Oxetane Ring Formation in Taxol Biosynthesis Is Catalyzed by a Bifunctional Cytochrome P450 Enzyme.

Taxol is a potent drug used in various cancer treatments. Its complex structure has prompted extensive research into its biosynthesis. However, certain critical steps, such as the formation of the oxetane ring, which is essential for its activity, have remained unclear. Previous proposals suggested that oxetane formation follows the acetylation of taxadien-5α-ol. Here, we proposed that the oxetane ring is formed by cytochrome P450-mediated oxidation events that occur prior to C5 acetylation. To test this hypothesis, we analyzed the genomic and transcriptomic information for Taxus species to identify cytochrome P450 candidates and employed two independent systems, yeast (Saccharomyces cerevisiae) and plant (Nicotiana benthamiana), for their characterization. We revealed that a single enzyme, CYP725A4, catalyzes two successive epoxidation events, leading to the formation of the oxetane ring. We further showed that both taxa-4(5)-11(12)-diene (endotaxadiene) and taxa-4(20)-11(12)-diene (exotaxadiene) are precursors to the key intermediate, taxologenic oxetane, indicating the potential existence of multiple routes in the Taxol pathway. Thus, we unveiled a long-elusive step in Taxol biosynthesis.

Biosynthesis and biotechnological production of the anti-obesity agent celastrol.

Obesity is a major health risk still lacking effective pharmacological treatment. A potent anti-obesity agent, celastrol, has been identified in the roots of Tripterygium wilfordii. However, an efficient synthetic method is required to better explore its biological utility. Here we elucidate the 11 missing steps for the celastrol biosynthetic route to enable its de novo biosynthesis in yeast. First, we reveal the cytochrome P450 enzymes that catalyse the four oxidation steps that produce the key intermediate celastrogenic acid. Subsequently, we show that non-enzymatic decarboxylation-triggered activation of celastrogenic acid leads to a cascade of tandem catechol oxidation-driven double-bond extension events that generate the characteristic quinone methide moiety of celastrol. Using this acquired knowledge, we have developed a method for producing celastrol starting from table sugar. This work highlights the effectiveness of combining plant biochemistry with metabolic engineering and chemistry for the scalable synthesis of complex specialized metabolites.

A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis.

The ginkgo tree (Ginkgo biloba) is considered a living fossil due to its 200 million year's history under morphological stasis. Its resilience is partly attributed to its unique set of specialized metabolites, in particular, ginkgolides and bilobalide, which are chemically complex terpene trilactones. Here, we use a gene cluster-guided mining approach in combination with co-expression analysis to reveal the primary steps in ginkgolide biosynthesis. We show that five multifunctional cytochrome P450s with atypical catalytic activities generate the tert-butyl group and one of the lactone rings, characteristic of all G. biloba trilactone terpenoids. The reactions include scarless C-C bond cleavage as well as carbon skeleton rearrangement (NIH shift) occurring on a previously unsuspected intermediate. The cytochrome P450s belong to CYP families that diversifies in pre-seed plants and gymnosperms, but are not preserved in angiosperms. Our work uncovers the early ginkgolide pathway and offers a glance into the biosynthesis of terpenoids of the Mesozoic Era.

Silencing of ascorbate oxidase results in reduced growth, altered ascorbic acid levels and ripening pattern in melon fruit.

Ascorbate oxidase (AO, EC 1.10.3.3) is a copper-containing enzyme localized at the apoplast, where it catalyzes the oxidation of ascorbic acid (AA) to dehydroascorbic acid (DHA) via monodehydroascorbic acid (MDHA) intermediate. Despite it has been extensively studied, no biological roles have been definitively ascribed. To understand the role of AO in plant metabolism, fruit growth and physiology, we suppressed AO expression in melon (Cucumis melo L.) fruit. Reduction of AO activity increased AA content in melon fruit, which is the result of repression of AA oxidation and simultaneous induction of certain biosynthetic and recycling genes. As a consequence, ascorbate redox state was altered in the apoplast. Interestingly, transgenic melon fruit displayed increased ethylene production rate coincided with elevated levels of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO, EC 1.14.17.4) activity and gene expression, which might contribute to earlier ripening. Moreover, AO suppressed transgenic melon fruit exhibited a dramatic arrest in fruit growth, due to a simultaneous decrease in fruit cell size and in plasmalemma (PM) ATPase activity. All the above, support for the first time, the in vivo AO participation in the rapid fruit growth of Cucurbitaceae and further suggest an alternative route for AA increase in ripening fruit.

