Discovery of a novel flavonol O-methyltransferase possessing sequential 4'- and 7-O-methyltransferase activity from Camptotheca acuminata Decne.

The biosynthetic route for flavonol in Camptotheca acuminata has been recently elucidated from a chemical point of view. However, the genes involved in flavonol methylation remain unclear. It is a critical step for fully uncovering the flavonol metabolism in this ancient plant. In this study, the multi-omics resource of this plant was utilized to perform flavonol O-methyltransferase-oriented mining and screening. Two genes, CaFOMT1 and CaFOMT2 are identified, and their recombinant CaFOMT proteins are purified to homogeneity. CaFOMT1 exhibits strict substrate and catalytic position specificity for quercetin, and selectively methylates only the 4'-OH group. CaFOMT2 possesses sequential O-methyltransferase activity for the 4'-OH and 7-OH of quercetin. These CaFOMT genes are enriched in the leaf and root tissues. The catalytic dyad and critical substrate-binding sites of the CaFOMTs are determined by molecular docking and further verified through site-mutation experiments. PHE181 and MET185 are designated as the critical sites for flavonol substrate selectivity. Genomic environment analysis indicates that CaFOMTs evolved independently and that their ancestral genes are different from that of the known Ca10OMT. This study provides molecular insights into the substrate-binding pockets of two new CaFOMTs responsible for flavonol metabolism in C. acuminata.

CaERF1- mediated ABA signal positively regulates camptothecin biosynthesis by activating the iridoid pathway in Camptotheca acuminata.

Camptotheca acuminata is one of the primary sources of camptothecin (CPT), which is widely used in the treatment of human malignancies because of its inhibitory activity against DNA topoisomerase I. Although several transcription factors have been identified for regulating CPT biosynthesis in other species, such as Ophiorrhiza pumila, the specific regulatory components controlling CPT biosynthesis in C. acuminata have yet to be definitively determined. In this study, CaERF1, an DREB subfamily of the APETALA2/ethylene response factors (AP2ERFs), was identified in C. acuminata. The transient overexpression and silencing of CaERF1 in C. acuminata leaves confirmed that it positively regulates the accumulation of CPT by inducing the expression of CaCYC1 and CaG8O in the iridoid pathway. Results of transient transcriptional activity assay and yeast one-hybrid assays have showed that CaERF1 transcriptionally activates the expression of CaCYC1 and CaG8O by binding to RAA and CEI elements in the promoter regions of these two genes. Furthermore, the expression of CaCYC1 and CaG8O in CaERF1-silenced leaves was less sensitive to ABA treatment, indicating that CaERF1 is a crucial component involved in ABA-regulated CPT biosynthesis in C. acuminata.

Integrated transcriptome and proteome analysis provides new insights into camptothecin biosynthesis and regulation in Camptotheca acuminata.

Camptotheca acuminata Decne., the main source of camptothecin (CPT), has received increasing attention for its remarkable antitumor activity. Many CPT derivatives are clinically used as effective anticancer agents worldwide. However, their biosynthesis mechanism remains unclear, and uncovering this pathway would greatly facilitate development of alternative CPT production methods to replace current inefficient plant-derived ones. The expression of >30,000 genes was accurately quantified using unique molecular identifier RNA sequencing in 10 C. acuminata tissues, and 7854 proteins from five tissues were quantified with label-free quantitative proteomics. Fifteen full-length transcriptomes were sequenced with long-read Oxford Nanopore Technologies, and 5692 alternative splicing events were discovered among 4746 genes. Integrated transcriptome and proteome analysis provided novel insights into CPT biosynthesis and its hierarchical regulation. Five cytochrome P450s and three O-methyltransferases were considered as candidates involved in the biosynthesis of CPT and its derivatives, while 15 transcription factors potentially regulating CPT biosynthesis were screened. These findings provide important clues for elucidating the biosynthetic mechanisms of CPT and its derivatives and substantially contribute to the future production of these anticancer agents with synthetic biology. The generated large-scale multiomics data also provide valuable resources for investigating the functional genomics of the most important CPT-producing plant species-C. acuminata.

