Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3β-tigloyloxytropane

Abstract

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3β-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3β-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3β-tigloyloxytropane synthase (TS), catalyzes 3β-tropanol and tigloyl-CoA to form 3β-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3β-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3β-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3β-tigloyloxytropane from tiglic acid and 3β-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3β-tigloyloxytropane.

Fig. 1. Proposed pathway for the biosynthesis of tropanol-derived alkaloids.

TRI tropine-forming reductase or tropinone reductase I, TRII pseudotropine-forming reductase or tropinone reductase II, LS littorine synthase, TS tigloidine synthase, the key finding of this study. The 3β-tropane alkaloid biosynthetic pathway is marked in blue, and the 3α-tropane alkaloid biosynthetic pathway is marked in green. P450-5021, 3β-tigloyloxytropane demethylase. P450-116623, tigloyl norpseudotropine hydroxylase.

Fig. 2. Metabolite, transcriptome, and phylogenetic association analysis.

ATissue profile of 3β-tigloyloxytropane. Three independent plants were used in the tissue profile analysis of 3β-tigloyloxytropane. The data are presented as means values ± s.d. **P = 0.0032. DW, dry weight. B Association analysis of the metabolites and BAHD gene family expression patterns. C Relative expression levels of TS in different organs, as indicated by qPCR. Three independent plants were used in the relative expression analysis of TS. The data are presented as means values ± s.d. **P = 0.0007. D Phylogenetic analysis of the BAHD-AT gene family. The phylogenetic tree was divided into clades 0–6. E BAHD acyltransferases and their substrates in clade 3: TS, the BAHD acyltransferase identified in this study; EcCS, Erythroxylum coca cocaine synthase, a BAHD acyltransferase of the coca tree; EcBAHD8, the homolog of EcCS in Erythroxylum coca; PaxASAT1-4, an acylsugar acyltransferase in Petunia axillaris; SlyASAT1-3, an acylsugar acyltransferase in Solanum lycopersicum; SsiASAT1-3, an acylsugar acyltransferase in Salpiglossis sinuata; ShaASAT2, an acylsugar acyltransferase in Solanum habrochaites; LeSAT1, a shikonin O-acyltransferase in Lithospermum erythrorhizon; LeAAT1, an alkannin O-acyltransferase in Lithospermum erythrorhizon; CrMAT, a minovincinine-19-hydroxy-O-acetyltransferase in Catharanthus roseus; and CrDAT, a deacetylvindoline 4-O-acetyltransferase in Catharanthus roseus. All functionally identified BAHD-ATs are labeled with their corresponding accession numbers. Statistical analysis was performed according to the two-sided independent sample t-test.

Fig. 3. Functional characterization of TS.

A TS enzymatic assays with tigloyl-CoA as the acyl donor and 3β-tropanol as the acyl acceptor. B Mass spectrometry (MS) data of 3β-tigloyloxytropane. C Relative expression levels of TS in VIGS-TS A. belladonna seedlings. **P < 0.0001. D Contents of 3β-tropanol in VIGS-TS A. belladonna seedlings. **P = 0.0024. E Contents of 3β-tigloyloxytropane in VIGS-TS A. belladonna seedlings. **P < 0.0001. F Contents of 3β-acetoxytropane in VIGS-TS A. belladonna seedlings. **P < 0.0001. G Contents of tigloyl norpseudotropine in VIGS-TS A. belladonna seedlings. **P < 0.0001. H Contents of tigloyl 1-hydroxynorpseudotropine in VIGS-TS A. belladonna seedlings. **P < 0.0001. I Contents of calystegine A3 in VIGS-TS A. belladonna seedlings. **P = 0.0002. Control, control line obtained by empty plasmid transformation. VIGS-TS, TS-silenced line. Fifteen independent plants each from the control group and VIGS-TS group were used in the VIGS assays. Center line of box plot denotes the median value; lower and upper bounds denote first and third quartile; whiskers extend to the smallest and maximum values. J Relative expression levels of TS in TS-overexpressing A. belladonna hairy root cultures. **P < 0.0001 (OE−1), **P < 0.0001 (OE-2), **P < 0.0001 (OE-3), **P < 0.0001 (OE-4). K Contents of 3β-tropanol in TS-overexpressing A. belladonna hairy root cultures. L Contents of 3β-tigloyloxytropane in TS-overexpressing A. belladonna hairy root cultures. **P = 0.0007 (OE−1), **P = 0.0024 (OE-2), **P = 0.0044 (OE-3), **P = 0.0025 (OE-4). M Contents of 3β-acetoxytropane in TS-overexpressing A. belladonna hairy root cultures. *P = 0.0136 (OE-1), **P =  0.0087 (OE-2), **P = 0.0048 (OE-3), **P  =  0.0039 (OE-4). N Contents of tigloyl norpseudotropine in TS-overexpressing A. belladonna hairy root cultures. O Contents of tigloyl 1-hydroxynorpseudotropine in TS-overexpressing A. belladonna hairy root cultures. P Contents of calystegine A3 in TS-overexpressing A. belladonna hairy root cultures. CK, eight independently transformed root culture lines transformed with pBI121. OE denotes all independently transformed root culture lines overexpressing TS (three biological replicates for each line), including OE-1, OE-2, OE-3, and OE-4. The data are presented as means values ± s.d. Statistical analysis was performed according to the two-sided independent sample t-test. DW, dry weight.

