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Abstract
Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-β-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside.
Highlights
The natural product 2-isopropyl-5-methylcyclohexanol is a monocyclic monoterpene alcohol that has three asymmetric carbon atoms and, occurs in eight stereoisomeric forms [1,2]
The coding sequences of the full-length candidate genes TEA009739 and TEA009753 were randomly selected. Their cDNA sequences were obtained from the leaves of the tea plant C. sinensis var. sinensis, cloned into the pGEX-4T1-vector and the encoded uridine diphosphate sugar-dependent enzymes (UGTs) were successfully produced as N-terminal glutathione S-transferase (GST) fusion proteins in Escherichia coli BL21(DE3)pLysS
UGT93Y1 and UGT93Y2 are 1404 and 1413 base pairs long, respectively, encode proteins with a length of 467 and 470 amino acids, respectively, and share a sequence identity of 74.3%. Both sequences show the characteristic features of functional UGTs of the carbohydrate active enzymes (CAZy) family 1, including the catalytically active amino acids histidine at position 29 [38], the activating aspartic acid and the 44 amino acid long plant secondary product glycosyltransferase (PSPG) box [19,31] (Supplementary material Figure S2)
Summary
The natural product 2-isopropyl-5-methylcyclohexanol is a monocyclic monoterpene alcohol that has three asymmetric carbon atoms and, occurs in eight stereoisomeric forms [1,2]. In addition to the enantiomers of menthol, three other pairs of diastereomers called isomeric menthols exist, such as neomenthol, isomenthol and neoisomenthol [3]. The natural (−)-menthol is the most abundant isomer and is found in many essential oils, especially mint oils of the genus Mentha [4,5,6,7]. In Japanese peppermint oil from field mint (Mentha arvensis) grown in Japan or China, it accounts for up to 90% of the essential oil, and (−)-menthol is the main component in the peppermint oil (Mentha piperita). The isomeric (+)-neomenthol is found in Japanese peppermint oil, while (−)-neoisomenthol makes up to one percent in geranium oil [3,4,7,8,9,10,11,12]
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