Nerylneryl diphosphate is the precursor of serrulatane, viscidane and cembrane-type diterpenoids in Eremophila species.

BACKGROUND: Eremophila R.Br. (Scrophulariaceae) is a diverse genus of plants with species distributed across semi-arid and arid Australia. It is an ecologically important genus that also holds cultural significance for many Indigenous Australians who traditionally use several species as sources of medicines. Structurally unusual diterpenoids, particularly serrulatane and viscidane-types, feature prominently in the chemical profile of many species and recent studies indicate that these compounds are responsible for much of the reported bioactivity. We have investigated the biosynthesis of diterpenoids in three species: Eremophila lucida, Eremophila drummondii and Eremophila denticulata subsp. trisulcata. RESULTS: In all studied species diterpenoids were localised to the leaf surface and associated with the occurrence of glandular trichomes. Trichome-enriched transcriptome databases were generated and mined for candidate terpene synthases (TPS). Four TPSs with diterpene biosynthesis activity were identified: ElTPS31 and ElTPS3 from E. lucida were found to produce (3Z,7Z,11Z)-cembratrien-15-ol and 5-hydroxyviscidane, respectively, and EdTPS22 and EdtTPS4, from E. drummondii and E. denticulata subsp. trisulcata, respectively, were found to produce 8,9-dihydroserrulat-14-ene which readily aromatized to serrulat-14-ene. In all cases, the identified TPSs used the cisoid substrate, nerylneryl diphosphate (NNPP), to form the observed products. Subsequently, cis-prenyl transferases (CPTs) capable of making NNPP were identified in each species. CONCLUSIONS: We have elucidated two biosynthetic steps towards three of the major diterpene backbones found in this genus. Serrulatane and viscidane-type diterpenoids are promising candidates for new drug leads. The identification of an enzymatic route to their synthesis opens up the possibility of biotechnological production, making accessible a ready source of scaffolds for further modification and bioactivity testing.

Characterization of CYP71AX36 from Sunflower (Helianthus annuus L., Asteraceae).

Sesquiterpene lactones (STL) are a subclass of isoprenoids with many known bioactivities frequently found in the Asteraceae family. In recent years, remarkable progress has been made regarding the biochemistry of STL, and today the biosynthetic pathway of the core backbones of many STLs has been elucidated. Consequently, the focus has shifted to the discovery of the decorating enzymes that can modify the core skeleton with functional hydroxy groups. Using in vivo pathway reconstruction assays in heterologous organisms such as Saccharomyces cerevisiae and Nicotiana benthamiana, we have analyzed several cytochrome P450 enzyme genes of the CYP71AX subfamily from Helianthus annuus clustered in close proximity to one another on the sunflower genome. We show that one member of this subfamily, CYP71AX36, can catalyze the conversion of costunolide to 14-hydroxycostunolide. The catalytic activity of CYP71AX36 may be of use for the chemoenzymatic production of antileukemic 14-hydroxycostunolide derivatives and other STLs of pharmaceutical interest. We also describe the full 2D-NMR assignment of 14-hydroxycostunolide and provide all 13C chemical shifts of the carbon skeleton for the first time.

Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

Kauniolide synthase is a P450 with unusual hydroxylation and cyclization-elimination activity.

Guaianolides are an important class of sesquiterpene lactones with unique biological and pharmaceutical properties. They have been postulated to be derived from germacranolides, but for years no progress has been made in the elucidation of their biosynthesis that requires an unknown cyclization mechanism. Here we demonstrate the isolation and characterization of a cytochrome P450 from feverfew (Tanacetum parthenium), kauniolide synthase. Kauniolide synthase catalyses the formation of the guaianolide kauniolide from the germacranolide substrate costunolide. Unlike most cytochrome P450s, kauniolide synthase combines stereoselective hydroxylation of costunolide at the C3 position, with water elimination, cyclization and regioselective deprotonation. This unique mechanism of action is supported by in silico modelling and docking experiments. The full kauniolide biosynthesis pathway is reconstructed in the heterologous hosts Nicotiana benthamiana and yeast, paving the way for biotechnological production of guaianolide-type sesquiterpene lactones.

Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.

