IMP: bridging the gap for medicinal plant genomics.

Medicinal plants have garnered significant attention in ethnomedicine and traditional medicine due to their potential antitumor, anti-inflammatory and antioxidant properties. Recent advancements in genome sequencing and synthetic biology have revitalized interest in natural products. Despite the availability of sequenced genomes and transcriptomes of these plants, the absence of publicly accessible gene annotations and tabular formatted gene expression data has hindered their effective utilization. To address this pressing issue, we have developed IMP (Integrated Medicinal Plantomics), a freely accessible platform at https://www.bic.ac.cn/IMP. IMP curated a total of 8 565 672 genes for 84 high-quality genome assemblies, and 2156 transcriptome sequencing samples encompassing various organs, tissues, developmental stages and stimulations. With the integrated 10 analysis modules, users could simply examine gene annotations, sequences, functions, distributions and expressions in IMP in a one-stop mode. We firmly believe that IMP will play a vital role in enhancing the understanding of molecular metabolic pathways in medicinal plants or plants with medicinal benefits, thereby driving advancements in synthetic biology, and facilitating the exploration of natural sources for valuable chemical constituents like drug discovery and drug production.

Adjusting Catalytic Activity of ß-Amyrin Synthase GgBAS by Utilizing the Plasticity Residues of an Active Site.

ß-Amyrin synthase (bAS) is a representative plant oxidosqualene cyclase (OSC), and previous studies have identified many functional residues and mutants that can alter its catalytic activity. However, the regulatory mechanism of the active site architecture for adjusting the catalytic activity remains unclear. In this study, we investigate the function of key residues and their regulatory effects on the catalytic activity of Glycyrrhiza glabra ß-amyrin synthase (GgbAS) through molecular dynamics simulations and site-directed mutagenesis experiments. We identified the plasticity residues located in two active site regions and explored the interactions between these residues and tetracyclic/pentacyclic intermediates. Based on computational and experimental results, we further categorize these plasticity residues into three types: effector, adjuster, and supporter residues, according to their functions in the catalytic process. This study provides valuable insights into the catalytic mechanism and active site plasticity of GgbAS, offering important references for the rational enzyme engineering of other OSC enzyme.

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.

Phylogenetic analysis and functional characterization of norcoclaurine synthase involved in benzylisoquinoline alkaloids biosynthesis in Stephania tetrandra.

Benzylisoquinoline alkaloids (BIAs) are a class of secondary metabolites that possess diverse pharmaceutical properties and are exclusively accumulated in specific plant genera. The Pictet-Spengler condensation, catalyzed by norcoclaurine synthase (NCS), represents a key enzymatic reaction in the biosynthetic pathway of BIAs. While NCS genes have been identified in several plant families such as Papaveraceae, Berberidaceae, and Ranunculaceae, no NCS genes have been reported in Menispermaceae, which is another genus known to accumulate BIAs. Here, NCSs were isolated and functionally characterized from the Menispermaceae family plant Stephania tetrandra. In vitro enzyme assay identified two functional StNCSs which could catalyze the formation of (S)-norcoclaurine. These functionally characterized genes were then integrated into engineered yeast to enable the production of norcoclaurine. Phylogenetic analysis of the NCS enzymes revealed that the StNCSs predominantly clustered into two clades. The functional StNCSs clustered with known NCSs, highlighting the presence of a specific NCS catalytic domain. This study not only provides additional genetic components for the synthetic biology-based production of BIAs in yeast but also contributes to the understanding of the phylogenetic relationships and structure-function relationship of NCS genes involved in the origin and production of BIAs.

Structural and mechanistic insights into the precise product synthesis by a bifunctional miltiradiene synthase.

Selaginella moellendorffii miltiradiene synthase (SmMDS) is a unique bifunctional diterpene synthase (diTPS) that catalyses the successive cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP) via (+)-copalyl diphosphate (CPP) to miltiradiene, which is a crucial precursor of important medicinal compounds, such as triptolide, ecabet sodium and carnosol. Miltiradiene synthetic processes have been studied in monofunctional diTPSs, while the precise mechanism by which active site amino acids determine product simplicity and the experimental evidence for reaction intermediates remain elusive. In addition, how bifunctional diTPSs work compared to monofunctional enzymes is attractive for detailed research. Here, by mutagenesis studies of SmMDS, we confirmed that pimar-15-en-8-yl+ is an intermediate in miltiradiene synthesis. Moreover, we determined the apo-state and the GGPP-bound state crystal structures of SmMDS. By structure analysis and mutagenesis experiments, possible contributions of key residues both in class I and II active sites were suggested. Based on the structural and functional analyses, we confirmed the copal-15-yl+ intermediate and unveiled more details of the catalysis process in the SmMDS class I active site. Moreover, the structural and experimental results suggest an internal channel for (+)-CPP produced in the class II active site moving towards the class I active site. Our research is a good example for intermediate identification of diTPSs and provides new insights into the product specificity determinants and intermediate transport, which should greatly facilitate the precise controlled synthesis of various diterpenes.

