Shengchao Yang

Engineering yeast for the production of breviscapine by genomic analysis and synthetic biology approaches

The flavonoid extract from Erigeron breviscapus, breviscapine, has increasingly been used to treat cardio- and cerebrovascular diseases in China for more than 30 years, and plant supply of E. breviscapus is becoming insufficient to satisfy the growing market demand. Here we report an alternative strategy for the supply of breviscapine by building a yeast cell factory using synthetic biology. We identify two key enzymes in the biosynthetic pathway (flavonoid-7-O-glucuronosyltransferase and flavone-6-hydroxylase) from E. breviscapus genome and engineer yeast to produce breviscapine from glucose. After metabolic engineering and optimization of fed-batch fermentation, scutellarin and apigenin-7-O-glucuronide, two major active ingredients of breviscapine, reach to 108 and 185 mg l–1, respectively. Our study not only introduces an alternative source of these valuable compounds, but also provides an example of integrating genomics and synthetic biology knowledge for metabolic engineering of natural compounds.Breviscapine is the flavonoid extract from medical plant Erigeron breviscapus for the treatment of cardio- and cerebrovascular disease. Here, the authors identify the key enzymes of the biosynthetic pathway from the plant genome and engineer yeast to produce breviscapine from glucose.

Analysis of the Transcriptome of Erigeron breviscapus Uncovers Putative Scutellarin and Chlorogenic Acids Biosynthetic Genes and Genetic Markers

Background Erigeron breviscapus (Vant.) Hand-Mazz. is a famous medicinal plant. Scutellarin and chlorogenic acids are the primary active components in this herb. However, the mechanisms of biosynthesis and regulation for scutellarin and chlorogenic acids in E. breviscapus are considerably unknown. In addition, genomic information of this herb is also unavailable. Principal Findings Using Illumina sequencing on GAIIx platform, a total of 64,605,972 raw sequencing reads were generated and assembled into 73,092 non-redundant unigenes. Among them, 44,855 unigenes (61.37%) were annotated in the public databases Nr, Swiss-Prot, KEGG, and COG. The transcripts encoding the known enzymes involved in flavonoids and in chlorogenic acids biosynthesis were discovered in the Illumina dataset. Three candidate cytochrome P450 genes were discovered which might encode flavone 6-hydroase converting apigenin to scutellarein. Furthermore, 4 unigenes encoding the homologues of maize P1 (R2R3-MYB transcription factors) were defined, which might regulate the biosynthesis of scutellarin. Additionally, a total of 11,077 simple sequence repeat (SSR) were identified from 9,255 unigenes. Of SSRs, tri-nucleotide motifs were the most abundant motif. Thirty-six primer pairs for SSRs were randomly selected for validation of the amplification and polymorphism. The result revealed that 34 (94.40%) primer pairs were successfully amplified and 19 (52.78%) primer pairs exhibited polymorphisms. Conclusion Using next generation sequencing (NGS) technology, this study firstly provides abundant genomic data for E. breviscapus. The candidate genes involved in the biosynthesis and transcriptional regulation of scutellarin and chlorogenic acids were obtained in this study. Additionally, a plenty of genetic makers were generated by identification of SSRs, which is a powerful tool for molecular breeding and genetics applications in this herb.

Building a Genetic Manipulation Tool Box for Orchid Biology: Identification of Constitutive Promoters and Application of CRISPR/Cas9 in the Orchid, Dendrobium officinale

Orchidaceae is the second largest family of flowering plants, which is highly valued for its ornamental purposes and medicinal uses. Dendrobium officinale is a special orchid species that can grow without seed vernalization. Because the whole-genome sequence of D. officinale is publicly available, this species is poised to become a convenient research model for the evolutionary, developmental, and genetic studies of Orchidaceae. Despite these advantages, the methods of genetic manipulation are poorly developed in D. officinale. In this study, based on the previously developed Agrobacterium-mediated gene transformation system, we identified several highly efficient promoters for exogenous gene expression and successfully applied the CRISPR/Cas9 system for editing endogenous genes in the genome of D. officinale. These two basic techniques contribute to the genetic manipulation toolbox of Orchidaceae. The pCambia-1301-35SN vector containing the CaMV 35S promoter and the β-glucuronidase (GUS) and Superfolder green fluorescence protein (SG) as reporter genes were introduced into the plant tissues by the Agrobacterium-mediated transformation system. Fluorescence emission from the transformed plants confirmed the successful transcription and translation of SG genes into functional proteins. We compared the GUS activity under different promoters including four commonly used promoters (MtHP, CVMV, MMV and PCISV) with CaMV 35S promoter and found that MMV, CVMV, and PCISV were as effective as the 35S promoter. Furthermore, we applied the CRISPR/Cas9-mediated genome editing system successfully in D. officinale. By selecting five target genes (C3H, C4H, 4CL, CCR, and IRX) in the lignocellulose biosynthesis pathway, we showed that, for a given target, this system can generate edits (insertions, deletions, or substitutions) at a rate of 10 to 100%. These results showed that our two genetic manipulation tools can efficiently express exogenous genes and edit endogenous genes in D. officinale. These efficient research tools will not only help create novel D. officinale varieties, but will also facilitate the molecular genetic investigation of orchid biology.

De novo Sequencing and Transcriptome Analysis of Pinellia ternata Identify the Candidate Genes Involved in the Biosynthesis of Benzoic Acid and Ephedrine

Background: The medicinal herb, Pinellia ternata, is purported to be an anti-emetic with analgesic and sedative effects. Alkaloids are the main biologically active compounds in P. ternata, especially ephedrine that is a phenylpropylamino alkaloid specifically produced by Ephedra and Catha edulis. However, how ephedrine is synthesized in plants is uncertain. Only the phenylalanine ammonia lyase (PAL) and relevant genes in this pathway have been characterized. Genomic information of P. ternata is also unavailable. Results: We analyzed the transcriptome of the tuber of P. ternata with the Illumina HiSeq™ 2000 sequencing platform. 66,813,052 high-quality reads were generated, and these reads were assembled de novo into 89,068 unigenes. Most known genes involved in benzoic acid biosynthesis were identified in the unigene dataset of P. ternata, and the expression patterns of some ephedrine biosynthesis-related genes were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR). Also, 14,468 simple sequence repeats (SSRs) were identified from 12,000 unigenes. Twenty primer pairs for SSRs were randomly selected for the validation of their amplification effect. Conclusion: RNA-seq data was used for the first time to provide a comprehensive gene information on P. ternata at the transcriptional level. These data will advance molecular genetics in this valuable medicinal plant.

Candidate Genes Involved in the Biosynthesis of Triterpenoid Saponins in Platycodon grandiflorum Identified by Transcriptome Analysis.

Background: Platycodon grandiflorum is the only species in the genus Platycodon of the family Campanulaceae, which has been traditionally used as a medicinal plant for its lung-heat-clearing, antitussive, and expectorant properties in China, Japanese, and Korean. Oleanane-type triterpenoid saponins were the main chemical components of P. grandiflorum and platycodin D was the abundant and main bioactive component, but little is known about their biosynthesis in plants. Hence, P. grandiflorum is an ideal medicinal plant for studying the biosynthesis of Oleanane-type saponins. In addition, the genomic information of this important herbal plant is unavailable. Principal findings: A total of 58,580,566 clean reads were obtained, which were assembled into 34,053 unigenes, with an average length of 936 bp and N50 of 1,661 bp by analyzing the transcriptome data of P. grandiflorum. Among these 34,053 unigenes, 22,409 unigenes (65.80%) were annotated based on the information available from public databases, including Nr, NCBI, Swiss-Prot, KOG, and KEGG. Furthermore, 21 candidate cytochrome P450 genes and 17 candidate UDP-glycosyltransferase genes most likely involved in triterpenoid saponins biosynthesis pathway were discovered from the transcriptome sequencing of P. grandiflorum. In addition, 10,626 SSRs were identified based on the transcriptome data, which would provide abundant candidates of molecular markers for genetic diversity and genetic map for this medicinal plant. Conclusion: The genomic data obtained from P. grandiflorum, especially the identification of putative genes involved in triterpenoid saponins biosynthesis pathway, will facilitate our understanding of the biosynthesis of triterpenoid saponins at molecular level.