An ABCG-Type Transporter Facilitates ABA Influx and Regulates Camptothecin Biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) and its derivatives from Camptotheca acuminata have antitumor effects as a DNA topoisomerase I inhibitor. Previous studies have shown that application of exogenous abscisic acid (ABA) significantly promoted the accumulation level of CPT and induced the expression of CPT biosynthetic genes, which revealed that ABA signaling is effectively involved in regulating CPT biosynthesis in C. acuminata. In this study, an ABA transporter, CaABAT, which encodes a plasma membrane protein belonging to the ABCG subfamily, was identified in C. acuminata, and its ABA import activity was confirmed by transport assay in yeast cells. Real-time PCR analysis showed that CaABAT was predominately expressed in C. acuminata leaves and its expression could be significantly upregulated by exogenous ABA treatment. Silencing of CaABAT down-regulated the expression of ABA response genes, which indicated that translocation of ABA by CaABAT should initiate changes in plant physiological status in response to ABA signaling, thus leading to decreased expression of CPT biosynthesis pathway genes and low accumulation levels of CPT in C. acuminata.

Functional characterization of secologanin synthase-like homologous genes suggests their involvement in the biosynthesis of diverse metabolites in the secoiridoid biosynthetic pathway of Camptotheca acuminata Decne.

The assignment of functions based on homology has recently been challenged by the frequent discovery of functional divergence among homologous gene family members of enzymes involved in plant secondary metabolism. Secologanin synthase (SLS) is the key CYP450 enzyme that acts critically in the biosynthesis of Strychnos alkaloid scaffold. In this study, to fully elucidate the mechanism that underlies metabolic variation, the CYP450 paralogs that participate in oxidative transformation of the secoiridoid pathway were functionally characterized by combining multitiered strategies of metabolite profiling, phylogenetic analyses, biochemistry assays and reverse genetics techniques. Five CaSLSs-like homologous genes were mined and isolated from an integrative multi-omics database of Camptotheca acuminata. Protein sequences, structural comparisons, and phylogenetic analyses confirmed that CaSLS1-2 and CaSLS4-5 were grouped into the SLS clade, and only CaSLS3 clustered into the 7DLH clade. Five homologs, including two previously identified enzymatic genes, were thus designated as CaSLAS1, CaSLAS2, Ca7DLH, CaSLS4 and CaSLS5. Enzymatic assays of the recombinant proteins in yeast showed that CaSLAS1 and CaSLAS2 displayed multi-catalytic activities of SLS, secologanic acid synthase (SLAS) and secoxyloganin synthase (SXS). Additionally, the reactions of CaSLASs enzymes generated stereospecific isomers of secoiridoid products, and a new product of secoxyloganin was observed. CaSLS5, a third SLS enzyme isoform that catalyzes the formation of secologanin, was reported for the first time. However, CaSXS enzymatic activities in vitro had little physiological impact on the biosynthesis of camptothecin (CPT) in Camptotheca acuminata. The primary and secondary roles of CaSLSs-like genes in secoiridoid metabolism were confirmed by virus-induced gene silencing (VIGS) in plant leaves. Efficient silencing and transcriptional downregulation of CaSLAS2, compared with the CaSLAS1 homologs, resulted in a greater reduction of the accumulation of CPT within silenced plants, and CaSLS5 had barely any effect on the contents of metabolites in planta. Thus, CaSLAS2, rather than CaSLAS1, appeared to function as a major participant in the biosynthesis of CPT, and there were redundant functions in the CaSLSs-like enzymes. Consistent with such roles, CaSLAS2 was ubiquitously expressed at very high levels in Camptotheca tissues, and CaSLAS2 was specifically expressed in young leaves. In contrast, CaSLS5 was poorly expressed in every tissue tested. Our findings demonstrate that homologs that belong to the CYP72 gene family are functionally diverse and exhibit divergence and thereby uncover an expanding group of enzymatic genes that determine the chemo-diversity of the iridoid pathway.

Single mutations toggle the substrate selectivity of multifunctional Camptotheca secologanic acid synthases.

