[Construction of cell factories for production of lupeol in Saccharomyces cerevisiae].

Lupane-type triterpenoids, such as betulinic acid, are derived from lupeol and have excellent properties in anti-HIV, anti-cancer activities and so on. For realizing heterogenous production of lupane-type triterpenoids, our research firstly integrated all the seven genes in the MVA pathway in Saccharomyces cerevisiae to increase the supply of squalene (triterpenoids universal precursor) in a single step using the DNA assembler method. Next, cell factories for production of lupeol was constructed by integrating Arabidopsis thaliana lupeol synthetic gene (AtLUP) into chromosome of triterpenoid chassis strain. Results showed that the MVA pathway, about 20 kb nucleotide length, could be assembled in one-pot process and the doubled MVA pathway could significantly improve squalene by 500-fold, reaching 354.00 mg•L⁻¹. NK2-LUP was obtained by introducing AtLUP gene on chromosome, and could produce 8.23 mg•L⁻¹ lupeol. This study supports the possibility of large-scale biosynthetic pathway assembly in S.cerevisiae and lays the foundation of obtaining cell factories for production of lupan-type triterpenoids at the same time.

Isotopologue profiling of triterpene formation under physiological conditions. Biosynthesis of lupeol-3-(3'-R-hydroxy)-stearate in Pentalinon andrieuxii.

The biosynthesis of lupeol-3-(3'R-hydroxy)-stearate (procrim b, 1) was investigated in the Mexican medicinal plant Pentalinon andrieuxii by (13)CO2 pulse-chase experiments. NMR analyses revealed positional enrichments of (13)C2-isotopologues in both the triterpenoid and the hydroxystearate moieties of 1. Five of the six isoprene units reflected a pattern with [1,2-(13)C2]- and [3,5-(13)C2]-isotopologues from the respective C5-precursors, IPP and DMAPP, whereas one isoprene unit in the ring E of 1 showed only the [3,5-(13)C2]-connectivity of the original C5-precursor, due to rearrangement of the dammarenyl cation intermediate during the cyclization process. The presence of (13)C2-isotopologues was indicative of [(13)C2]acetyl-CoA being the precursor units in the formation of the fatty acid moiety and of the triterpene via the mevalonate route. The observed labeling pattern was in agreement with a chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor during the cyclization process, suggesting that the lupeol synthase from P. andrieuxii is of the same type as that from Olea europea and Taraxacum officinale, but different from that of Arabidopsis thaliana. The study shows that (13)CO2 pulse-chase experiments are powerful in elucidating, under in vivo conditions and in a single experiment, the biosynthesis of complex plant products including higher terpenes.

Methyl jasmonate-elicited transcriptional responses and pentacyclic triterpene biosynthesis in sweet basil.

Sweet basil (Ocimum basilicum) is well known for its diverse pharmacological properties and has been widely used in traditional medicine for the treatment of various ailments. Although a variety of secondary metabolites with potent biological activities are identified, our understanding of the biosynthetic pathways that produce them has remained largely incomplete. We studied transcriptional changes in sweet basil after methyl jasmonate (MeJA) treatment, which is considered an elicitor of secondary metabolites, and identified 388 candidate MeJA-responsive unique transcripts. Transcript analysis suggests that in addition to controlling its own biosynthesis and stress responses, MeJA up-regulates transcripts of the various secondary metabolic pathways, including terpenoids and phenylpropanoids/flavonoids. Furthermore, combined transcript and metabolite analysis revealed MeJA-induced biosynthesis of the medicinally important ursane-type and oleanane-type pentacyclic triterpenes. Two MeJA-responsive oxidosqualene cyclases (ObAS1 and ObAS2) that encode for 761- and 765-amino acid proteins, respectively, were identified and characterized. Functional expressions of ObAS1 and ObAS2 in Saccharomyces cerevisiae led to the production of ß-amyrin and α-amyrin, the direct precursors of oleanane-type and ursane-type pentacyclic triterpenes, respectively. ObAS1 was identified as a ß-amyrin synthase, whereas ObAS2 was a mixed amyrin synthase that produced both α-amyrin and ß-amyrin but had a product preference for α-amyrin. Moreover, transcript and metabolite analysis shed light on the spatiotemporal regulation of pentacyclic triterpene biosynthesis in sweet basil. Taken together, these results will be helpful in elucidating the secondary metabolic pathways of sweet basil and developing metabolic engineering strategies for enhanced production of pentacyclic triterpenes.

Oxidation of Fe(II) leads to increased C-2 methylation of pentacyclic triterpenoids in the anoxygenic phototrophic bacterium Rhodopseudomonas palustris strain TIE-1.

Hopanoids are among the most widespread biomarkers of bacteria that are used as indicators for past and present bacterial activity. Our understanding of the production, function, and distribution of hopanoids in bacteria has improved greatly, partly due to genetic, culture-independent studies. Culture-based studies are important to determine hopanoid function and the environmental conditions under which these compounds are produced. This study compares the lipid inventory of Rhodopseudomonas palustris strain TIE-1 under anoxic photoautotrophic conditions using either H2 or Fe(II) as electron donor. The high amount to which adenosylhopane is produced irrespective of the used electron donor suggests a specific function of this compound rather than its exclusive role as an intermediate in bacteriohopanepolyol biosynthesis. C-2 methylated hopanoids and tetrahymanol account for as much as 59% of the respective C-2 methylated/non-methylated homologs during growth with Fe(II) as electron donor, as compared with 24% C-2 methylation for growth with H2 . This observation reveals that C-2 methylated hopanoids have a specific function and are preferentially synthesized in response to elevated Fe(II) concentrations. The presence of C-2 methylated pentacyclic triterpenoids has commonly been used as a biosignature for the interpretation of paleoenvironments. These new findings suggest that increased C-2 methylation may indicate anoxic ferrous conditions, in addition to other environmental stressors that have been previously reported.

Discovery, taxonomic distribution, and phenotypic characterization of a gene required for 3-methylhopanoid production.

Hopanoids methylated at the C-3 position are a subset of bacterial triterpenoids that are readily preserved in modern and ancient sediments and in petroleum. The production of 3-methylhopanoids by extant aerobic methanotrophs and their common occurrence in modern and fossil methane seep communities, in conjunction with carbon isotope analysis, has led to their use as biomarker proxies for aerobic methanotrophy. In addition, these lipids are also produced by aerobic acetic acid bacteria and, lacking carbon isotope analysis, are more generally used as indicators for aerobiosis in ancient ecosystems. However, recent genetic studies have brought into question our current understanding of the taxonomic diversity of methylhopanoid-producing bacteria and have highlighted that a proper interpretation of methylhopanes in the rock record requires a deeper understanding of their cellular function. In this study, we identified and deleted a gene, hpnR, required for methylation of hopanoids at the C-3 position in the obligate methanotroph Methylococcus capsulatus strain Bath. Bioinformatics analysis revealed that the taxonomic distribution of HpnR extends beyond methanotrophic and acetic acid bacteria. Phenotypic analysis of the M. capsulatus hpnR deletion mutant demonstrated a potential physiological role for 3-methylhopanoids; they appear to be required for the maintenance of intracytoplasmic membranes and cell survival in late stationary phase. Therefore, 3-methylhopanoids may prove more useful as proxies for specific environmental conditions encountered during stationary phase rather than a particular bacterial group.

Molecular cloning and expression of squalene synthase and 2,3-oxidosqualene cyclase genes in persimmon (Diospyros kaki L.) fruits.

Oleanolic acid (OA) and ursolic acid (UA) are the main triterpene acids in persimmon fruit, and squalene synthase and 2,3-oxidosqualene cyclases are important enzymes in pentacyclic triterpene biosynthesis. In order to study their relationship, DkSQS and DkOSC were cloned from persimmon fruits in the present study. The full-length cDNA of DkSQS was 1647 bp, containing an open reading frame (ORF) of 1245 bp that encoded a peptide of 415 amino acids (AA). The 3'-end of DkOSC cDNA fragment contained 522 bp, including a partial ORF of 298 bp, a full poly A tail that encoded 98 AA. Two cultivars of persimmon, i.e. cv. Nishimurawase and cv. Niuxinshi, were used to study the content of OA and UA and the related gene expression. Results showed that OA and UA contents changed in both cultivars during fruit development, the difference in cv. Nishimurawase was greater than that in cv. Niuxinshi. The expression of DkSQS and DkOSC had no obvious correlation with the biosynthesis of OA and UA in the flesh. There may be two main reasons. Firstly, different enzymes involved in the biosynthesis of triterpenes and mutual adjustment were existed in different gene expressions. Secondly, it was not clear that the DkOSC cloned in this research belonged to which subfamily. Therefore, the real relationship between triterpenes and DkSQS and DkOSC in persimmon fruits is still to be revealed.

Cloning and characterization of oxidosqualene cyclases from Kalanchoe daigremontiana: enzymes catalyzing up to 10 rearrangement steps yielding friedelin and other triterpenoids.

The first committed step in triterpenoid biosynthesis is the cyclization of oxidosqualene to polycyclic alcohols or ketones C(30)H(50)O. It is catalyzed by single oxidosqualene cyclase (OSC) enzymes that can carry out varying numbers of carbocation rearrangements and, thus, generate triterpenoids with diverse carbon skeletons. OSCs from diverse plant species have been cloned and characterized, the large majority of them catalyzing relatively few rearrangement steps. It was recently predicted that special OSCs must exist that can form friedelin, the pentacyclic triterpenoid whose formation involves the maximum possible number of rearrangement steps. The goal of the present study, therefore, was to clone a friedelin synthase from Kalanchoe daigremontiana, a plant species known to accumulate this triterpenoid in its leaf surface waxes. Five OSC cDNAs were isolated, encoding proteins with 761-779 amino acids and sharing between 57.4 and 94.3% nucleotide sequence identity. Heterologous expression in yeast and GC-MS analyses showed that one of the OSCs generated the steroid cycloartenol together with minor side products, whereas the other four enzymes produced mixtures of pentacyclic triterpenoids dominated by lupeol (93%), taraxerol (60%), glutinol (66%), and friedelin (71%), respectively. The cycloartenol synthase was found expressed in all leaf tissues, whereas the lupeol, taraxerol, glutinol, and friedelin synthases were expressed only in the epidermis layers lining the upper and lower surfaces of the leaf blade. It is concluded that the function of these enzymes is to form respective triterpenoid aglycones destined to coat the leaf exterior, probably as defense compounds against pathogens or herbivores.