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.

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-infammatory 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 highquality, pseudochromosome-level genome of the DA-rich species Aconitum vilmorinianum(A.vilmorinianum)(5.76 Gb). An A. vilmorinianumspecific 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.

Functional identification of the terpene synthase family involved in diterpenoid alkaloids biosynthesis in Aconitum carmichaelii

Aconitum carmichaelii is a high-value medicinal herb widely used across China,Japan,and other Asian countries.Aconitine-type diterpene alkaloids(DAs)are the characteristic compounds in Aconitum.Although six transcriptomes,based on short-read next generation sequencing technology,have been reported from the Aconitum species,the terpene synthase(TPS)corresponding to DAs biosynthesis remains unidentified.We apply a combination of Pacbio isoform sequencing and RNA sequencing to provide a comprehensive view of the A.carmichaelii transcriptome.Nineteen TPSs and five alternative splicing isoforms belonging to TPS-b,TPS-c,and TPS-e/f subfamilies were identified.In vitro enzyme reaction analysis functional identified two sesqui-TPSs and twelve di TPSs.Seven of the TPS-c subfamily genes reacted with GGPP to produce the intermediate ent-copalyl diphosphate.Five Ac KSLs separately reacted with ent-CPP to produce ent-kaurene,ent-atiserene,and ent-13-epi-sandaracopimaradie:a new diterpene found in Aconitum.Ac TPSs gene expression in conjunction DAs content analysis in different tissues validated that ent-CPP is the sole precursor to all DAs biosynthesis,with Ac KSL1,Ac KSL2 s and Ac KSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis,respectively.These data clarified the molecular basis for the C20-DAs biosynthetic pathway in A.carmichaelii and pave the way for further exploration of C19-DAs biosynthesis in the Aconitum species.

aunalysis of the Genome Sequence of the Medicinal Plant Salvia miltiorrhiza

Dear Editor Salvia miltiorrhiza Bunge （Danshen） is a medicinal plant of the Lamiaceae family, and its dried roots have long been used in traditional Chinese medicine with hydrophilic phenolic acids and tanshinones as pharmaceutically active components （Zhang et al., 2014; Xu et al., 2016）. The first step of tanshinone biosynthesis is bicyclization of the general diterpene precursor （E,E,E）-geranylgeranyl diphosphate （GGPP） to copalyl diphosphate （CPP） by CPP synthases （CPSs）, which is followed by a cyclization or rearrangement reaction catalyzed by kaurene synthase-like enzymes （KSL）. The resulting intermediate is usually an olefin, which requires the insertion of oxygen by cytochrome P450 mono-oxygenases （CYPs） for the final production of diterpenoids （Zi et al., 2014）. While the CPS, KSL, and several early acting CYPs （CYP76AH1, CYP76AH3, and CYP76AK1） for tanshinone biosynthesis have been identified in S. miltiorrhiza （Gao et al., 2009; Guo et al., 2013, 2016; Zi and Peters, 2013）, the majority of the overall biosynthetic pathway, as well as the relevant regulatory factors associated with tanshinone production, remains elusive （Figure 1B）.

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±1.38μM 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 ERG20 F96W-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.