Evolution of TPS20-related terpene synthases influences chemical diversity in the glandular trichomes of the wild tomato relative Solanum habrochaites

A systematic screen of volatile terpene production in the glandular trichomes of 79 accessions of Solanum habrochaites was conducted and revealed the presence of 21 mono‐ and sesquiterpenes that exhibit a range of qualitative and quantitative variation. Hierarchical clustering identified distinct terpene phenotypic modules with shared patterns of terpene accumulation across accessions. Several terpene modules could be assigned to previously identified terpene synthase (TPS) activities that included members of the TPS‐e/f subfamily that utilize the unusual cis‐prenyl diphosphate substrates neryl diphosphate and 2z,6z‐farnesyl diphosphate. DNA sequencing and in vitro enzyme activity analysis of TPS‐e/f members from S. habrochaites identified three previously unassigned enzyme activities that utilize these cisoid substrates. These produce either the monoterpenes α‐pinene and limonene, or the sesquiterpene 7‐epizingiberene, with the in vitro analyses that recapitulated the trichome chemistry found in planta. Comparison of the distribution of S. habrochaites accessions with terpene content revealed a strong preference for the presence of particular TPS20 alleles at distinct geographic locations. This study reveals that the unusually high intra‐specific variation of volatile terpene synthesis in glandular trichomes of S. habrochaites is due at least in part to evolution at the TPS20 locus.     

The tomato terpene synthase gene family

Compounds of the terpenoid class play numerous roles in the interactions of plants with their environment, such as attracting pollinators and defending the plant against pests. We show here that the genome of cultivated tomato (Solanum lycopersicum) contains 44 terpene synthase (TPS) genes, including 29 that are functional or potentially functional. Of these 29 TPS genes, 26 were expressed in at least some organs or tissues of the plant. The enzymatic functions of eight of the TPS proteins were previously reported, and here we report the specific in vitro catalytic activity of 10 additional tomato terpene synthases. Many of the tomato TPS genes are found in clusters, notably on chromosomes 1, 2, 6, 8, and 10. All TPS family clades previously identified in angiosperms are also present in tomato. The largest clade of functional TPS genes found in tomato, with 12 members, is the TPS-a clade, and it appears to encode only sesquiterpene synthases, one of which is localized to the mitochondria, while the rest are likely cytosolic. A few additional sesquiterpene synthases are encoded by TPS-b clade genes. Some of the tomato sesquiterpene synthases use z,z-farnesyl diphosphate in vitro as well, or more efficiently than, the e,e-farnesyl diphosphate substrate. Genes encoding monoterpene synthases are also prevalent, and they fall into three clades: TPS-b, TPS-g, and TPS-e/f. With the exception of two enzymes involved in the synthesis of ent-kaurene, the precursor of gibberellins, no other tomato TPS genes could be demonstrated to encode diterpene synthases so far.     

The evolution of foliar terpene diversity in Myrtaceae

Plant terpenes play many roles in natural systems, from altering plant–animal interactions, to altering the local abiotic environment. Additionally, many industries depend on terpenes. For example, commercially used essential oils, including tea tree oil and lavender oil, are a mixture of terpenes. Many species of the family Myrtaceae form a key resource for these industries due to the high concentration of terpenes found predominately in their leaves. The frequency of chemotypic differences within many species and populations can lead to costly errors in industry. Terpene diversity in Myrtaceae is driven by variation in the terpene synthase enzymes, which catalyse the conversion a few common substrates into thousands of terpene structures. We review terpene diversity within and between species of Myrtaceae and relate this to variation in the terpene synthase enzymes to reconstruct the evolution of foliar terpene diversity in Myrtaceae. We found that (1) high inter- and intra-species variation exists in terpene profile and that α-pinene the most likely ancestral foliar terpene, and (2) that high concentration of 1,8-cineole (a compound which is regarded as the signature compound of Myrtaceae) is limited to just four Myrtaceae sub-families. We suggest that the terpene synthase enzymes do not limit terpene diversity in this family and variation in these enzymes suggests a mode of enzymatic evolution that could lead to high 1,8-cineole production. Our analysis highlights the need to standardise methods for collecting and reporting foliar terpene data, and we discuss some methods and issues here. Although there are many gaps in the published data, our large scale analysis using the results of many studies, shows the value of a family wide analysis for understanding both the evolution and industrial potential of terpene-producing plants.     

