BACKGROUND: Rose myrtle (Rhodomyrtus tomentosa (Ait.) Hassk), is an evergreen shrub species belonging to the family Myrtaceae, which is enriched with bioactive volatiles (α-pinene and β-caryophyllene) with medicinal and industrial applications. However, the mechanism underlying the volatile accumulation in the rose myrtle is still unclear.
RESULTS: Here, we present a chromosome-level genomic assembly of rose myrtle (genome size = 466 Mb, scaffold N50 = 43.7 Mb) with 35,554 protein-coding genes predicted. Through comparative genomic analysis, we found that gene expansion and duplication had a potential contribution to the accumulation of volatile substances. We proposed that the action of positive selection was significantly involved in volatile accumulation. We identified 43 TPS genes in R. tomentosa. Further transcriptomic and TPS gene family analyses demonstrated that the distinct gene subgroups of TPS may contribute greatly to the biosynthesis and accumulation of different volatiles in the Myrtle family of shrubs and trees. The results suggested that the diversity of TPS-a subgroups led to the accumulation of special sesquiterpenes in different plants of the Myrtaceae family.
CONCLUSIONS: The high quality chromosome-level rose myrtle genome and the comparative analysis of TPS gene family open new avenues for obtaining a higher commercial value of essential oils in medical plants.

The phlebotomine sandfly, Lutzomyia longipalpis, a major vector of the Leishmania parasite, uses terpene pheromones to attract conspecifics for mating. Examination of the L. longipalpis genome revealed a putative terpene synthase (TPS), which-upon heterologous expression in, and purification from, Escherichia coli-yielded a functional enzyme. The TPS, termed LlTPS, converted geranyl diphosphate (GPP) into a mixture of monoterpenes with low efficiency, of which β-ocimene was the major product. (E,E)-farnesyl diphosphate (FPP) principally produced small amounts of (E)-β-farnesene, while (Z,E)- and (Z,Z)-FPP yielded a mixture of bisabolene isomers. None of these mono- and sesquiterpenes are known volatiles of L. longipalpis. Notably, however, when provided with (E,E,E)-geranylgeranyl diphosphate (GGPP), LlTPS gave sobralene as its major product. This diterpene pheromone is released by certain chemotypes of L. longipalpis, in particular those found in the Ceará state of Brazil. Minor diterpene components were also seen as products of the enzyme that matched those seen in a sandfly pheromone extract.

Enzymatic terpene functionalization is an essential part of plant secondary metabolite diversity. Within this, multiple terpene-modifying enzymes are required to enable the chemical diversity of volatile compounds essential in plant communication and defense. This work sheds light on the differentially transcribed genes within Caryopteris × clandonensis that are capable of functionalizing cyclic terpene scaffolds, which are the product of terpene cyclase action. The available genomic reference was subjected to further improvements to provide a comprehensive basis, where the number of contigs was minimized. RNA-Seq data of six cultivars, Dark Knight, Grand Bleu, Good as Gold, Hint of Gold, Pink Perfection, and Sunny Blue, were mapped on the reference, and their distinct transcription profile investigated. Within this data resource, we detected interesting variations and additionally genes with high and low transcript abundancies in leaves of Caryopteris × clandonensis related to terpene functionalization. As previously described, different cultivars vary in their modification of monoterpenes, especially limonene, resulting in different limonene-derived molecules. This study focuses on predicting the cytochrome p450 enzymes underlying this varied transcription pattern between investigated samples. Thus, making them a reasonable explanation for terpenoid differences between these plants. Furthermore, these data provide the basis for functional assays and the verification of putative enzyme activities.