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.

Genome-Wide Characterization and Expression of the Hsf Gene Family in Salvia miltiorrhiza (Danshen) and the Potential Thermotolerance of SmHsf1 and SmHsf7 in Yeast.

Salvia miltiorrhiza Bunge (Danshen) is a traditional Chinese herb with significant medicinal value. The yield and quality of Danshen are greatly affected by climatic conditions, in particular high temperatures. Heat shock factors (Hsfs) play important regulatory roles in plant response to heat and other environmental stresses. However, little is currently known about the role played by the Hsf gene family in S. miltiorrhiza. Here, we identified 35 SmHsf genes and classified them into three major groups: SmHsfA (n = 22), SmHsfB (n = 11), and SmHsfC (n = 2) using phylogenetic analysis. The gene structure and protein motifs were relatively conserved within subgroups but diverged among the different groups. The expansion of the SmHsf gene family was mainly driven by whole-genome/segmental and dispersed gene duplications. The expression profile of SmHsfs in four distinct organs revealed its members (23/35) are predominantly expressed in the root. The expression of a large number of SmHsfs was regulated by drought, ultraviolet, heat and exogenous hormones. Notably, the SmHsf1 and SmHsf7 genes in SmHsfB2 were the most responsive to heat and are conserved between dicots and monocots. Finally, heterologous expression analysis showed that SmHsf1 and SmHsf7 enhance thermotolerance in yeast. Our results provide a solid foundation for further functional investigation of SmHsfs in Danshen plants as a response to abiotic stresses.

Multi-omics analysis reveals the evolutionary origin of diterpenoid alkaloid biosynthesis pathways in Aconitum.

Diterpenoid alkaloids (DAs) have been often utilized in clinical practice due to their analgesic and anti-inflammatory properties. Natural DAs are prevalent in the family Ranunculaceae, notably in the Aconitum genus. Nevertheless, the evolutionary origin of the biosynthesis pathway responsible for DA production remains unknown. In this study, we successfully assembled a high-quality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum (A. vilmorinianum) (5.76 Gb). An A. vilmorinianum-specific whole-genome duplication event was discovered using comparative genomic analysis, which may aid in the evolution of the DA biosynthesis pathway. We identified several genes involved in DA biosynthesis via integrated genomic, transcriptomic, and metabolomic analyses. These genes included enzymes encoding target ent-kaurene oxidases and aminotransferases, which facilitated the activation of diterpenes and insertion of nitrogen atoms into diterpene skeletons, thereby mediating the transformation of diterpenes into DAs. The divergence periods of these genes in A. vilmorinianum were further assessed, and it was shown that two major types of genes were involved in the establishment of the DA biosynthesis pathway. Our integrated analysis offers fresh insights into the evolutionary origin of DAs in A. vilmorinianum as well as suggestions for engineering the biosynthetic pathways to obtain desired DAs.

[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.

The ERF-VII transcription factor SmERF73 coordinately regulates tanshinone biosynthesis in response to stress elicitors in Salvia miltiorrhiza.

Here, we investigate the role of SmERF73, a group VII ETHYLENE RESPONSE FACTOR stress response transcription factor, in the regulation of post-modification of the skeleton precursors of diterpene tanshinones in Salvia miltiorrhiza. Most genes found to be involved in tanshinone biosynthesis are located on chromosome 6, and five of these genes comprise a large gene cluster in S. miltiorrhiza. We found that SmERF73 overexpression in S. miltiorrhiza coordinately up-regulated the transcription of seven tanshinone biosynthetic genes, four of which were located in the tanshinone gene cluster, consequently increasing tanshinone accumulation, while SmERF73 silencing reduced corresponding gene transcription and tanshinone accumulation. SmERF73 recognizes GCC-box promoter elements of four tanshinone-associated genes (DXR1, CPS1, KSL1 and CYP76AH3) and activates their expression. Moreover, SmERF73 and its targets were up-regulated by stress elicitors; SmERF73 appears to be at least partly mediated by the jasmonic acid (JA) signaling pathway via interaction with SmJAZ3. SmERF73 coordinately regulates tanshinone biosynthetic gene expression, suggesting a potential link between tanshinone production and plant stress responses.

Chemical-activity-based quality marker screening strategy for Viscum articulatum.

Viscum articulatum Burm. f. is a parasitic plant rich in flavonoids, triterpenoids, and catechins and has a high nutritional value. It has been reported that consuming V. articulatum can prevent cardiac diseases. In this study, six bioactive compounds, including catechins, triterpenoids, and phenylpropanoid glycosides, were determined in alcohol extracts of the plant using HPLC. The anti-inflammatory and antioxidant activities of three catechins, two triterpenoids, and three combination drugs were measured in cardiomyocytes, and the results showed that the anti-inflammatory activity was significantly enhanced while retaining strong antioxidant activity when epicatechin and ursolic acid were used in combination. The main quality markers epicatechin and ursolic acid were screened based on the specificity of the genuine herb and a potent synergistic effect, and the lowest limitation contents of V. articulatum which could discriminate it from some other taxonomically similar materials were accordingly determined. This self-built lowest limitation content of the two screened quality markers could quickly and accurately reflect the efficacy in terms of chemical composition and reverse the disorderly market use of nongenuine herbs or confusing species for adulteration. This study is of some significance for market regulation, drug development, and clinical medication.

[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.

CYP76B74 Catalyzes the 3''-Hydroxylation of Geranylhydroquinone in Shikonin Biosynthesis.

Shikonin and its derivatives are the most abundant naphthoquinone pigments formed in species of the medicinally and economically valuable Boraginaceae. A key step in the shikonin biosynthetic pathway, namely the C-3'' hydroxylation of the prenylated phenolic intermediate geranylhydroquinone, is expected to be catalyzed by a cytochrome P450. To identify cytochrome P450 candidates with transcription profiles similar to those of genes known to be involved in shikonin biosynthesis, we carried out coexpression analysis of transcriptome data sets of shikonin-proficient and shikonin-deficient cell lines and examined the spatial expression of candidate genes in different organs of Arnebia euchroma In biochemical assays using geranylhydroquinone as the substrate, CYP76B74 exhibited geranylhydroquinone 3''-hydroxylase activity and produced 3''-hydroxy-geranylhydroquinone. In CYP76B74 RNA interference A. euchroma hairy roots, shikonin derivative accumulation decreased dramatically, which demonstrated that CYP76B74 is required for shikonin biosynthesis in the plant. Phylogenetic analysis confirmed that CYP76B74 belonged to the CYP76B subfamily and was most likely derived from an ancestral geraniol 10-hydroxylase. In a subcellular localization analysis, a GFP-CYP76B74 fusion localized to endoplasmic reticulum membranes. Our results demonstrate that CYP76B74 catalyzes the key hydroxylation step in shikonin biosynthesis with high efficiency. The characterization of the CYP76B74 described here paves the way for further exploration of the ring closure reactions generating the naphthoquinone skeleton as well as for the alternative metabolism of geranylhydroquinone 3''-hydroxylase to dihydroechinofuran.

[Research progress of tissue culture of Aconitum].

Aconitum is a kind of important medicinal plant, which has been used in China for more than 2 000 years, with both a good medicinal and ornamental value. However, due to the lack of effective breeding methods and low seed and root propagation coefficients, the comprehensive development and utilization of Aconitum were greatly restricted. Tissue culture is an important basis for seed selection, germplasm conservation and genetic engineering. Therefore, this paper summarized the research on tissue culture of Aconitum, put forward the main problems and corresponding countermeasures, and provided important references for accelerating the seedling breeding of Aconitum and conducting the basic research of molecular biology.

An integrated strategy to identify genes responsible for sesquiterpene biosynthesis in turmeric.

KEY MESSAGE: Metabolic module, gene expression pattern and PLS modeling were integrated to precisely identify the terpene synthase responsible for sesquiterpene formation. Functional characterization confirmed the feasibility and sensitivity of this strategy. Plant secondary metabolite biosynthetic pathway elucidation is crucial for the production of these compounds with metabolic engineering. In this study, an integrated strategy was employed to predict the gene function of sesquiterpene synthase (STS) genes using turmeric as a model. Parallel analysis of gene expression patterns and metabolite modules narrowed the candidates into an STS group in which the STSs showed a similar expression pattern. The projections to latent structures by means of partial least squares model was further employed to establish a clear relationship between the candidate STS genes and metabolites and to predict three STSs (ClTPS16, ClTPS15 and ClTPS14) involved in the biosynthesis of several sesquiterpene skeletons. Functional characterization revealed that zingiberene and ß-sesquiphellandrene were the major products of ClTPS16, and ß-eudesmol was produced by ClTPS15, both of which indicated the accuracy of the prediction. Functional characterization of a control STS, ClTPS1, produced a small amount of ß-sesquiphellandrene, as predicted, which confirmed the sensitivity of metabolite module analysis. This integrated strategy provides a methodology for gene function predictions, which represents a substantial improvement in the elucidation of biosynthetic pathways in nonmodel plants.

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.