The Eucalyptus terpene synthase gene family

Background

Terpenoids are abundant in the foliage of Eucalyptus, providing the characteristic smell as well as being valuable economically and influencing ecological interactions. Quantitative and qualitative inter- and intra- specific variation of terpenes is common in eucalypts.

Results

The genome sequences of Eucalyptus grandis and E. globulus were mined for terpene synthase genes (TPS) and compared to other plant species. We investigated the relative expression of TPS in seven plant tissues and functionally characterized five TPS genes from E. grandis. Compared to other sequenced plant genomes, Eucalyptus grandis has the largest number of putative functional TPS genes of any sequenced plant. We discovered 113 and 106 putative functional TPS genes in E. grandis and E. globulus, respectively. All but one TPS from E. grandis were expressed in at least one of seven plant tissues examined. Genomic clusters of up to 20 genes were identified. Many TPS are expressed in tissues other than leaves which invites a re-evaluation of the function of terpenes in Eucalyptus.

Conclusions

Our data indicate that terpenes in Eucalyptus may play a wider role in biotic and abiotic interactions than previously thought. Tissue specific expression is common and the possibility of stress induction needs further investigation. Phylogenetic comparison of the two investigated Eucalyptus species gives insight about recent evolution of different clades within the TPS gene family. While the majority of TPS genes occur in orthologous pairs some clades show evidence of recent gene duplication, as well as loss of function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1598-x) contains supplementary material, which is available to authorized users.     

Reduced terpene levels in cottonseed add food to fiber

Using RNA interference (RNAi) technology, the levels of a toxic phytoprotectant have recently been reduced specifically in the seeds of cotton to generate a novel dual-purpose crop. By engineering an endogenous terpene pathway, there is now the exciting potential for an added-value, genetically modified crop with the cash value of the fiber supported by the improved nutritional value and expanded food and feed use for the cottonseed, which is normally a low-value by-product.     

Variation in leaf oil terpene composition of Sitka spruce

The composition of the leaf oil of Sitka spruce was determined and the quantitative variation within trees, within populations and amongst 3 southern, 5 central and 2 northern populations was recorded. No regional or geographic differences were found. In addition to the relatively large amounts of myrcene, piperitone, 1:8-cineole, and relatively low camphene and bornyl acetate percentages, the apparently unique isoamyl and isopentenyl isovalerate may serve to distinguish hybrids in areas of introgression with white spruce.     

Lipase-catalyzed synthesis of terpene esters by transesterification in n-hexane

Summary Short chain fatty acid esters of geraniol and citronellol were synthesized by lipase-catalyzèd transesterification with yields as high as 98% molar conversion. Triacylglycerols were the best substrates and immobilized Candida antarctica lipase, SP435 gave the highest overall yields. The lipases tested successfully accommodated novel acyl donors such as isopropenyl acetate and glycidyl butyrate.     

Progress in terpene synthesis strategies through engineering of Saccharomyces cerevisiae

Terpenes are natural products with a remarkable diversity in their chemical structures and they hold a significant market share commercially owing to their distinct applications. These potential molecules are usually derived from terrestrial plants, marine and microbial sources. In vitro production of terpenes using plant tissue culture and plant metabolic engineering, although receiving some success, the complexity in downstream processing because of the interference of phenolics and product commercialization due to regulations that are significant concerns. Industrial workhorses’ viz., Escherichia coli and Saccharomyces cerevisiae have become microorganisms to produce non-native terpenes in order to address critical issues such as demand-supply imbalance, sustainability and commercial viability. S. cerevisiae enjoys several advantages for synthesizing non-native terpenes with the most significant being the compatibility for expressing cytochrome P450 enzymes from plant origin. Moreover, achievement of high titers such as 40?g/l of amorphadiene, a sesquiterpene, boosts commercial interest and encourages the researchers to envisage both molecular and process strategies for developing yeast cell factories to produce these compounds. This review contains a brief consideration of existing strategies to engineer S. cerevisiae toward the synthesis of terpene molecules. Some of the common targets for synthesis of terpenes in S. cerevisiae are as follows: overexpression of tHMG1, ERG20, upc2-1 in case of all classes of terpenes; repression of ERG9 by replacement of the native promoter with a repressive methionine promoter in case of mono-, di- and sesquiterpenes; overexpression of BTS1 in case of di- and tetraterpenes. Site-directed mutagenesis such as Upc2p (G888A) in case of all classes of terpenes, ERG20p (K197G) in case of monoterpenes, HMG2p (K6R) in case of mono-, di- and sesquiterpenes could be some generic targets. Efforts are made to consolidate various studies (including patents) on this subject to understand the similarities, to identify novel strategies and to contemplate potential possibilities to build a robust yeast cell factory for terpene or terpenoid production. Emphasis is not restricted to metabolic engineering strategies pertaining to sterol and mevalonate pathway, but also other holistic approaches for elsewhere exploitation in the S. cerevisiae genome are discussed. This review also focuses on process considerations and challenges during the mass production of these potential compounds from the engineered strain for commercial exploitation.     