BACKGROUND: Forskolin is a high-value diterpenoid produced exclusively by the Lamiaceae plant Coleus forskohlii. Today forskolin is used pharmaceutically for its adenyl-cyclase activating properties. The limited availability of pure  forskolin is currently hindering its full utilization, thus a new environmentally friendly, scalable and sustainable strategy is needed for forskolin production. Recently, the entire biosynthetic pathway leading to forskolin was elucidated. The key steps of the pathway are catalyzed by cytochrome P450 enzymes (CYPs), which have been shown to be the limiting steps of the pathway. Here we study whether protein engineering of the substrate recognition sites (SRSs) of CYPs can improve their efficiency towards forskolin biosynthesis in yeast. RESULTS: As a proof of concept, we engineered the enzyme responsible for the first putative oxygenation step of the forskolin pathway: the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide, catalyzed by the CYP76AH15. Four CYP76AH15 variants-engineered in the SRS regions-yielded at least a twofold increase of 11-oxo-13R-manoyl oxide when expressed in yeast cells grown in microtiter plates. The highest titers (5.6-fold increase) were observed with the variant A99I, mutated in the SRS1 region. Double or triple CYP76AH15 mutant variants resulted in additional enzymes with optimized performances. Moreover, in planta CYP76AH15 can synthesize ferruginol from miltiradiene. In this work, we showed that the mutants affecting 11-oxo-13R-manoyl oxide synthesis, do not affect ferruginol production, and vice versa. The best performing variant, A99I, was utilized to reconstruct the forskolin biosynthetic pathway in yeast cells. Although these strains showed increased 11-oxo-manoyl oxide production and higher accumulation of other pathway intermediates compared to the native CYP76AH15, lower production of forskolin was observed. CONCLUSIONS: As demonstrated for CYP76AH15, site-directed mutagenesis of SRS regions of plant CYPs may be an efficient and targeted approach to increase the performance of these enzymes. Although in this work we have managed to achieve higher efficiency and specificity of the first CYP of the pathway, further work is necessary in order to increase the overall production of forskolin in yeast cells.

Phototrophic production of heterologous diterpenoids and a hydroxy-functionalized derivative from Chlamydomonas reinhardtii.

Photosynthetic microalgae harbor enormous potential as light-driven green-cell factories for sustainable bio-production of a range of natural and heterologous products such as isoprenoids. Their capacity for photosynthesis and rapid low-input growth with (sun)light and CO2 is coupled to a robust metabolic architecture structured toward the generation of isoprenoid pigments and compounds involved in light capture, electron transfer, and radical scavenging. Metabolic engineering approaches using eukaryotic green microalgae have previously been hampered mainly by low-levels of nuclear transgene expression. Here, we employed a strategy of optimized transgene design which couples codon optimization and synthetic intron spreading for the expression of heterologous plant enzymes from the algal nuclear genome. The diterpenoids casbene, taxadiene, and 13R(+) manoyl oxide were produced after expressing heterologous diterpene synthases and enzymes participating in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway which were all targeted to the algal chloroplast. Additionally, a truncated and soluble plant microsomal cytochrome P450 monooxygenase was functionally expressed and able to hydroxylate 13R(+) manoyl oxide when directed into the chloroplasts. The heterologous diterpenoids were found to be excreted from the cells and accumulate in dodecane solvent-culture overlays. It was shown that the algal cell could tolerate significant metabolic pull towards diterpenoids without loss of native pigments. Using an algal strain producing 13R(+) manoyl oxide as a model, diterpenoid production was shown to be highest in photoautotrophic cultivations using CO2 as the sole carbon source and day:night illumination cycles. Up to 80 mg 13R(+) manoyl oxide per gram cell dry mass (CDM) could be produced from C. reinhardtii in a 7 day batch cultivation with a sustained maximal productivity of 22.5 mg gcdm-1 d-1 over 3 consecutive days. Collectively the results presented here suggest that green algal cells have remarkable potential for the heterologous production of non-native isoprenoids and support the use of these hosts for (sun)light driven bioproduction concepts.

Biosynthesis of Eupatolide-A Metabolic Route for Sesquiterpene Lactone Formation Involving the P450 Enzyme CYP71DD6.

