Atisine Biosynthesis in Aconitum gymnandrum Maxim.

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum, which are classified into four skeletal types: C18, C19, C20, and bisditerpenoid alkaloids. C20-DAs are thought to be the precursor of the other types. Their biosynthetic pathway, however, is largely unclear. Herein, we combine metabolomics and transcriptomics to unveil the methyl jasmonate (MJ) inducible biosynthesis of DAs in the sterile seedling of A. gymnandrum, the only species in the Subgenus Gymnaconitum (Stapf) Rapaics. Target metabolomics based on root and aerial portions identified 51 C19-DAs and 15 C20-DAs, with 40 inducible compounds. The highest content of C20-DA atisine was selected for further network analysis. PacBio Isoform sequencing integrated with RNA sequencing not only provided the full-length transcriptome but also their response to induction, revealing 1994 genes that exhibited up-regulated expression. Further, 38 genes involved in terpenoid biosynthesis were identified, including 7 diterpene synthases. In addition to the expected function of the four diterpene synthases, AgCPS5 was identified to be a new ent-8,13-CPP synthase in Aconitum and could also combine with AgKSL1 to form the C20-DAs precursor ent-atiserene. Combined with multiple network analyses, six CYP450 and seven 2-ODD genes predicted to be involved in the biosynthesis of atisine were also identified. This study not only sheds light on diterpene synthase evolution in Aconitum but also provides a rich dataset of full-length transcriptomes, systemic metabolomes, and gene expression profiles, setting the groundwork for further investigation of the C20-DAs biosynthesis pathway.

Recent progress and new perspectives for diterpenoid biosynthesis in medicinal plants.

Diterpenoids, including more than 18,000 compounds, represent an important class of metabolites that encompass both phytohormones and some industrially relevant compounds. These molecules with complex, diverse structures and physiological activities, have high value in the pharmaceutical industry. Most medicinal diterpenoids are extracted from plants. Major advances in understanding the biosynthetic pathways of these active compounds are providing unprecedented opportunities for the industrial production of diterpenoids by metabolic engineering and synthetic biology. Here, we summarize recent developments in the field of diterpenoid biosynthesis from medicinal herbs. An overview of the pathways and known biosynthetic enzymes is presented. In particular, we look at the main findings from the past decade and review recent progress in the biosynthesis of different groups of ringed compounds. We also discuss diterpenoid production using synthetic biology and metabolic engineering strategies, and draw on new technologies and discoveries to bring together many components into a useful framework for diterpenoid production.

[Research progress of natural borneol resources].

Natural borneol is an important traditional Chinese medicine herb with resuscitation-inducing, antipyretic and analgesic effects, and has been widely used in the fields of medicine, perfume and chemical industry. At present, natural borneol is short supply, with promising market development prospects. This paper summarized the distribution of borneol plant resources, cultivation status and molecular biological research progress, in the expectation of providing basis and ideas for the research and application of natural borneol.

[Study on tanshinones regulating root-associated microbiomes of Salvia miltiorrhiza].

The plant root-associated microbiomes include root microbiome and rhizosphere microbiome, which are closely related to plant life activities. Nearly 30% of photosynthesis products of plants are used to synthesize root compounds, there is evidence that root compounds regulate and significantly affect the root microbiome Tanshinones are the main hydrophobic components in Salvia miltiorrhiza. In order to study whether these compounds can regulate the root-associated microbiomes of S. miltiorrhiza, our study first identified a white root S. miltiorrhiza(BG) which contains little tanshinones. Retain of the fifth intron of tanshinones synthesis key enzyme gene SmCPS1 leading to the early termination of the SmCPS1 gene, and a stable white root phenotype. Further, wild type(WT) and BG were planted in greenhouse with nutrient soil(Pindstrup, Denmark) and Shandong soil(collected from the S. miltiorrhiza base in Weifang, Shandong), then high-throughput sequencing was used to analyze the root-associated microbiomes. The results showed that the tanshinones significantly affected the root-associated microbiomes of S. miltiorrhiza, and the impact on root microbiomes was more significant. There are significant differences between WT and BG root microbiomes in species richness, dominant strains and co-occurrence network. Tanshinones have a certain repelling effect on Bacilli which belongs to Gram-positive, while specifically attract some Gram-negative bacteria such as Betaproteobacteria and some specific genus of Alphaproteobacteria. This study determined the important role of tanshinones in regulating the structure of root-associated microbiomes from multiple angles, and shed a light for further improving the quality and yield of S. miltiorrhiza through microenvironment regulation.

