Plant Metabolic Gene Clusters: Evolution, Organization, and Their Applications in Synthetic Biology.

Plants are a remarkable source of high-value specialized metabolites having significant physiological and ecological functions. Genes responsible for synthesizing specialized metabolites are often clustered together for a coordinated expression, which is commonly observed in bacteria and filamentous fungi. Similar to prokaryotic gene clustering, plants do have gene clusters encoding enzymes involved in the biosynthesis of specialized metabolites. More than 20 gene clusters involved in the biosynthesis of diverse metabolites have been identified across the plant kingdom. Recent studies demonstrate that gene clusters are evolved through gene duplications and neofunctionalization of primary metabolic pathway genes. Often, these clusters are tightly regulated at nucleosome level. The prevalence of gene clusters related to specialized metabolites offers an attractive possibility of an untapped source of highly useful biomolecules. Accordingly, the identification and functional characterization of novel biosynthetic pathways in plants need to be worked out. In this review, we summarize insights into the evolution of gene clusters and discuss the organization and importance of specific gene clusters in the biosynthesis of specialized metabolites. Regulatory mechanisms which operate in some of the important gene clusters have also been briefly described. Finally, we highlight the importance of gene clusters to develop future metabolic engineering or synthetic biology strategies for the heterologous production of novel metabolites.

Functional characterization of a defense-responsive bulnesol/elemol synthase from potato.

Terpene synthases (TPSs) produce a variety of terpenoids that play numerous functional roles in primary and secondary metabolism, as well as in ecological interactions. Here, we report the functional characterization of an inducible potato TPS gene encoding bulnesol/elemol synthase (StBUS/ELS). The expression of StBUS/ELS in potato leaves was significantly induced in response to both bacterial (Pseudomonas syringae) and fungal (Alternaria solani) infection as well as methyl jasmonate treatment, indicating its role in defense. The leaves exhibited the highest StBUS/ELS expression followed by the stem with least and similar expression in tuber, sprout and root. Recombinant StBUS/ELS catalyzed the formation of different sesquiterpenes by utilizing farnesyl diphosphate as substrate, and the monoterpene geraniol from geranyl diphosphate. Among the sesquiterpenes formed by StBUS/ELS, elemol was the predominant product followed by α-bulnesene, bulnesol and ß-elemene. Further gas chromatography-mass spectrometry (GC-MS) analysis of StBUS/ELS assay products at different injection temperatures revealed elemol and bulnesol as the major products at 275 and 200/150°C, respectively, without much change in the levels of minor products. This indicated thermal rearrangement of bulnesol into elemol at higher temperatures. Transient overexpression of StBUS/ELS in potato leaves conferred tolerance against the growth of bacteria P. syringae and Ralstonia solanacearum, and the fungus A. solani. Further, expression analysis of pathogenesis-related (PR) genes in StBUS/ELS overexpressing leaves showed no significant change in comparison to control, indicating a direct involvement of StBUS/ELS enzymatic products against pathogens. Overall, our study suggested that StBUS/ELS is a pathogen-inducible TPS encoding bulnesol/elemol synthase and could provide a direct role in defense against biotic stress in potato.

Terpene Moiety Enhancement by Overexpression of Geranyl(geranyl) Diphosphate Synthase and Geraniol Synthase Elevates Monomeric and Dimeric Monoterpene Indole Alkaloids in Transgenic Catharanthus roseus.

Catharanthus roseus is the sole source of two of the most important anticancer monoterpene indole alkaloids (MIAs), vinblastine and vincristine and their precursors, vindoline and catharanthine. The MIAs are produced from the condensation of precursors derived from indole and terpene secoiridoid pathways. It has been previously reported that the terpene moiety limits MIA biosynthesis in C. roseus. Here, to overcome this limitation and enhance MIAs levels in C. roseus, bifunctional geranyl(geranyl) diphosphate synthase [G(G)PPS] and geraniol synthase (GES) that provide precursors for early steps of terpene moiety (secologanin) formation, were overexpressed transiently by agroinfiltration and stably by Agrobacterium-mediated transformation. Both transient and stable overexpression of G(G)PPS and co-expression of G(G)PPS+GES significantly enhanced the accumulation of secologanin, which in turn elevated the levels of monomeric MIAs. In addition, transgenic C. roseus plants exhibited increased levels of root alkaloid ajmalicine. The dimeric alkaloid vinblastine was enhanced only in G(G)PPS but not in G(G)PPS+GES transgenic lines that correlated with transcript levels of peroxidase-1 (PRX1) involved in coupling of vindoline and catharanthine into 3',4'-anhydrovinblastine, the immediate precursor of vinblastine. Moreover, first generation (T1) lines exhibited comparable transcript and metabolite levels to that of T0 lines. In addition, transgenic lines displayed normal growth similar to wild-type plants indicating that the bifunctional G(G)PPS enhanced flux toward both primary and secondary metabolism. These results revealed that improved availability of early precursors for terpene moiety biosynthesis enhanced production of MIAs in C. roseus at the whole plant level. This is the first report demonstrating enhanced accumulation of monomeric and dimeric MIAs including root MIA ajmalicine in C. roseus through transgenic approaches.

De Novo Sequencing and Analysis of Lemongrass Transcriptome Provide First Insights into the Essential Oil Biosynthesis of Aromatic Grasses.

Aromatic grasses of the genus Cymbopogon (Poaceae family) represent unique group of plants that produce diverse composition of monoterpene rich essential oils, which have great value in flavor, fragrance, cosmetic, and aromatherapy industries. Despite the commercial importance of these natural aromatic oils, their biosynthesis at the molecular level remains unexplored. As the first step toward understanding the essential oil biosynthesis, we performed de novo transcriptome assembly and analysis of C. flexuosus (lemongrass) by employing Illumina sequencing. Mining of transcriptome data and subsequent phylogenetic analysis led to identification of terpene synthases, pyrophosphatases, alcohol dehydrogenases, aldo-keto reductases, carotenoid cleavage dioxygenases, alcohol acetyltransferases, and aldehyde dehydrogenases, which are potentially involved in essential oil biosynthesis. Comparative essential oil profiling and mRNA expression analysis in three Cymbopogon species (C. flexuosus, aldehyde type; C. martinii, alcohol type; and C. winterianus, intermediate type) with varying essential oil composition indicated the involvement of identified candidate genes in the formation of alcohols, aldehydes, and acetates. Molecular modeling and docking further supported the role of identified protein sequences in aroma formation in Cymbopogon. Also, simple sequence repeats were found in the transcriptome with many linked to terpene pathway genes including the genes potentially involved in aroma biosynthesis. This work provides the first insights into the essential oil biosynthesis of aromatic grasses, and the identified candidate genes and markers can be a great resource for biotechnological and molecular breeding approaches to modulate the essential oil composition.

Stress-Induced Accumulation of DcAOX1 and DcAOX2a Transcripts Coincides with Critical Time Point for Structural Biomass Prediction in Carrot Primary Cultures (Daucus carota L.).

Stress-adaptive cell plasticity in target tissues and cells for plant biomass growth is important for yield stability. In vitro systems with reproducible cell plasticity can help to identify relevant metabolic and molecular events during early cell reprogramming. In carrot, regulation of the central root meristem is a critical target for yield-determining secondary growth. Calorespirometry, a tool previously identified as promising for predictive growth phenotyping has been applied to measure the respiration rate in carrot meristem. In a carrot primary culture system (PCS), this tool allowed identifying an early peak related with structural biomass formation during lag phase of growth, around the 4th day of culture. In the present study, we report a dynamic and correlated expression of carrot AOX genes (DcAOX1 and DcAOX2a) during PCS lag phase and during exponential growth. Both genes showed an increase in transcript levels until 36 h after explant inoculation, and a subsequent down-regulation, before the initiation of exponential growth. In PCS growing at two different temperatures (21°C and 28°C), DcAOX1 was also found to be more expressed in the highest temperature. DcAOX genes' were further explored in a plant pot experiment in response to chilling, which confirmed the early AOX transcript increase prior to the induction of a specific anti-freezing gene. Our findings point to DcAOX1 and DcAOX2a as being reasonable candidates for functional marker development related to early cell reprogramming. While the genomic sequence of DcAOX2a was previously described, we characterize here the complete genomic sequence of DcAOX1.