Sesquiterpene lactones are a class of natural compounds well-known for their bioactivity and are characteristic for the Asteraceae family. Most sesquiterpene lactones are considered derivatives of germacrene A acid (GAA). GAA can be stereospecifically hydroxylated by the cytochrome P450 enzymes (CYP) Lactuca sativa costunolide synthase CYP71BL2 (LsCOS) and Helianthus annuus GAA 8ß-hydroxylase CYP71BL1 (HaG8H) at C6 (in α-orientation) or C8 (in ß-orientation), respectively. Spontaneous subsequent lactonization of the resulting 6α-hydroxy-GAA leads to costunolide, whereas 8ß-hydroxy-GAA has not yet been reported to cyclize to a sesquiterpene lactone. Sunflower and related species of the Heliantheae tribe contain sesquiterpene lactones mainly derived from inunolide (7,8-cis lactone) and eupatolide (8ß-hydroxy-costunolide) precursors. However, the mechanism of 7,8-cis lactonization in general, and the 6,7-trans lactone formation in the sunflower tribe, remain elusive. Here, we show that, in plant cells, heterologous expression of CYP71BL1 leads to the formation of inunolide. Using a phylogenetic analysis of enzymes from the CYP71 family involved in sesquiterpenoid metabolism, we identified the CYP71DD6 gene, which was able to catalyze the 6,7-trans lactonization in sunflowers, using as a substrate 8ß-hydroxy-GAA. Consequently, CYP71DD6 resulted in the synthesis of eupatolide, thus called HaES ( Helianthus annuus eupatolide synthase). Thus, our study shows the entry point for the biosynthesis of two distinct types of sesquiterpene lactones in sunflowers: the 6,7-trans lactones derived from eupatolide and the 7,8-cis lactones derived from inunolide. The implications for tissue-specific localization, based on expression studies, are discussed.

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.

Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii.

Forskolin is a unique structurally complex labdane-type diterpenoid used in the treatment of glaucoma and heart failure based on its activity as a cyclic AMP booster. Commercial production of forskolin relies exclusively on extraction from its only known natural source, the plant Coleus forskohlii, in which forskolin accumulates in the root cork. Here, we report the discovery of five cytochrome P450s and two acetyltransferases which catalyze a cascade of reactions converting the forskolin precursor 13R-manoyl oxide into forskolin and a diverse array of additional labdane-type diterpenoids. A minimal set of three P450s in combination with a single acetyl transferase was identified that catalyzes the conversion of 13R-manoyl oxide into forskolin as demonstrated by transient expression in Nicotiana benthamiana. The entire pathway for forskolin production from glucose encompassing expression of nine genes was stably integrated into Saccharomyces cerevisiae and afforded forskolin titers of 40 mg/L.

Sunlight-driven Environmental Benign Production of Bioactive Natural Products with Focus on Diterpenoids and the Pathways Involved in their Formation.

Diterpenoids are high value compounds characterized by high structural complexity. They constitute the largest class of specialized metabolites produced by plants. Diterpenoids are flexible molecules able to engage in specific binding to drug targets like receptors and transporters. In this review we provide an account on how the complex pathways for diterpenoids may be elucidated. Following plant pathway discovery, the compounds may be produced in heterologous hosts like yeasts and E. coli. Environmentally contained production in photosynthetic cells like cyanobacteria, green algae or mosses are envisioned as the ultimate future production system.

Identification of PTP1B and α-Glucosidase Inhibitory Serrulatanes from Eremophila spp. by Combined use of Dual High-Resolution PTP1B and α-Glucosidase Inhibition Profiling and HPLC-HRMS-SPE-NMR.

According to the International Diabetes Federation, type 2 diabetes (T2D) has reached epidemic proportions, affecting more than 382 million people worldwide. Inhibition of protein tyrosine phosphatase-1B (PTP1B) and α-glucosidase is a recognized therapeutic approach for management of T2D and its associated complications. The lack of clinical drugs targeting PTP1B and side effects of the existing α-glucosidase drugs, emphasize the need for new drug leads for these T2D targets. In the present work, dual high-resolution PTP1B and α-glucosidase inhibition profiles of Eremophila gibbosa, E. glabra, and E. aff. drummondii "Kalgoorlie" were used for pinpointing α-glucosidase and/or PTP1B inhibitory constituents directly from the crude extracts. A subsequent targeted high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy (HPLC-HRMS-SPE-NMR) analysis and preparative-scale HPLC isolation led to identification of 21 metabolites from the three species, of which 16 were serrulatane-type diterpenoids (12 new) associated with either α-glucosidase and/or PTP1B inhibition. This is the first report of serrulatane-type diterpenoids as potential α-glucosidase and/or PTP1B inhibitors.

High-resolution PTP1B inhibition profiling combined with high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy: Proof-of-concept and antidiabetic constituents in crude extract of Eremophila lucida.

