Functional Identification of Triterpene Methyltransferases from Botryococcus braunii Race B

Tom D Niehaus,Scott Kinison,Shigeru Okada,Yun-Soo Yeo,Stephen A Bell,Ping Cui,Timothy P Devarenne,Joe Chappell

doi:10.1074/jbc.m111.316059

Abstract

Botryococcus braunii race B is a colony-forming, green algae that accumulates triterpene oils in excess of 30% of its dry weight. The composition of the triterpene oils is dominated by dimethylated to tetramethylated forms of botryococcene and squalene. Although unusual mechanisms for the biosynthesis of botryococcene and squalene were recently described, the enzyme(s) responsible for decorating these triterpene scaffolds with methyl substituents were unknown. A transcriptome of B. braunii was screened computationally assuming that the triterpene methyltransferases (TMTs) might resemble the S-adenosyl methionine-dependent enzymes described for methylating the side chain of sterols. Six sterol methyltransferase-like genes were isolated and functionally characterized. Three of these genes when co-expressed in yeast with complementary squalene synthase or botryococcene synthase expression cassettes resulted in the accumulation of mono- and dimethylated forms of both triterpene scaffolds. Surprisingly, TMT-1 and TMT-2 exhibited preference for squalene as the methyl acceptor substrate, whereas TMT-3 showed a striking preference for botryococcene as its methyl acceptor substrate. These in vivo preferences were confirmed with in vitro assays utilizing microsomal preparations from yeast overexpressing the respective genes, which encode for membrane-associated enzymes. Structural examination of the in vivo yeast generated mono- and dimethylated products by NMR identified terminal carbons, C-3 and C-22/C-20, as the atomic acceptor sites for the methyl additions to squalene and botryococcene, respectively. These sites are identical to those previously reported for the triterpenes extracted from the algae. The availability of closely related triterpene methyltransferases exhibiting distinct substrate selectivity and successive catalytic activities provides important tools for investigating the molecular mechanisms responsible for the specificities exhibited by these unique enzymes.

Highlights

Botryococcus braunii accumulates high levels of methylated triterpenes that contribute to the buoyancy of the algae and serve as highly valued chemical feedstocks
Identification of Triterpene Methyltransferase Candidate Genes—We predicted that a methyltransferase that could act on squalene or botryococcene might resemble a C-24 sterol methyltransferase (SMT) because these enzymes act on the linear side chain of sterols
Amino acid alignments revealed that all six candidate genes share three conserved adenosyl methionine (AdoMet)-binding sites as identified by Kagen and Clarke [28]; the sterolbinding domain SMT-2, which is invariant in all known plant SMTs [31, 32], is absolutely conserved in three of the candidates (SMT-1, -2, and -3), but not so in the other three (TMT-1, -2, and -3) (Fig. 1)

Summary

Background

Botryococcus braunii accumulates high levels of methylated triterpenes that contribute to the buoyancy of the algae and serve as highly valued chemical feedstocks. It was suggested that the squalene and botryococcene produced by the SSL enzymes were channeled into the production of the liquid oils and the biosynthesis of squalene derivatives [17], such as the extracellular matrix, whereas the conventional B. braunii squalene synthase [18] appears to synthesize squalene destined for sterol biosynthesis It is not botryococcene and squalene, that accumulate to substantial levels in this algae, but the methylated forms of these triterpenes. Unlike many green algae that are flagellated and phototaxic [22], the buoyancy characteristic of Botryococcus provides a means for it to float in its normal aqueous habitats and to intercept a greater amount of photosynthetic light In addition to this purported physiological role, the methylated forms of botryococcene and squalene enhance their utility as feedstocks for petrochemical processing and chemical manufacturing [23]. We searched for homologs of sterol MTs [31, 32] within a transcriptome of B. braunii and provide here the first functional identification of several unique triterpene C-methyltransferases

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION