Novel trends for producing plant triterpenoids in yeast

Triterpenoids possess versatile biological activities including antiviral, anticancer, and hepatoprotective activities. They are widely used in medicine and other health-related fields. However, current production of such compounds relies on plant culture and extraction, which brings about concerns for environmental, ecological, and infield problems. With increasing awareness of environmental sustainability, various microbes have been engineered to produce natural products, in which yeast turned out to be feasible for the heterologous biosynthesis of triterpenoids on account of its inherent advantages such as the robustness, safety, and sufficient precursor supplementation. This review has focused on recent progress regarding the biosynthesis of triterpenoids in yeast. The key enzymes to reconstruct the triterpenoid pathways in yeast, include: oxidosqualene cyclases, cytochrome P450s and UDP-glycosyltransferases are systematically presented. We then discuss recent metabolic engineering strategies and future prospects of protein engineering, pathway compartmentalization, product transportation, and other aspects for triterpenoid production in yeast.     

The triterpene cyclase protein family: a systematic analysis

Triterpene cyclases catalyze a broad range of cyclization reactions to form polycyclic triterpenes. Triterpene cyclases that convert squalene to hopene are named squalene-hopene cyclases (SHC) and triterpene cyclases that convert oxidosqualene are named oxidosqualene cyclases (OSC). Many sequences have been published, but there is only one structure available for each of SHCs and OSCs. Although they catalyze a similar reaction, the sequence similarity between SHCs and OSCs is low. A family classification based on phylogenetic analysis revealed 20 homologous families which are grouped into two superfamilies, SHCs and OSCs. Based on this family assignment, the Triterpene Cyclase Engineering Database (TTCED) was established. It integrates available information on sequence and structure of 639 triterpene cyclases as well as on structurally and functionally relevant amino acids. Family specific multiple sequence alignments were generated to identify the functionally relevant residues. Based on sequence alignments, conserved residues in SHCs and OSCs were analyzed and compared to experimentally confirmed mutational data. Functional schematic models of the central cavities of OSCs and SHCs were derived from structure comparison and sequence conservation analysis. These models demonstrate the high similarity of the substrate binding cavity of SHCs and OSCs and the equivalences of the respective residues. The TTCED is a novel source for comprehensive information on the triterpene cyclase family, including a compilation of previously described mutational data. The schematic models present the conservation analysis in a readily available fashion and facilitate the correlation of residues to a specific function or substrate interaction.     

Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng

Panax ginseng is one of the most highly valued herbal medicines in the Orient, where it has gained an almost magical reputation for being able to maintain the quality of life. The root of ginseng contains noble tetracyclic triterpenenoid saponins, which are thought to be the major effective ingredients in P. ginseng. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by oxidosqualene cyclase, dammarenediol synthase (DDS). The gene encoding DDS has been characterized. Here, we investigated the expression of the DDS gene together with the genes involved in ginsenoside biosynthesis (SS, SE, PNX, PNY, PNY2 and PNZ). Expression of DDS mRNA was higher in flower buds compared with root, leaf and petiole of ginseng plants. Elicitor (methyl jasmonate) treatment up-regulated the expression of DDS mRNA. Ectopic expression of DDS in a yeast mutant (erg7) lacking lanosterol synthase resulted in the production of dammarenediol and hydroxydammarenone which were confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression which leads to a reduction of ginsenoside production to 84.5% in roots. These results indicate that expression of DDS played a vital role in the biosynthesis of ginsenosides in P. ginseng.     

Squalene cyclase and oxidosqualene cyclase from a fern

Ferns are the most primitive vascular plants. The phytosterols of ferns are the same as those of higher plants, but they produce characteristic triterpenes. The most distinct feature is the lack of oxygen functionality at C-3, suggesting that the triterpenes of ferns may be biosynthesized by direct cyclization of squalene. To obtain some insights into the molecular bases for the biosynthesis of triterpenes in ferns, we cloned ACX, an oxidosqualene cyclase homologue, encoding a cycloartenol synthase (CAS) and ACH, a squalene cyclase homologue, encoding a 22-hydroxyhopane synthase from Adiantum capillus-veneris. Phylogenetic analysis revealed that ACH is located in the cluster of bacterial SCs, while ACX is in the cluster of higher plant CASs.     

Altering product outcome in Abies grandis (-)-limonene synthase and (-)-limonene/(-)-alpha-pinene synthase by domain swapping and directed mutagenesis

(-)-(4S)-limonene synthase (LS) and (-)-(4S)-limonene/(-)-(1S, 5S)-alpha-pinene synthase (LPS) from grand fir (Abies grandis) exhibit nearly 91% sequence identity (93% similarity) at the amino acid level, yet produce very different mixtures of monoterpene olefins. To elucidate critical amino acids involved in determining monoterpene product distribution, a combination of domain swapping and reciprocal site-directed mutagenesis was carried out between these two enzymes. Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome, whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS, and resulted in a 5-fold increase in relative velocity. These results, in conjunction with modeling of the two enzymes, suggest that amino acids in the predicted D through F helix regions are critical for product determination.     

Cyclic nucleotides

The natural occurrence of cyclic nucleotides in higher plants, formerly a topic of fierce debate, is now established, as is the presence of nucleotidyl cyclases and cyclic nucleotide phosphodiesterases capable of their synthesis and breakdown. Here we describe the significant properties of cyclic nucleotides, also outlining their second messenger functions and the history of plant cyclic nucleotide research over its first three decades. Findings of the last five years are detailed within the context of the functional role of cyclic nucleotides in higher plants, with particular emphasis upon nucleotidyl cyclases and cyclic nucleotide-responsive protein kinases, -binding proteins and -gated ion channels, with future objectives and strategies discussed.     

Molecular cloning and expression in yeast of 2,3–oxidosqualene– triterpenoid cyclases from Arabidopsis thaliana

A vast array of triterpenes are found in living organisms in addition to lanosterol and cycloartenol, which are involved in sterol biosynthesis in non–photosynthetic and photosynthetic eukaryotes respectively. The chemical structure of these triterpenes is determined by a single step catalysed by 2,3–oxidosqualene–triterpene cyclases. The present study describes cloning and functional expression in yeast of several OS–triterpene cyclases. Three Arabidopsis thaliana cDNAs encoding proteins (ATLUP1, ATLUP2, ATPEN1) 57%, 58% and 49% identical to cycloartenol synthase from the same plant were isolated. Expression of these cDNAs in yeast showed that the recombinant proteins catalyse the synthesis of various pentacyclic triterpenes. Whereas ATLUP1 is essentially involved in the synthesis of lupeol, ATLUP2 catalyses the production of lupeol, – and –amyrin (in a 15:55:30 ratio). ATLUP2 is therefore a typical multifunctional enzyme. Under the same conditions, ATPEN1 did not lead to any product. Systematic sequencing of the Arabidopsis genome has led to genomic sequences encoding proteins identical to the above triterpene synthases. ATLUP1 and ATLUP2 are representative of a small subfamily (A) of at least five genes, whereas ATPEN1 is representative of a subfamily (B) of at least seven genes. The number of introns is characteristic of each subfamily. Whereas genes of family A possess 17 exons and 16 introns, genes of the subfamily B contain 14 exons and 13 introns. The size of each exon is remarkably conserved within each subfamily whereas that of each intron appears to be highly variable. Organization of the genes, sequences and functions of the deduced proteins are discussed in evolutionary terms.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.