An alternative splicing alters the product outcome of a class I terpene synthase in Isodon rubescens.

The labdane-related diterpenoids are an important superfamily of natural products. Their structural diversity mainly depends on diterpene synthases, which generate the hydrocarbon skeletal structures. Isodon rubescens contains an expanded family of class I terpene synthases with different functions. Here we report a novel class I terpene synthase cDNA (IrKSL3a) with loss of 18 nucleotides compared with the reported cDNA sequence (IrKSL3). Inspection of IrKSL3 genomic sequence indicated that IrKSL3a and IrKSL3 transcripts may be generated by an alternative splicing event that utilizes different 3' splice site. In vitro assays showed that IrKSL3a produced isopimaradiene and miltiradiene, while IrKSL3 only produced miltiradiene. Protein sequence alignment found the six residues encoded by the alternative exon was unique to IrKSL3, which are 17 residues away from the conserved DDXXD motif. A deletion mutant of IrKSL3 showed that maintaining two residues within the six-amino acid is sufficient for miltiradiene production, while the other mutants lost nearly all enzymatic function. Our results illustrated how product outcomes can be changed by alternative splicing, and further gave an interesting example for studying the loop conformation in tuning product outcome in class I terpene synthase.

Functional Diversification of Kaurene Synthase-Like Genes in Isodon rubescens.

Ent-kaurene diterpenoids are the largest group of known Isodon diterpenoids. Among them, oridonin is accumulated in the leaves, and is the most frequently studied compound because of its antitumor and antibacterial activities. We have identified five copalyl diphosphate synthase (CPS) and six kaurene synthase-like (KSL) genes by transcriptome profiling of Isodon rubescens leaves. An in vitro assay assigns ten of them to five different diterpene biosynthesis pathways, except IrCPS3 that has a mutation in the catalytic motif. The Lamiaceae-specific clade genes (IrCPS1 and IrCPS2) synthesize the intermediate copalyl diphosphate (normal-CPP), while IrCPS4 and IrCPS5 synthesize the intermediate ent-copalyl diphosphate (ent-CPP). IrKSL2, IrKSL4, and IrKSL5 react with ent-CPP to produce an ent-isopimaradiene-like compound, ent-atiserene and ent-kaurene, respectively. Correspondingly, the Lamiaceae-specific clade genes IrKSL1 or IrKSL3 combined with normal-CPP led to the formation of miltiradiene. The compound then underwent aromatization and oxidization with a cytochrome P450 forming two related compounds, abietatriene and ferruginol, which were detected in the root bark. IrKSL6 reacts with normal-CPP to produce isopimaradiene. IrKSL3 and IrKSL6 have the γßα tridomain structure, as these proteins tend to possess the bidomain structure of IrKSL1, highlighting the evolutionary history of KSL gene domain loss and further elucidating chemical diversity evolution from a macroevolutionary stance in Lamiaceae.

Transcriptomic Insight into Terpenoid Biosynthesis and Functional Characterization of Three Diterpene Synthases in Scutellaria barbata.