BACKGROUND: Flooding is among the most severe abiotic stresses in plant growth and development. The mechanism of submergence tolerance of cotton in response to submergence stress is unknown.
RESULTS: The transcriptome results showed that a total of 6,893 differentially expressed genes (DEGs) were discovered under submergence stress. Gene Ontology (GO) enrichment analysis showed that DEGs were involved in various stress or stimulus responses. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that DEGs related to plant hormone signal transduction, starch and sucrose metabolism, glycolysis and the biosynthesis of secondary metabolites were regulated by submergence stress. Eight DEGs related to ethylene signaling and 3 ethylene synthesis genes were identified in the hormone signal transduction. For respiratory metabolism, alcohol dehydrogenase (ADH, GH_A02G0728) and pyruvate decarboxylase (PDC, GH_D09G1778) were significantly upregulated but 6-phosphofructokinase (PFK, GH_D05G0280), phosphoglycerate kinase (PGK, GH_A01G0945 and GH_D01G0967) and sucrose synthase genes (SUS, GH_A06G0873 and GH_D06G0851) were significantly downregulated in the submergence treatment. Terpene biosynthetic pathway-related genes in the secondary metabolites were regulated in submergence stress.
CONCLUSIONS: Regulation of terpene biosynthesis by respiratory metabolism may play a role in enhancing the tolerance of cotton to submergence under flooding. Our findings showed that the mevalonate pathway, which occurs in the cytoplasm of the terpenoid backbone biosynthesis pathway (ko00900), may be the main response to submergence stress.

Liquid-liquid phase separation forms condensates that feature a highly concentrated liquid phase, a defined yet dynamic boundary, and dynamic exchange at and across the boundary. Phase transition drives the formation of dynamic multienzyme complexes in cells, and understanding how phase separation regulates multienzyme catalysis may need the help of in vitro investigations. Recently we have constructed synthetic versions of multienzyme biosynthetic systems by assembling enzymes in protein condensates. Here, we describe the methods for checking the enzyme assembly using fluorescent microscopy and centrifugation assay. We further provide steps for analysis of the cascade enzyme catalytic efficiencies inside the condensates, using enzymes from terpene biosynthesis pathway.

Azadirachta indica (neem), an evergreen tree of the Meliaceae family, is a source of the potent biopesticide azadirachtin. The lack of a chromosome-level assembly impedes an in-depth understanding of its genome architecture and the comparative genomic analysis of A. indica. Here, a high-quality genome assembly of A. indica was constructed using a combination of data from Illumina, PacBio, and Hi-C technology, which is the first chromosome-scale genome assembly of A. indica. Based on the length of our assembly, the genome size of A. indica is estimated to be 281 Mb anchored to 14 chromosomes (contig N50 = 6 Mb and scaffold N50 = 19 Mb). The genome assembly contained 115 Mb repetitive elements and 25,767 protein-coding genes. Evolutional analysis revealed that A. indica didn\'t experience any whole-genome duplication (WGD) event after the core eudicot γ event, but some genes and genome segment might likely experienced recent duplications. The secondary metabolite clusters, TPS genes, and CYP genes were also identified. Comparative genomic analysis revealed that most of the A. indica-specific TPS genes and CYP genes were located on the terpene-related clusters on chromosome 13. It is suggested that chromosome 13 may play an important role in the specific terpene biosynthesis of A. indica. The gene duplication events may be responsible for the terpene biosynthesis expansion in A. indica. The genomic dataset and genomic analysis created for A. indica will shed light on terpene biosynthesis in A. indica and facilitate comparative genomic research of the family Meliaceae.

Terpenoids are a large group of secondary metabolites with broad industrial applications. Engineering cyanobacteria is an attractive route for the sustainable production of commodity terpenoids. Currently, a major obstacle lies in the low productivity attained in engineered cyanobacterial strains. Traditional metabolic engineering to improve pathway kinetics has led to limited success in enhancing terpenoid productivity. In this study, we reveal thermodynamics as the main determinant for high limonene productivity in cyanobacteria. Through overexpressing the primary sigma factor, a higher photosynthetic rate was achieved in an engineered strain of Synechococcus elongatus PCC 7942. Computational modeling and wet lab analyses showed an increased flux toward both native carbon sink glycogen synthesis and the non-native limonene synthesis from photosynthate output. On the other hand, comparative proteomics showed decreased expression of terpene pathway enzymes, revealing their limited role in determining terpene flux. Lastly, growth optimization by enhancing photosynthesis has led to a limonene titer of 19 mg/L in 7 days with a maximum productivity of 4.3 mg/L/day. This study highlights the importance of enhancing photosynthesis and substrate input for the high productivity of secondary metabolic pathways, providing a new strategy for future terpenoid engineering in phototrophs.

Sindora glabra is an economically important tree that produces abundant oleoresin in the trunk. Here, we present a high-quality chromosome-scale assembly of S. glabra genome by combining Illumina HiSeq, Pacific Biosciences sequencing, and Hi-C technologies. The size of S. glabra genome was 1.11 Gb, with a contig N50 of 1.27 Mb and 31,944 predicted genes. This is the first sequenced genome of the subfamily Caesalpinioideae. As a sister taxon to Papilionoideae, S. glabra underwent an ancient genome triplication shared by core eudicots and further whole-genome duplication shared by early-legume in the last 73.3 million years. S. glabra harbors specific genes and expanded genes largely involved in stress responses and biosynthesis of secondary metabolites. Moreover, 59 terpene backbone biosynthesis genes and 64 terpene synthase genes were identified, which together with co-expressed transcription factors could contribute to the diversity and specificity of terpene compounds and high terpene content in S. glabra stem. In addition, 63 disease resistance NBS-LRR genes were found to be unique in S. glabra genome and their expression levels were correlated with the accumulation of terpene profiles, suggesting potential defense function of terpenes in S. glabra. These together provide new resources for understanding genome evolution and oleoresin production.

Serrulatane diterpenoids are natural products found in plants from a subset of genera within the figwort family (Scrophulariaceae). Many of these compounds have been characterized as having anti-microbial properties and share a common diterpene backbone. One example, leubethanol from Texas sage (Leucophyllum frutescens) has demonstrated activity against multi-drug-resistant tuberculosis. Leubethanol is the only serrulatane diterpenoid identified from this genus; however, a range of such compounds have been found throughout the closely related Eremophila genus. Despite their potential therapeutic relevance, the biosynthesis of serrulatane diterpenoids has not been previously reported. Here we leverage the simple product profile and high accumulation of leubethanol in the roots of L. frutescens and compare tissue-specific transcriptomes with existing data from Eremophila serrulata to decipher the biosynthesis of leubethanol. A short-chain cis-prenyl transferase (LfCPT1) first produces the rare diterpene precursor nerylneryl diphosphate, which is cyclized by an unusual plastidial terpene synthase (LfTPS1) into the characteristic serrulatane diterpene backbone. Final conversion to leubethanol is catalyzed by a cytochrome P450 (CYP71D616) of the CYP71 clan. This pathway documents the presence of a short-chain cis-prenyl diphosphate synthase, previously only found in Solanaceae, which is likely involved in the biosynthesis of other known diterpene backbones in Eremophila. LfTPS1 represents neofunctionalization of a compartment-switching terpene synthase accepting a novel substrate in the plastid. Biosynthetic access to leubethanol will enable pathway discovery to more complex serrulatane diterpenoids which share this common starting structure and provide a platform for the production and diversification of this class of promising anti-microbial therapeutics in heterologous systems.

Terpenoids are the largest and structurally most diverse class of natural products. They possess potent and specific biological activity in multiple assays and against diseases, including cancer and malaria as notable examples. Although the number of characterized terpenoid molecules is huge, our knowledge of how they are biosynthesized is limited, particularly when compared to the well-studied thiotemplate assembly lines. Bacteria have only recently been recognized as having the genetic potential to biosynthesize a large number of complex terpenoids, but our current ability to associate genetic potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This functional promiscuity of terpene biosynthetic pathways renders terpene biosynthesis susceptible to rational pathway engineering using the latest developments in the field of synthetic biology. These engineered pathways will not only facilitate the rational creation of both known and novel terpenoids, their development will deepen our understanding of a significant branch of biosynthesis. The biosynthetic insights gained will likely empower a greater degree of engineering proficiency for non-natural terpene biosynthetic pathways and pave the way towards the biotechnological production of high value terpenoids.