Hybrid de novo genome assembly of the Chinese herbal fleabane Erigeron breviscapus

Abstract Background: The plants in the Erigeron genus of the Compositae (Asteraceae) family are commonly called fleabanes, possibly due to the belief that certain chemicals in these plants repel fleas. In the traditional Chinese medicine, Erigeron breviscapus, which is native to China, was widely used in the treatment of cerebrovascular disease. A handful of bioactive compounds, including scutellarin, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid, have been isolated from the plant. With the purpose of finding novel medicinal compounds and understanding their biosynthetic pathways, we propose to sequence the genome of E. breviscapus. Findings: We assembled the highly heterozygous E. breviscapus genome using a combination of PacBio single-molecular real-time sequencing and next-generation sequencing methods on the Illumina HiSeq platform. The final draft genome is approximately 1.2 Gb, with contig and scaffold N50 sizes of 18.8 kb and 31.5 kb, respectively. Further analyses predicted 37 504 protein-coding genes in the E. breviscapus genome and 8172 shared gene families among Compositae species. Conclusions: The E. breviscapus genome provides a valuable resource for the investigation of novel bioactive compounds in this Chinese herb.

Genome-Wide Transcriptional Excavation of Dipsacus asperoides Unmasked both Cryptic Asperosaponin Biosynthetic Genes and SSR Markers

Background: Dipsacus asperoides is a traditional Chinese medicinal crop. The root is generally used as a medicine and is frequently prescribed by Chinese doctors for the treatment of back pain, limb paralysis, flutter trauma, tendon injuries, and fractures. With the rapid development of bioinformatics, research has been focused on this species at the gene or molecular level. For purpose of fleshing out genome information about D. asperoides, in this paper we conducted transcriptome analysis of this species. Principal Findings: To date, many genes encoding enzymes involved in the biosynthesis of triterpenoid saponins in D.asperoides have not been elucidated. Illumina paired-end sequencing was employed to probe D. asperoidess various enzymes associated with the relevant mesostate. A total of 30, 832,805 clean reads and de novo spliced 43,243 unigenes were obtained. Of all unigenes, only 8.27% (3578) were successfully annotated in total of seven public databases: Nr, Nt, Swiss-Prot, GO, KOG, KEGG, and Pfam, which might be attributed to the poor studies on D. asperoides. The candidate genes encoding enzymes involved in triterpenoid saponin biosynthesis were identified and experimentally verified by reverse transcription qPCR, encompassing nine cytochrome P450s and 17 UDP-glucosyltransferases. Specifically, unearthly putative genes involved in the glycosylation of hederagenin were acquired. Simultaneously, 4490 SSRs from 43,243 examined sequences were determined via bioinformatics analysis. Conclusion: This study represents the first report on the use of the Illumina sequence platform on this crop at the transcriptome level. Our findings of candidate genes encoding enzymes involved in Dipsacus saponin VI biosynthes is provide novel information in efforts to further understand the triterpenoid metabolic pathway on this species. The initial genetics resources in this study will contribute significantly to the genetic breeding program of D. asperoides, and are beneficial for clinical diagnosis and treatment.