Scutellaria barbata (Lamiaceae) is an important medicinal herb widely used in China, Korea, India, and other Asian countries. Neo-clerodane diterpenoids are the largest known group of Scutellaria diterpenoids and show promising cytotoxic activity against several cancer cell lines. Here, Illumina-based deep transcriptome analysis of flowers, the aerial parts (leaf and stem), and roots of S. barbata was used to explore terpenoid-related genes. In total, 121,958,564 clean RNA-sequence reads were assembled into 88,980 transcripts, with an average length of 1370 nt and N50 length of 2144 nt, indicating high assembly quality. We identified nearly all known terpenoid-related genes (33 genes) involved in biosynthesis of the terpenoid backbone and 14 terpene synthase genes which generate skeletons for different terpenoids. Three full length diterpene synthase genes were functionally identified using an in vitro assay. SbTPS8 and SbTPS9 were identified as normal-CPP and ent-CPP synthase, respectively. SbTPS12 reacts with SbTPS8 to produce miltiradiene. Furthermore, SbTPS12 was proven to be a less promiscuous class I diterpene synthase. These results give a comprehensive understanding of the terpenoid biosynthesis in S. barbata and provide useful information for enhancing the production of bioactive neo-clerodane diterpenoids through genetic engineering.

Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis.

Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza.

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such as vasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase (CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, which has led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerial tissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 and S. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormone biosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome of the CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues, may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functional diterpenoids in plants.

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

OBJECTIVES: To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. RESULTS: Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. CONCLUSIONS: This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza.

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

Functional diversity of diterpene synthases in Aconitum plants.

Members of the Aconitum genus within the Ranunculaceae family are known to accumulate a broad array of medicinal and toxic diterpenoid alkaloids (DAs). Historically, ent-copalyl diphosphate (ent-CPP) was considered the sole precursor in DAs biosynthesis. However, the recent discovery of ent-8,13-CPP synthase in A. gymnandrum Maxim., which participates in ent-atiserene biosynthesis, raises the question of whether this gene is conserved throughout the Aconitum genus. In this study, RNA sequencing and PacBio Iso-sequencing were employed to identify diterpene synthases (diTPSs) in four additional Aconitum species with distinct DA compositions. In vitro and in vivo analyses functionally characterized a diverse array of 10 class II and 9 class I diTPSs. In addition to the identification of seven class II diTPSs as ent-CPP synthases, three other synthases generating ent-8,13-CPP, 8,13-CPP, and 8α-hydroxy-CPP were also discovered. Four class I kaurene synthases-like (KSLs) were observed to react with ent-CPP to yield ent-kaurene. Three KSLs not only reacted with ent-CPP but also ent-8,13-CPP to produce ent-atiserene. AsiKSL2-1 was found to react with 8α-hydroxy-CPP to produce Z-abienol and AsiKSL2-2 exhibited no activity with any of the four intermediates. This research delineates the known diterpene biosynthesis pathways in six Aconitum species and explores the highly divergent diterpene synthases within the genus, which are consistent with their phylogeny and may be responsible for the differential distribution of diterpenoid alkaloids in root and aerial parts. These findings contribute valuable insights into the diversification of diterpene biosynthesis and establish a solid foundation for future investigation into DA biosynthetic pathways in Aconitum.

Functional Characterization of a 2OGD Involved in Abietane-Type Diterpenoids Biosynthetic Pathway in Salvia miltiorrhiza.

Salvia miltiorrhiza is one of the most commonly used Chinese medicinal herbs. Tanshinones, the most abundant lipid-soluble bioactive constituents of S. miltiorrhiza, are a class of structural highly oxidized abietane-type diterpenoids with multiple pharmacological activities. Although several enzymes, including diterpene synthase, cytochrome P450, and Fe(II)/2-oxoglutarate-dependent dioxygenase (2OGD), have been functionally characterized in biosynthesis of abietane-type diterpenoids, the highly oxidized structure and complex secondary metabolic network of tanshinones imply that more oxidases should be characterized. Here, we identified a new 2OGD (Sm2OGD25) from S. miltiorrhiza. Molecular cloning and functional studies in vitro showed that Sm2OGD25 could catalyze the hydroxylation of sugiol at C-15 and C-16 positions to produce hypargenin B and crossogumerin C, respectively. The phylogenetic analysis of the DOXC family demonstrated that Sm2OGD25 belongs to the DOXC54 clade. Furthermore, structural modeling and site-directed mutagenesis characterization revealed the importance of the hydrogen-bonding residue Y339 and the hydrophobic residues (V122, F129, A144, A208, F303, and L344) in substrate binding and enzyme activity. This study will promote further studies on the catalytic characterization of plant 2OGDs and the secondary metabolic biosynthesis network of diterpenoids.

