BAHD acetyltransferase contributes to wound‐induced biosynthesis of oleo‐gum resin triterpenes in Boswellia

Abstract

SUMMARY Triterpenes (30‐carbon isoprene compounds) represent a large and highly diverse class of natural products that play various physiological functions in plants. The triterpene biosynthetic enzymes, particularly those catalyzing the late‐stage regio‐selective modifications are not well characterized. The bark of select Boswellia trees, e.g., B. serrata exudes specialized oleo‐gum resin in response to wounding, which is enriched with boswellic acids (BAs), a unique class of C3α‐epimeric pentacyclic triterpenes with medicinal properties. The bark possesses a network of resin secretory structures comprised of vertical and horizontal resin canals, and amount of BAs in bark increases considerably in response to wounding. To investigate BA biosynthetic enzymes, we conducted tissue‐specific transcriptome profiling and identified a wound‐responsive BAHD acetyltransferase (BsAT1) of B. serrata catalyzing the late‐stage C3α‐O‐acetylation reactions in the BA biosynthetic pathway. BsAT1 catalyzed C3α‐O‐acetylation of αBA, βBA, and 11‐keto‐βBA in vitro and in planta assays to produce all the major C3α‐O‐acetyl‐BAs (3‐acetyl‐αBA, 3‐acetyl‐βBA, and 3‐acetyl‐11‐keto‐βBA) found in B. serrata bark and oleo‐gum resin. BsAT1 showed strict specificity for BA scaffold, whereas it did not acetylate the more common C3β‐epimeric pentacyclic triterpenes. The analysis of steady‐state kinetics using various BAs revealed distinct substrate affinity and catalytic efficiency. BsAT1 transcript expression coincides with increased levels of C3α‐O‐acetyl‐BAs in bark in response to wounding, suggesting a role of BsAT1 in wound‐induced biosynthesis of C3α‐O‐acetyl‐BAs. Overall, the results provide new insights into the biosynthesis of principal chemical constituents of Boswellia oleo‐gum resin.

A recently evolved BAHD acetyltransferase, responsible for bitter soyasaponin A production, is indispensable for soybean seed germination

“…We determined the pH optimum for GmSSAcT1 activity over a pH range from 5.0 to 9.0 using null‐acetyl soyasaponin A and acetyl‐CoA as substrates. We measured a pH optimum of about pH 7.5, which matches values for most known BAHD terpenoid acyltransferases (Walker et al, 2002; Yan et al, 2020; Kumar et al, 2021). The K m for null‐acetyl Aa and Ab were 24.87 and 48.8 μmol/L ( n = 3), respectively, when acetyl‐CoA was used as the acyl donor (Table 1).…”

Identification and Functional Characterization of a Novel Sinapyl Alcohol Acyltransferase from Euphorbia lathyris L.

“…Most of the reported BAHD acyltransferases are expressed in the cytoplasm. 20,33 Interestingly, the two kinds of acyltransferases are expressed differently in Echinacea purpurea: EpHQT and EpHCT (BAHD) are expressed in the cytoplasm, while EpCAS (SCLP) is localized in the vacuole. 34 Software predictions and observations confirmed that ElBAHD10 functions in the cytoplasm, possibly caused by a lack of transport peptides or other sequences in acyltransferase.…”

UGT86C11 is a novel plant UDP-glycosyltransferase involved in labdane diterpene biosynthesis

“…The soluble protein fraction was obtained after centrifugation (18,000xg at 4°C for 30 min) and subjected to affinity chromatography using Ni-NTA agarose (Qiagen). To prepare enriched fraction of recombinant UGT for use in initial screening of UGT activity, the soluble protein of 100 ml bacterial culture was bound to pre-equilibrated Ni-NTA agarose (100 μl) for 2-3 hr at 4°C, passed through a 10 ml gravity column (Bio-Rad), washed with lysis buffer containing 20 mM imidazole and eluted with lysis buffer containing 250 mM imidazole (68). For purification of ApUGT12 to electrophoretic homogeneity, soluble protein of 1 litre bacterial culture was bound to pre-equilibrated Ni-NTA agarose (1 ml), passed through a 1 ml chromatography column (Bio-Rad) equipped with a peristaltic pump (Miclins India) and a fraction collector (Bio-Rad).…”

“…ApUGT12 was amplified using Phusion high-fidelity DNA polymerase J o u r n a l P r e -p r o o f with ORF-specific primers (Table S2), inserted into pENTR D-TOPO (Invitrogen) and finally cloned into pGWB441 and pGWB442 following LR clonase reaction (70). Empty pGWB441 and pGWB454 for expressing free EYFP and mRFP were prepared by recombining empty pENTR/D-TOPO plasmid with pGWB441 and pGWB454 (68). The recombinant clones were selected in LB media containing spectinomycin (100 μg/ml).…”

“…After 36 to 48 hr of agroinfiltration, leaf sections were analyzed and images were captured under a Carl Zeiss LSM880 laser scanning confocal microscope using 63X (numerical aperture 1.4) oil-immersion objective. YFP was excited at 514 nm and detected in 525-562 nm range while RFP was excited at 561 nm and detected in 570-652 nm range (68). The simultaneous detection of RFP and YFP was done by combining the settings mentioned above in the sequential scanning function of the microscope as per manufacturer instructions.…”