Transcriptome analysis of Panax zingiberensis identifies genes encoding oleanolic acid glucuronosyltransferase involved in the biosynthesis of oleanane-type ginsenosides

Main conclusionOleanolic acid glucuronosyltransferase (OAGT) genes synthesizing the direct precursor of oleanane-type ginsenosides were discovered. The four recombinant proteins of OAGT were able to transfer glucuronic acid at C-3 of oleanolic acid that yields oleanolic acid 3-O-β-glucuronide.Ginsenosides are the primary active components in the genus Panax, and great efforts have been made to elucidate the mechanisms underlying dammarane-type ginsenoside biosynthesis. However, there is limited information on oleanane-type ginsenosides. Here, high-performance liquid chromatography analysis demonstrated that oleanane-type ginsenosides (particularly ginsenoside Ro and chikusetsusaponin IV and IVa) are the abundant ginsenosides in Panax zingiberensis, an extremely endangered Panax species in southwest China. These ginsenosides are derived from oleanolic acid 3-O-β-glucuronide, which may be formed from oleanolic acid catalyzed by an unknown oleanolic acid glucuronosyltransferase (OAGT). Transcriptomic analysis of leaves, stems, main roots, and fibrous roots of P. zingiberensis was performed, and a total of 46,098 unigenes were obtained, including all the identified homologous genes involved in ginsenoside biosynthesis. The most upstream genes were highly expressed in the leaves, and the UDP-glucosyltransferase genes were highly expressed in the roots. This finding indicated that the precursors of ginsenosides are mainly synthesized in the leaves and transported to different parts for the formation of particular ginsenosides. For the first time, enzyme activity assay characterized four genes (three from P. zingiberensis and one from P. japonicus var. major, another Panax species with oleanane-type ginsenosides) encoding OAGT, which particularly transfer glucuronic acid at C-3 of oleanolic acid to form oleanolic acid 3-O-β-glucuronide. Taken together, our study provides valuable genetic information for P. zingiberensis and the genes responsible for synthesizing the direct precursor of oleanane-type ginsenosides.

Polygonatone H, a new homoisoflavanone with cytotoxicity from Polygonatum Cyrtonema Hua.

Abstract A new homoisoflavonoid, (3R)-5,7-dihydroxy-6-methyl-3-(2′-hydroxy-4′-methoxybenzyl)-chroman-4-one (1), namely polygonatone H, in addition to fourteen known homoisoflavones (2–15) were isolated from the rhizome of Polygonatum Cyrtonema Hua. The structures were identified with the aid of 1D/2D NMR spectroscopic technologies. Compounds 2, 6, 8, 10, 11, 13, and 15 were isolated from P. Cyrtonema for the first time. Compound 1 showed cytotoxicities to human cancer cell lines with IC50 values to comparable those of cisplatin.

Identification and Characterization of Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in Coptis Species

The dried rhizomes of Coptis chinensis have been extensively used in heat clearing, dampness drying, fire draining, and detoxification by virtue of their major bioactive components, benzylisoquinoline alkaloids (BIAs). However, C. teeta and C. chinensis are occasionally interchanged, and current understanding of the molecular basis of BIA biosynthesis in these two species is limited. Here, berberine, coptisine, jatrorrhizine, and palmatine were detected in two species, and showed the highest contents in the roots, while epiberberine were found only in C. chinensis. Comprehensive transcriptome analysis of the roots and leaves of C. teeta and C. chinensis, respectively, identified 53 and 52 unigenes encoding enzymes potentially involved in BIA biosynthesis. By integrating probable biosynthetic pathways for BIAs, the jatrorrhizine biosynthesis ill-informed previously was further characterized. Two genes encoding norcoclaurine/norlaudanosoline 6-O-methyltransferases (Cc6OMT1 and Cc6OMT2) and one gene encoding norcoclaurine-7OMT (Ct7OMT) catalyzed enzymatically O-methylate (S)-norcoclaurine at C6 that yield (S)-coclaurine, along with a smaller amount of O-methylation occurred at C7, thereby forming its isomer (isococlaurine). In addition, scoulerine 9-OMT (CtSOMT) was determined to show strict substrate specificity, targeting (S)-scoulerine to yield (S)-tetrahydrocolumbamine. Taken together, the integration of the transcriptome and enzyme activity assays further provides new insight into molecular mechanisms underlying BIA biosynthesis in plants and identifies candidate genes for the study of synthetic biology in microorganisms.