Type 2 diabetes (T2D) constituted 90% of the global 387 million diabetes cases in 2014. The enzyme protein-tyrosine phosphatase 1B (PTP1B) has been recognized as a therapeutic target for treatment of T2D and its adverse complications. With the aim of accelerating the investigation of complex natural sources, such as crude plant extracts, for potential PTP1B inhibitors, we have developed a bio-analytical platform combining high-resolution PTP1B inhibition profiling and high-performance liquid chromatography-high-resolution mass spectrometry-solid-phase extraction-nuclear magnetic resonance spectroscopy, i.e., HR-bioassay/HPLC-HRMS-SPE-NMR. Human recombinant PTP1B enzyme was used for the microplate-based PTP1B inhibition assay, which was optimized for pH and substrate concentration to be compatible with rate measurements within the 10 min incubation time. Subsequently, analytical-scale HPLC-based microfractionation followed by colorimetric microplate-based PTP1B bioassaying enabled construction of a high-resolution inhibition profile corresponding to the HPLC profile. The high-resolution PTP1B inhibition profiling was validated using an artificial mixture of known PTP1B inhibitors and non-inhibiting compounds as negative controls. Finally, a proof-of-concept study with a real sample was performed using crude ethyl acetate extract of the phytochemically hitherto unexplored plant Eremophila lucida. This led to the identification of the first viscidane type diterpene, i.e., 5-hydroxyviscida-3,14-dien-20-oic acid (9) as PTP1B inhibitor with an IC50 value of 42.0 ± 5.9 µM. In addition, a series of flavonoids, i.e., luteolin (1), dinatin (3a), tricin (3b), 3,6-dimethoxyapigenin (4), jaceidin (5), and cirsimaritin (6) as well as a cembrene diterpene, (3Z, 7E, 11Z)-15-hydroxycembra-3,7,11-trien-19-oic acid (8), were also identified for the first time from E. lucida.

Expanding the Landscape of Diterpene Structural Diversity through Stereochemically Controlled Combinatorial Biosynthesis.

Plant-derived diterpenoids serve as important pharmaceuticals, food additives, and fragrances, yet their low natural abundance and high structural complexity limits their broader industrial utilization. By mimicking the modularity of diterpene biosynthesis in plants, we constructed 51 functional combinations of class I and II diterpene synthases, 41 of which are "new-to-nature". Stereoselective biosynthesis of over 50 diterpene skeletons was demonstrated, including natural variants and novel enantiomeric or diastereomeric counterparts. Scalable biotechnological production for four industrially relevant targets was accomplished in engineered strains of Saccharomyces cerevisiae.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes. Two putative diterpene synthase genes, namely SfCPS and SfKSL, showing similarities to copalyl diphosphate synthase and kaurene synthase-like genes, respectively, were isolated and functionally characterized. Recombinant expression in Escherichia coli followed by in vitro enzyme activity assays confirmed that SfCPS is a copalyl diphosphate synthase. Coupling of SfCPS with SfKSL, both in vitro and in yeast, resulted in the synthesis miltiradiene, as confirmed by 1D and 2D NMR analyses (1H, 13C, DEPT, COSY H-H, HMQC and HMBC). Coupled transient in vivo assays of SfCPS and SfKSL in Nicotiana benthamiana further confirmed production of miltiradiene in planta. To elucidate the subsequent biosynthetic step, RNA-Seq data of S. fruticosa and R. officinalis were searched for cytochrome P450 (CYP) encoding genes potentially involved in the synthesis of the first phenolic compound in the CA pathway, ferruginol. Three candidate genes were selected, SfFS, RoFS1 and RoFS2. Using yeast and N. benthamiana expression systems, all three where confirmed to be coding for ferruginol synthases, thus revealing the enzymatic activities responsible for the first three steps leading to CA in two Lamiaceae genera.

Cytochromes P450 for terpene functionalisation and metabolic engineering.

Plants have evolved the capacity to produce a striking array of specialised metabolites. Terpenoids are the oldest and most diverse class of such compounds and have attracted interest for industrial and pharmaceutical applications. The development of biotechnological alternatives for their production is the focus of intense research. Photosynthetic systems provide new strategies for autotrophic metabolic engineering. Focusing on cytochromes P450, involved in the functionalisation of the core terpene molecules, this review highlights the latest approaches in this field and looks towards recent discoveries that have the potential to shape the future of terpenoid bioengineering.

Tomato fruit chromoplasts behave as respiratory bioenergetic organelles during ripening.

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b6f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c6 during ripening suggests that it could act as electron acceptor of the cytochrome b6f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages.