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

Abstract

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.

Keywords: Coptis; O-methyltransferases; benzylisoquinoline alkaloid; biosynthesis; transcriptome.

FIGURE 1

Putative pathways for BIA biosynthesis in C. teeta and C. chinensis. Enzymes found in this study are boxed. Abbreviations: TyrAT, L-tyrosine aminotransferase; 4HPPDC, 4-hydroxyphenylpuruvate decarboxylase; TYDC, tyrosine decarboxylase; 3OHase, tyrosine/tyramine 3-hydroxylase; NCS, (S)-norcoclaurine synthase; 6OMT, norcoclaurine 6-O-methyltransferase; CNMT, (S)-coclaurine N-methyltransferase; NMCH, N-methylcoclaurine 3^′-hydroxylase; 4^′OMT, 3^′-hydroxy-N-methylcoclaurine 4^′-O-methyltransferase; BBE, berberine bridge enzyme; CTS, corytuberine synthase; SCNMT, (S)-corytuberine-N-methyltransferase; SOMT, (S)-scoulerine 9-O-methyltransferase; CAS, (S)-canadine synthase; STOX, (S)-tetrahydroprotoberberine oxidase; CoOMT, columbamine O-methyltransferase; CFS, (S)-cheilanthifoline synthase; SPS, (S)-stylopine synthase.

FIGURE 2

Morphology comparison of roots and shoots between C. teeta and C. chinensis. Three-year-old C. teeta and C. chinensis were collected from Yunnan and Guangxi, respectively.

FIGURE 3

The contents of five main BIAs in three organs of C. teeta (A) and C. chinensis (B). ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 4

Phylogenetic tree of OMTs. Phylogenetic tree was constructed based on the deduced amino acid sequences from C. teeta and C. chinensis OMTs (triangles of different colors) and other plant OMTs. Abbreviations and GenBank accession numbers for the sequences used are as follows: EcOMT, putative E. californica OMT (ACO90220.1); CjCoOMT, C. japonica columbamine OMT (Q8H9A8.1); TtOMT, Thalictrum tuberosum catechol OMT (AAD29845.1); TtCaOMT, T. tuberosum catechol OMT (AAD29843.1); PsCaOMT, P. somniferum catechol OMT (AAQ01670.1); PsN7OMT, P. somniferum norreticuline 7OMT (ACN88562.1); Ps6OMT, P. somniferum norcoclaurine 6OMT (AAP45315.1); Ps4^′OMT2, P. somniferum 3^′-hydroxy-N-methylcoclaurine 4^′OMT2 (AAP45314.1); Ps4^′OMT1, P. somniferum 3^′-hydroxy-N-methylcoclaurine 4^′OMT1 (AAP45314.1); Cc4^′OMT, C. chinensis 3^′-hydroxy-N-methylcoclaurine 4^′OMT (ABY75613.1); Cj4^′OMT, C. japonica 3^′-hydroxy-N-methylcoclaurine 4^′OMT (Q9LEL5.1); Tf4^′OMT, T. flavum 3^′-hydroxy-N-methylcoclaurine 4^′OMT (AAU20768.1); Cj6OMT, C. japonica norcoclaurine 6OMT (Q9LEL6.1); Tf6OMT, T. flavum norcoclaurine 6OMT (AAU20765.1); VvReOMT, Vitis vinifera resveratrol OMT (CAQ76879.1); PtFlOMT, Populus trichocarpa flavonoid OMT predicted protein (XP_002312933.1); CjSOMT, C. japonica scoulerine 9OMT (Q39522.1); TfSOMT, T. flavum scoulerine 9OMT(AAU20770.1); PaCafOMT, Picea abies caffeate OMT (CAI30878.1); CaCafOMT, Capsicum annuum caffeate OMT (AAG43822.1); PsOMT1, P. somniferum SOMT1 (JN185323); PsOMT2, P. somniferum SOMT2 (JN185324); PsOMT3, P. somniferum SOMT3 (JN185325); ObEuOMT, Ocimum basilicum eugenol OMT (AAL30424.1); MpFlOMT, Mentha X piperita flavonoid 8OMT (AAR09600.1); ObCafOMT, O. basilicum caffeate OMT (AAD38189.1); CbEuOMT, Clarkia breweri (iso)eugenol OMT (AAC01533.1); CbCafOMT, C. breweri caffeate OMT (O23760.1); AmCafOMT, Ammi majus caffeate OMT (AAR24095.1); Ec7OMT, E. californica reticuline 7OMT (BAE79723.1); Ps7OMT, P. somniferum reticuline 7OMT (AAQ01668.1); Ec4^′OMT, E. californica 3^′-hydroxy-N-methylcoclaurine 4^′OMT (BAM37633.1); and Ec6OMT, E. californica OMT (BAM37634.1).

FIGURE 5

Extract ion chromatograms showing the O-methylation activity of recombinant OMTs on various substrates. (A) His-tag purified recombinant OMTs on 12% SDS–PAGE gel, Lane M: Protein Marker, Lane 1: Unpurified Cc6OMT1, Lane 2: Flow through Cc6OMT1, Lane 3: Elution Cc6OMT1; Lane 4: Unpurified Cc6OMT2, Lane 5: Flow through Cc6OMT2, Lane 6: Elution Cc6OMT2; Lane 7: Unpurified Ct7OMT, Lane 8: Flow through Ct7OMT, Lane 9: Elution Ct7OMT. Verified reaction equation, the top (control) substrate with denatured purified OMTs proteins, in vitro assay products of Cc6OMT1, Cc6OMT2, Ct7OMT with (S)-norcoclaurine, respectively. (B) His-tag purified recombinant OMT on 12% SDS–PAGE gel, Lane M: Protein Marker, Lane 1: Unpurified CtSOMT, Lane 2: Flow through CtSOMT, Lane 3: Elution CtSOMT; verified reaction equation; the top (control) substrate with denatured purified OMT protein, in vitro assay product of CtSOMT with (S)-scoulerine. Products were identified based on retention times and ESI[+]-CID spectra using authentic standards, 1. (S)-norcoclaurine, 2. (S)-coclaurine, 3. (S)-scoulerine, 4. (S)-tetrahydrocolumbamine.

FIGURE 6

Expression patterns of candidate unigenes involved in BIA biosynthesis in C. teeta and C. chinensis. The transcripts were analyzed by qRT-PCR, with actin as an internal standard.