Terpene indole alkaloids (TIAs) are plant-derived specialized metabolites with widespread use in medicine. Species-specific pathways derive various TIAs from common intermediates, strictosidine or strictosidinic acid, produced by coupling tryptamine with secologanin or secologanic acid. The penultimate reaction in this pathway is catalyzed by either secologanin synthase (SLS) or secologanic acid synthase (SLAS) according to whether plants produce secologanin from loganin or secologanic acid from loganic acid. Previous work has identified SLSs and SLASs from different species, but the determinants of selectivity remain unclear. Here, combining molecular modeling, ancestral sequence reconstruction, and biochemical methodologies, we identified key residues that toggle SLS and SLAS selectivity in two CYP72A (cytochrome P450) subfamily enzymes from Camptotheca acuminata. We found that the positions of foremost importance are in substrate recognition sequence 1 (SRS1), where mutations to either of two adjacent histidine residues switched selectivity; His131Phe selects for and increases secologanin production whereas His132Asp selects for secologanic acid production. Furthermore, a change in SRS3 in the predicted substrate entry channel (Arg/Lys270Thr) and another in SRS4 at the start of the I-helix (Ser324Glu) decreased enzyme activity toward either substrate. We propose that the Camptotheca SLASs have maintained the broadened activities found in a common asterid ancestor, even as the Camptotheca lineage lost its ability to produce loganin while the campanulid and lamiid lineages specialized to produce secologanin by acquiring mutations in SRS1. The identification here of the residues essential for the broad substrate scope of SLASs presents opportunities for more tailored heterologous production of TIAs.

A chromosome-level Camptotheca acuminata genome assembly provides insights into the evolutionary origin of camptothecin biosynthesis.

Camptothecin and its derivatives are widely used for treating malignant tumors. Previous studies revealed only a limited number of candidate genes for camptothecin biosynthesis in Camptotheca acuminata, and it is still poorly understood how its biosynthesis of camptothecin has evolved. Here, we report a high-quality, chromosome-level C. acuminata genome assembly. We find that C. acuminata experiences an independent whole-genome duplication and numerous genes derive from it are related to camptothecin biosynthesis. Comparing with Catharanthus roseus, the loganic acid O-methyltransferase (LAMT) in C. acuminata fails to convert loganic acid into loganin. Instead, two secologanic acid synthases (SLASs) convert loganic acid to secologanic acid. The functional divergence of the LAMT gene and positive evolution of two SLAS genes, therefore, both contribute greatly to the camptothecin biosynthesis in C. acuminata. Our results emphasize the importance of high-quality genome assembly in identifying genetic changes in the evolutionary origin of a secondary metabolite.

Genome-wide identification and analysis of AP2/ERF transcription factors related to camptothecin biosynthesis in Camptotheca acuminata.

Camptotheca acuminata produces camptothecin (CPT), a monoterpene indole alkaloid (MIA) that is widely used in the treatment of lung, colorectal, cervical, and ovarian cancers. Its biosynthesis pathway has attracted significant attention, but the regulation of CPT biosynthesis by the APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors (TFs) remains unclear. In this study, a systematic analysis of the AP2/ERF TFs family in C. acuminata was performed, including phylogeny, gene structure, conserved motifs, and gene expression profiles in different tissues and organs (immature bark, cotyledons, young flower, immature fruit, mature fruit, mature leaf, roots, upper stem, and lower stem) of C. acuminata. A total of 198 AP2/ERF genes were identified and divided into five relatively conserved subfamilies, including AP2 (26 genes), DREB (61 genes), ERF (92 genes), RAV (18 genes), and Soloist (one gene). The combination of gene expression patterns in different C. acuminata tissues and organs, the phylogenetic tree, the co-expression analysis with biosynthetic genes, and the analysis of promoter sequences of key enzymes genes involved in CPT biosynthesis pathways revealed that eight AP2/ERF TFs in C. acuminata might be involved in CPT synthesis regulation, which exhibit relatively high expression levels in the upper stem or immature bark. Among these, four genes (CacAP2/ERF123, CacAP2/ERF125, CacAP2/ERF126, and CacAP2/ERF127) belong to the ERF-B2 subgroup; two genes (CacAP2/ERF149 and CacAP2/ERF152) belong to the ERF-B3 subgroup; and two more genes (CacAP2/ERF095 and CacAP2/ERF096) belong to the DREB-A6 subgroup. These results provide a foundation for future functional characterization of the AP2/ERF genes to enhance the biosynthesis of CPT compounds of C. acuminata.

Characterization of Camptotheca acuminata 10-hydroxygeraniol oxidoreductase and iridoid synthase and their application in biological preparation of nepetalactol in Escherichia coli featuring NADP+ - NADPH cofactors recycling.

Nepetalactol, an iridoid with four chiral carbons, is a crucial component of aphid sex pheromones that have been employed with great success to control the insect-related diseases. Despite of agricultural usage as end products, iridoids are fundamental biosynthetic intermediates for pharmaceutically important monoterpenoid indole alkaloids such as camptothecin (CAM) and vinca alkaloids. Herein we characterized 10-hydroxygeraniol oxidoreductase (10HGO) and iridoid synthase (IS) from Camptotheca acuminata, a CAM-producing plant, and reported their application in biological preparation of nepetalactol. Ca10HGO and CaIS were respectively cloned from C. acuminata, overexpressed in Escherichia coli, and purified to homogeneity. Ca10HGO catalyzes the oxidation of 10-hydroxygeraniol into 10-oxogeranial, in which NADP+ was reduced to NADPH. CaIS catalyzes nepetalactol formation from 10-oxogeranial using NADPH cofactor. The net outcome of the two reactions generate nepetalactol from 10-hydroxygeraniol efficiently, indicating NADP+ - NADPH recycling. Ca10HGO and CaIS were co-overexpressed in E. coli under optimized fermentation conditions to prepare cell-based catalysts that catalyze the conversion of 10-hydroxygeraniol into nepetalactol. The present work shows the enzymatic conversion of 10-hydroxygeraniol into nepetalactol involved in CAM biosynthesis. Co-overexpression of Ca10HGO and CaIS in E. coli is an alternative valuable cell-based biotransformation process with regenerating recycling of NADP+ - NADPH cofactors for nepetalactol preparation.

Two classes of cytochrome P450 reductase genes and their divergent functions in Camptotheca acuminata Decne.

Cytochrome P450 monooxygenases (CYP450s) and their auxiliary cytochrome P450 reductases (CPRs) are important and commonly involved in the biosynthesis of camptothecin (CPT). To better understand the possible functions of CPRs in planta, we first isolated two CaCPRs genes from Camptotheca acuminata. Sequence analysis revealed the presence of common conserved FMN-, FAD- and NADPH-binding motifs in putative CaCPR1/CaCPR2 proteins. The two CaCPR paralogs were assigned to the Class I and Class II of CPRs, respectively, according to phylogenetic tree. The recombinant CrCYP72-CaCPR1 and CrCYP72-CaCPR2 enzymes and their substrate bioconversion rates of 23.09% and 35.23% demonstrated that both CaCPRs could support the enzyme activities of CrSLS1. Gene silencing of CaCPRs by VIGS led to downregulation of CaCPR1/CaCPR2 expression by 50-67%, accompanied with 10-15% slight decrease and 57-63% dramatic reduction for CaCPR1 and CaCPR2 individually in CPT accumulations. Moreover, CaCPR1/CaCPR2 displayed almost omnipresent expression patterns across Camptotheca tissues. While in comparison to constitutive expression of CaCPR1 gene, CaCPR2 and CYP450 genes were all dramatically phytohormone-induced expressed in leaves which were main tissues for isoprenoid and CPT biosynthesis. Our results suggested that, in Camptotheca seedlings, CaCPR2 had a distinct function from CaCPR1 that was clearly involved in the inducible specialized metabolism for CPT biosynthesis.

A bZIP transcription factor, CaLMF, mediated light-regulated camptothecin biosynthesis in Camptotheca acuminata.

Camptothecin (CPT) has powerful biological activities and its analogs, irinothecan and topothecan, are effective anti-cancer drugs for clinical therapy. Camptothecin was first isolated from Camptotheca acuminata and its low accumulation in planta limits drug supply in the market. Previous works have confirmed that many environmental factors and plant hormones/elicitors could regulate CPT biosynthesis, but only light irradiance has a negative effect on CPT production in C. acuminata. Although light irradiance has been identified as a negative CPT biosynthesis regulator in C. acuminata for many years, the mechanisms of this regulation are still unknown. In order to search possible signal components involved in the process of light-regulated CPT biosynthesis, coexpression analysis was carried out according to the transcriptome database of Camptotheca above-ground green tissues. From coexpression analysis, a light-responsive bZIP transcription factor, CaLMF (Light-Mediated CPT biosynthesis Factor), was identified and further investigations showed that overexpression of CaLMF down-regulated the expression of CPT biosynthesis genes and decreased the accumulation of CPT in leaves, while light-regulated expression of CPT biosynthesis genes and CPT production were abolished in CaLMF silencing leaves under shading treatment. Our results show that CaLMF is a significant light signaling component, which mediates light-regulated CPT biosynthesis in C. acuminata.

Camptotheca acuminata 10-hydroxycamptothecin O-methyltransferase: an alkaloid biosynthetic enzyme co-opted from flavonoid metabolism.

The medicinal plant Camptotheca acuminata accumulates camptothecin, 10-hydroxycamptothecin, and 10-methoxycamptothecin as its major bioactive monoterpene indole alkaloids. Here, we describe identification and functional characterization of 10-hydroxycamptothecin O-methyltransferase (Ca10OMT), a member of the Diverse subclade of class II OMTs. Ca10OMT is highly active toward both its alkaloid substrate and a wide range of flavonoids in vitro and in this way contrasts with other alkaloid OMTs in the subclade that only utilize alkaloid substrates. Ca10OMT shows a strong preference for the A-ring 7-OH of flavonoids, which is structurally equivalent to the 10-OH of 10-hydroxycamptothecin. The substrates of other alkaloid OMTs in the subclade bear little similarity to flavonoids, but the 3-D positioning of the 7-OH, A- and C-rings of flavonoids is nearly identical to the 10-OH, A- and B-rings of 10-hydroxycamptothecin. This structural similarity likely explains the retention of flavonoid OMT activity by Ca10OMT and also why kaempferol and quercetin aglycones are potent inhibitors of its 10-hydroxycamptothecin activity. The catalytic promiscuity and strong inhibition of Ca10OMT by flavonoid aglycones in vitro prompted us to investigate the potential physiological roles of the enzyme in vivo. Based on its regioselectivity, kinetic parameters and absence of 7-OMT flavonoids in vivo, we conclude that the major and likely only substrate of Ca10OMTin vivo is 10-hydroxycamptothecin. This is likely accomplished by Ca10OMT being kept spatially separated at the tissue levels from potentially inhibitory flavonoid aglycones, and flavonoid aglycones being rapidly glycosylated to non-inhibitory flavonoid glycosides.

De novo genome assembly of Camptotheca acuminata, a natural source of the anti-cancer compound camptothecin.

Camptotheca acuminata is 1 of a limited number of species that produce camptothecin, a pentacyclic quinoline alkaloid with anti-cancer activity due to its ability to inhibit DNA topoisomerase. While transcriptome studies have been performed previously with various camptothecin-producing species, no genome sequence for a camptothecin-producing species is available to date. We generated a high-quality de novo genome assembly for C. acuminata representing 403 174 860 bp on 1394 scaffolds with an N50 scaffold size of 1752 kbp. Quality assessments of the assembly revealed robust representation of the genome sequence including genic regions. Using a novel genome annotation method, we annotated 31 825 genes encoding 40 332 gene models. Based on sequence identity and orthology with validated genes from Catharanthus roseus as well as Pfam searches, we identified candidate orthologs for genes potentially involved in camptothecin biosynthesis. Extensive gene duplication including tandem duplication was widespread in the C. acuminata genome, with 2571 genes belonging to 997 tandem duplicated gene clusters. To our knowledge, this is the first genome sequence for a camptothecin-producing species, and access to the C. acuminata genome will permit not only discovery of genes encoding the camptothecin biosynthetic pathway but also reagents that can be used for heterologous expression of camptothecin and camptothecin analogs with novel pharmaceutical applications.

A homomeric geranyl diphosphate synthase-encoding gene from Camptotheca acuminata and its combinatorial optimization for production of geraniol in Escherichia coli.