Induction of terpene biosynthesis in dinoflagellate symbionts of Caribbean gorgonians

This report describes a series of experiments designed to determine if terpene biosynthesis is inducible in two families of marine terpenes, pseudopterosins from the gorgonian coral Pseudopterogorgia elisabethae and fuscol from Eunicea fusca. Since we have recently shown that terpene biosynthesis is not under the control of the invertebrate host, but rather occurs within a dinoflagellate preparation, we examined the terpene content of the dinoflagellate symbiont following a decrease in UV/vis radiation as well as in response to the addition of methyl jasmonate, salicylic acid and gibberellic acid. We demonstrated that pseudopterosin and fuscol biosynthesis can be markedly increased through the addition of the plant bioactive substances. We also demonstrated that, while the terpene content of P. elisabethae increases in response to decreased UV/vis light, this is due primarily to an increase in the concentration of the dinoflagellate rather than simply an induction of terpene biosynthesis.     

Effects of light on terpene hydrocarbon synthesis in Pinus pinaster

The biosynthesis of terpene hydrocarbons has been investigated in maritime pine (Pinus pinaster Ait.) seedling primary leaves under light and darkness and with different precursors. Impossible in darkness, the synthesis of monoterpenes (mainly α- and β-pinene) is strongly activated by light. Only ¹⁴C-carbonate and ¹⁴C-acetate can be incorporated into monoterpenes. Activation by light is comparatively much more effective for seedling leaves previously cultivated under short days than in leaves from seedlings given long days. The spectral bands which are efficient for the synthesis of monoterpenes are located around 480 and 685 nm with ¹⁴C-carbonate and 480 and 630 nm with l-¹⁴C-acetate. Furthermore, this light activation does not occur if leaf pieces instead of whole leaves are used for the incorporation experiments. When 2-¹⁴C-mevalonic acid and 1-¹⁴C-isopentenyl pyrosphosphate are applied as precursors, no radioactivity is recorded in monoterpene hydrocarbons even after light exposures. In contrast, sesquiterpene hydrocarbons (caryophyllene and humulene) are easily synthesized under light or darkness in intact or fragmented leaves from the different precursors of photosynthetic or exogenous origin. From these results the compartmentalization in the synthesis of C₁₀ and C₁₅ hydrocarbons appears clear. There is a metabolic cooperation between the photosynthetic tissues and the specific site of elaboration of C₁₀ hydrocarbons, which site is located in the parts where the epithelial cells of resin ducts are functional. The synthesis of sesquiterpene hydrocarbons takes place in the whole leaf without activation by light.     

高通量技术在萜类合成酶筛选中的应用

萜类化合物种类繁多,生物活性多样,在食品、药品与化妆品等行业中具有广泛的应用。萜类化合物多来源于植物,然而随着合成生物学的快速发展,相较于传统的天然植物提取与化学合成方法,利用工程微生物进行萜类化合物异源合成的方法显得更为经济与环保。萜类合成酶的催化活性及合成产物的结构特性是萜类化合物异源合成的关键。通过蛋白定向进化与理性设计可以有针对性地优化萜类合成酶的催化性能及产物专一性,但该方案需要一个特异的筛选方法来实现蛋白突变体库的高通量筛选。近年来,一系列高通量筛选方法的建立使得萜类合成酶的筛选变得更加灵敏与高效。本文对近期建立的萜类合成酶高通量筛选方法进行了综述,简要概述了各种筛选方法的基本原理与优缺点,并对高通量筛选技术在萜类合成酶改造中的应用做出了展望。