Fig. 4. Subcellular localization analysis of TS.

A The localization of TS-YFP and N32-YFP in tobacco protoplasts was observed by confocal microscopy. YFP yellow fluorescence from YFP. MitoTracker Red, MitoTracker Red fluorescence-labeled mitochondria. Merge YFP + RED, the merged images for the yellow fluorescence and MitoTracker Red fluorescence. Chlorophyll, chlorophyll spontaneous fluorescence. Bright, bright field image. Overlapping images of all the channels mentioned above were merged. TS-YFP, TS fused with YFP. N32-YFP, mitochondrion-localizing signal peptide (the 32 amino acids at the N-terminus) of TS fused with YFP. All tobacco transformation and microscopic analyses were independently conducted three times with different plants. B Determination of A. belladonna mitochondria by Western blot analysis with an antibody against VDAC. Western blot analysis was independently conducted two times with different plants. C Crude protein from A. belladonna mitochondria-catalyzed tigloyl-CoA and 3β-tropanol to form 3β-tigloyloxytropane.

Fig. 5. Catalytic mechanism of TS.

A Schematic model of the catalytic pocket that contains 3β-tropanol and tigloyl-CoA. B Comparison of the relative activities of TS and TS^H162A using tigloyl-CoA as the acyl donor. C Key residues in the substrate pocket combined with 3β-tropanol. D Comparison of the relative activity of TS and TS mutants (3β-tropanol binding residues mutated to alanine) using tigloyl-CoA as the acyl donor. **P < 0.0001 (TS^I35A), **P < 0.0001 (TS^Q39A), **P < 0.0001 (TS^Y280A), **P < 0.0001 (TS^N298A), **P < 0.0001 (TS^L300A), **P < 0.0001 (TS^W340A). E 3β-Tropanol and acyl donors are converted to 3β-tropanol esters via the catalysis of TS. The data are presented as means values ± s.d. Recombinant protein obtained from three independent transformants of TS and each mutant for activity test. Statistical analysis was performed according to the two-sided independent sample t-test.

Fig. 6. Improvement in the substrate promiscuity of TS.

A Catalytic pocket of TS that contains benzoyl-CoA. B Catalytic pocket of TS^F46I that contains benzoyl-CoA. C Catalytic pocket of EcCS that contains benzoyl-CoA. D Catalytic pocket of TS that contains tigloyl-CoA. E Catalytic pocket of TS that contains acetyl-CoA. F Catalytic pocket of TS^F46I that contains tigloyl-CoA. G Catalytic pocket of TS^F46I that contains acetyl-CoA. H Enzymatic assays of TS and TS^F46I with benzoyl-CoA as the acyl donor.

Fig. 7. De novo synthesis of 3β-tigloyloxytropane in N. benthamiana.

A Reconstruction of the 3β-tigloyloxytropane biosynthetic pathway in N. benthamiana. B LC-MS detection of target metabolites in tobacco leaves. C The contents of tropinone. D The contents of hygrine. E The contents of 3β-tropanol. F The contents of 3β-tigloyloxytropane. **P = 0.003. G The contents of 3β-acetoxytropane. **P = 0.0068. The control represents tobacco leaves expressing YFP. TS (TS^S40T) represents tobacco leaves coexpressing six TA genes and TS (TS^S40T). The data are presented as means values ± s.d. Leaves from three independent plants of each line were used for metabolite analysis. Statistical analysis was performed according to the two-sided independent sample t-test.

Fig. 8. E. coli bioreactors for the biosynthesis of 3β-tropanol esters.

A PcICS enzymatic assays. B MS data of tigloyl-CoA. C Semi-biosynthetic route of 3β-tigloyloxytropane. D Production of 3β-tigloyloxytropane. Left Y-axis, the content of 3β-tigloyloxytropane. Right Y-axis, the conversion rate from 3β-tropanol to 3β-tigloyloxytropane. HS-CoA means coenzyme A (CoA). Three independent transformants of each engineered E. coli were used in the semi-biosynthesis of 3β-tigloyloxytropane. The data are presented as means values ± s.d.