Functional Characterization of UDP-Glycosyltransferases Involved in Anti-viral Lignan Glycosides Biosynthesis in Isatis indigotica.

Isatis indigotica is a popular herbal medicine with its noticeable antiviral properties, which are primarily due to its lignan glycosides such as lariciresinol-4-O-ß-D-glucoside and lariciresinol-4,4'-bis-O-ß-D-glucosides (also called clemastanin B). UDP-glucose-dependent glycosyltransferases are the key enzymes involved in the biosynthesis of these antiviral metabolites. In this study, we systematically characterized the UGT72 family gene IiUGT1 and two UGT71B family genes, IiUGT4 and IiUGT71B5a, with similar enzymatic functions. Kinetic analysis showed that IiUGT4 was more efficient than IiUGT1 or IiUGT71B5a for the glycosylation of lariciresinol. Further knock-down and overexpression of these IiUGTs in I. indigotica's hairy roots indicates that they play different roles in planta: IiUGT71B5a primarily participates in the biosynthesis of coniferin not pinoresinol diglucoside, and IiUGT1 primarily participates in the biosynthesis of pinoresinol diglucoside, while IiUGT4 is responsible for the glycosylation of lariciresinol and plays a dominant role in the biosynthesis of lariciresinol glycosides in I. indigotica. Analysis of the molecular docking and site-mutagenesis of IiUGT4 have found that key residues for its catalytic activity are H373, W376, E397, and that F151 could be associated with substrate preference. This study elucidates the biosynthetic route of anti-viral lignan glycosides in I. indigotica, and provides the foundation for the production of anti-viral lignan glycosides via synthetic biology under the heterologous model.

Identification of (-)-bornyl diphosphate synthase from Blumea balsamifera and its application for (-)-borneol biosynthesis in Saccharomyces cerevisiae.

Borneol is a precious monoterpenoid with two chiral structures, (-)-borneol and (+)-borneol. Bornyl diphosphate synthase is the key enzyme in the borneol biosynthesis pathway. Many (+)-bornyl diphosphate synthases have been reported, but no (-)-bornyl diphosphate synthases have been identified. Blumea balsamifera leaves are rich in borneol, almost all of which is (-)-borneol. In this study, we identified a high-efficiency (-)-bornyl diphosphate synthase (BbTPS3) from B. balsamifera that converts geranyl diphosphate (GPP) to (-)-bornyl diphosphate, which is then converted to (-)-borneol after dephosphorylation in vitro. BbTPS3 exhibited a K m value of 4.93 ± 1.38 µM for GPP, and the corresponding k cat value was 1.49 s-1. Multiple strategies were applied to obtain a high-yielding (-)-borneol producing yeast strain. A codon-optimized BbTPS3 protein was introduced into the GPP high-yield strain MD, and the resulting MD-B1 strain produced 1.24 mg·L-1 (-)-borneol. After truncating the N-terminus of BbTPS3 and adding a Kozak sequence, the (-)-borneol yield was further improved by 4-fold to 4.87 mg·L-1. Moreover, the (-)-borneol yield was improved by expressing the fusion protein module of ERG20F96W-N127W-YRSQI-t14-BbTPS3K2, resulting in a final yield of 12.68 mg·L-1 in shake flasks and 148.59 mg·L-1 in a 5-L bioreactor. This work is the first reported attempt to produce (-)-borneol by microbial fermentation.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.