Geranyl diphosphate (GPP), the unique precursor for all monoterpenoids, is biosynthesized from isopentenyl diphosphate and dimethylallyl diphosphate via the head-to-tail condensation reaction catalyzed by GPP synthase (GPPS). Herein a homomeric GPPS from Camptotheca acuminata, a camptothecin-producing plant, was obtained from 5'- and 3'-rapid amplification of cDNA ends and subsequent overlap extension and convenient PCR amplifications. The truncate CaGPPS was introduced to replace ispA of pBbA5c-MevT(CO)-MBIS(CO, ispA), a de novo biosynthetic construct for farnesyl diphosphate generation, and overexpressed in Escherichia coli, together with the truncate geraniol synthase-encoding gene from C. acuminata (tCaGES), to confirm CaGPPS-catalyzed reaction in vivo. A 24.0 ± 1.3 mg L-1 of geraniol was produced in the recombinant E. coli. The production of GPP was also validated by the direct UPLC-HRMSE analyses. The tCaGPPS and tCaGES genes with different copy numbers were introduced into E. coli to balance their catalytic potential for high-yield geraniol production. A 1.6-fold increase of geraniol production was obtained when four copies of tCaGPPS and one copy of tCaGES were introduced into E. coli. The following fermentation conditions optimization, including removal of organic layers and addition of new n-decane, led to a 74.6 ± 6.5 mg L-1 of geraniol production. The present study suggested that the gene copy number optimization, i.e., the ratio of tCaGPPS and tCaGES, plays an important role in geraniol production in the recombinant E. coli. The removal and addition of organic solvent are very useful for sustainable high-yield production of geraniol in the recombinant E. coli in view of that the solubility of geraniol is limited in the fermentation broth and/or n-decane.

Cross-Kingdom Regulation of Putative miRNAs Derived from Happy Tree in Cancer Pathway: A Systems Biology Approach.

MicroRNAs (miRNAs) are well-known key regulators of gene expression primarily at the post-transcriptional level. Plant-derived miRNAs may pass through the gastrointestinal tract, entering into the body fluid and regulate the expression of endogenous mRNAs. Camptotheca acuminata, a highly important medicinal plant known for its anti-cancer potential was selected to investigate cross-kingdom regulatory mechanism and involvement of miRNAs derived from this plant in cancer-associated pathways through in silico systems biology approach. In this study, total 33 highly stable putative novel miRNAs were predicted from the publically available 53,294 ESTs of C. acuminata, out of which 14 miRNAs were found to be regulating 152 target genes in human. Functional enrichment, gene-disease associations and network analysis of these target genes were carried out and the results revealed their association with prominent types of cancers like breast cancer, leukemia and lung cancer. Pathways like focal adhesion, regulation of lipolysis in adipocytes and mTOR signaling pathways were found significantly associated with the target genes. The regulatory network analysis showed the association of some important hub proteins like GSK3B, NUMB, PEG3, ITGA2 and DLG2 with cancer-associated pathways. Based on the analysis results, it can be suggested that the ingestion of the C. acuminata miRNAs may have a functional impact on tumorigenesis in a cross-kingdom way and may affect the physiological condition at genetic level. Thus, the predicted miRNAs seem to hold potentially significant role in cancer pathway regulation and therefore, may be further validated using in vivo experiments for a better insight into their mechanism of epigenetic action of miRNA.

Functional characterization of a geraniol synthase-encoding gene from Camptotheca acuminata and its application in production of geraniol in Escherichia coli.

Geraniol synthase (GES) catalyzes the conversion of geranyl diphosphate (GPP) into geraniol, an acyclic monoterpene alcohol that has been widely used in many industries. Here we report the functional characterization of CaGES from Camptotheca acuminata, a camptothecin-producing plant, and its application in production of geraniol in Escherichia coli. The full-length cDNA of CaGES was obtained from overlap extension PCR amplification. The intact and N-terminus-truncated CaGESs were overexpressed in E. coli and purified to homogeneity. Recombinant CaGES showed the conversion activity from GPP to geraniol. To produce geraniol in E. coli using tCaGES, the biosynthetic precursor GPP should be supplied and transferred to the catalytic pocket of tCaGES. Thus, ispA(S80F), a mutant of farnesyl diphosphate (FPP) synthase, was prepared to produce GPP via the head-to-tail condensation of isoprenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). A slight increase of geraniol production was observed in the fermentation broth of the recombinant E. coli harboring tCaGES and ispA(S80F). To enhance the supply of IPP and DMAPP, the encoding genes involved in the whole mevalonic acid biosynthetic pathway were introduced to the E. coli harboring tCaGES and the ispA(S80F) and a significant increase of geraniol yield was observed. The geraniol production was enhanced to 5.85 ± 0.46 mg L(-1) when another copy of ispA(S80F) was introduced to the above recombinant strain. The following optimization of medium composition, fermentation time, and addition of metal ions led to the geraniol production of 48.5 ± 0.9 mg L(-1). The present study will be helpful to uncover the biosynthetic enigma of camptothecin and tCaGES will be an alternative to selectively produce geraniol in E. coli with other metabolic engineering approaches.

Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant.

Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that CamCPR was expressed in the roots, stems, and leaves of C. acuminata seedlings. The relative transcript level of CamCPR in leaves was 2.2-fold higher than that of roots and the stems showed 1.5-fold transcript level higher than the roots. The functional characterization of CamCPR will be helpful to disclose the mysterious mechanisms of the biosynthesis of CAM. The present study established a platform to characterize the P450 enzymes involved in the growth, development, and metabolism of eukaryotic organisms.

Effect of precursors feeding and media manipulation on production of novel anticancer pro-drug camptothecin from endophytic fungus

We have established methodology for the isolation and characterization of a novel endophytic fungus from the inner bark of medicinal plant Nothapodytes foetida, which produced camptothecin in Sabouraud broth (SB) under shake flask conditions. Camptothecin and its related compounds are at present obtained by extraction from intact plants, but fungal endopytes may be an alternative source of production. In present study we have observed the effect of different nutrient combinations and precursors (tryptophan, tryptamine, geraniol, citral, mevalonic acid and leucine) on the accumulation of camptothecin by endophytic fungus Entrophospora infrequens. The precursors were fed either alone or in combinations (tryptophan and geraniol, tryptophan and citral, tryptophan and mevalonic acid, tryptophan and leucine). The highest camptothecin content was observed in the range of 503 ± 25µg/100g dry cell mass in Sabouraud medium. Camptothecin content in the medium was increased by 2.5 folds by the presence of tryptophan and leucine whereas the production with trytophan was also significantly different from other treatments. Furthermore, the effect of fungal camptothecin on the morphology of human cancer cell lines was also studied. The treated cells showed reduction in size, condensation of nucleus and the protoplasmic extensions were reduced. All these characteristics are found in apoptotic cells.

Pyrosequencing of the Camptotheca acuminata transcriptome reveals putative genes involved in camptothecin biosynthesis and transport.

BACKGROUND: Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database. RESULTS: From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis. CONCLUSION: This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata.

Effect of artificial reconstitution of the interaction between the plant Camptotheca acuminata and the fungal endophyte Fusarium solani on camptothecin biosynthesis.

Fungal endophytes inhabit healthy tissues of all terrestrial plant taxa studied and occasionally produce host-specific compounds. We recently isolated an endophytic fungus, Fusarium solani, from Camptotheca acuminata, capable of biosynthesizing camptothecin (CPT, 1), but this capability substantially decreased on repeated subculturing. The endophyte with an impaired 1 biosynthetic capability was artificially inoculated into the living host plants and then recovered after colonization. Although the host-endophyte interaction could be reconstituted, biosynthesis of 1 could not be restored. Using a homology-based approach and high-precision isotope-ratio mass spectrometry (HP-IRMS), a cross-species biosynthetic pathway is proposed where the endophyte utilizes indigenous G10H (geraniol 10-hydroxylase), SLS (secologanin synthase), and TDC (tryptophan decarboxylase) to biosynthesize precursors of 1. However, the endophyte requires host STR (strictosidine synthase) in order to condense the nitrogen-containing moiety (tryptamine, 2) with the carbon-containing moiety (secologanin, 3) to form strictosidine (4) and complete the biosynthesis of 1. Biosynthetic genes of 1 in the seventh subculture generation of the endophyte revealed random and unpredictable nonsynonymous mutations. These random base substitutions led to dysfunction at the amino acid level. The controls, Top1 gene and rDNA, remained intact over subculturing, revealing that instability of biosynthetic genes of 1 was not reflected in the primary metabolic processes and functioning of the housekeeping genes. The present results reveal the causes of decreased production of 1 on subculturing, which could not be reversed by host-endophyte reassociation.