New sweet diterpene glucosides from Stevia rebaudiana

From the leaves of Stevia rebaudiana, two new sweet glucosides, rebaudiosides A and B, were isolated besides the known glucosides, stevioside and steviolbioside. On the basis of IR, MS, ¹H and ¹³C NMR as well as chemical evidences, the structure of rebaudioside B was assigned as 13-O-[β-glucosyl(1-2)-β-glucosyl(1-3)]-β-glucosyl-steviol and rebaudioside A was formulated as its β-glucosyl ester.     

Diterpene cyclases and the nature of the isoprene fold

The structures and mechanism of action of many terpene cyclases are known, but no structures of diterpene cyclases have yet been reported. Here, we propose structural models based on bioinformatics, site‐directed mutagenesis, domain swapping, enzyme inhibition, and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ～20 α‐helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ‐domain structure together with a third, α‐domain, forming an αβγ structure, and in H⁺‐initiated cyclases, there is an EDxxD‐like Mg²⁺/diphosphate binding motif located in the γ‐domain. The results support a new view of terpene cyclase structure and function and suggest evolution from ancient (βγ) bacterial triterpene cyclases to (βγ) bacterial and thence to (αβγ) plant diterpene cyclases. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Casbene: An anti-fungal diterpene produced in cell-free extracts of Ricinus communis seedlings

The capacity of cell-free extracts of 2·5-day-old castor bean seedlings for synthesis of casbene from mevalonic acid were compared for seedings which had been germinated under sterile conditions and seedlings which were intentionally exposed to fungal cultures. Extracts from seedlings exposed to cultures of Rhizopus stolonifer, Aspergillus niger or Fusarium moniliforme produced much higher levels of casbene than extracts from sterile controls; the initial rates of casbene synthesis were 20–40 times higher in the extracts of seedlings that had been exposed to fungus. Although some variation in the capacity for synthesis of other diterpene hydrocarbons from mevalonic acid was seen in the two types of extracts, no consistent or striking stimulation in the synthesis of any of these was noted under these conditions of exposure of the seedlings to fungi. The potato-dectrose agar used as a fungal growth medium did not itself evoke the increase in casbene synthesis. Intact mycelia and cell-free extracts of mycelia of Rhizopus stolonifer gave no indication of diterpene biosynthesis from mevalonic acid. Purified casbene at concentrations of 10 μg ml⁻¹ or greater retarded the development of A. niger on potato-dextrose medium. Casbene was also found to inhibit the endogenous and gibberellic acid-stimulated growth of leaf sheaths of the dwarf-5 mutant of Zea mays and of the growth of the K-12 strain of Escherichia coli on glucose-minerals medium. It is suggested that casbene may serve the castor bean plant as a phytoalexin.     

De novo production of versatile oxidized kaurene diterpenes in Escherichia coli

The oxidized kaurene (Ox-Kau) compounds are the core structures of many important diterpenoids with biological activities and economical values. However, easy access to diverse Ox-Kau products is still limited by low natural abundance, and large-scale manufacture remain challenging due to lack of proper heterologous production. To achieve an abundant source alternative to natural extracts, we here report a highly effective Escherichia coli-based platform for the de novo production of multiple Ox-Kau molecules from simple carbon source. Pathway optimization in prokaryotic cells through modification of transmembrane CYP450 oxidases, cytochrome b₅ co-expression and AlphaFold-based protein engineering improved a 50-fold yield of steviol (1.07 g L⁻¹), a key intermediate in the kaurenoid biosynthesis. Combinatorial biosynthetic strategy further led to a series of oxidized derivatives (20–600 mg L⁻¹) with rich oxygenated functional groups on C3, C7, C16 and C19 previously hard to be introduced. Our engineered strains not only laid a foundation for realizing the industrial fermentation of gram-scale ent-kaurene diterpenoids, but also provided a reliable platform for characterization and utilization of kaurene-modifying oxidases, which may generate naturally rare or unnatural ent-kaurenoids with potential bioactivity.     

The in vivo synthesis of plant sesquiterpenes by Escherichia coli.

Three plant genes encoding (+)-delta-cadinene, 5-epi-aristolochene, and vetispiradiene cyclases were expressed in Escherichia coli to evaluate the potential of this bacterium to synthesize sesquiterpenes in vivo. Various growth temperatures, carbon sources, and host strains were examined to optimize terpene production. The highest levels of sesquiterpene production occurred when the enzymes were expressed in strain DH5alpha from the trc promoter (Ptrc) of the high-copy plasmidpTrc99A in M9 medium supplemented with 0.2% (v/v) glycerol at 30 degrees C for 5-epi-aristolochene and vetispiradiene and 37 degrees C for (+)-delta-cadinene. The highest concentrations of sesquiterpenes observed were 10.3 microg of (+)-delta-cadinene, 0.24 microg of 5-epi-aristolochene (measured as (+)-delta-cadinene equivalents), and 6.4 microg of vetispiradiene (measured as (+)-delta-cadinene equivalents) per liter of culture. These sesquiterpene production levels are >500-fold lower than carotenoid production, both of which are synthesized from endogenous trans-farnesyl diphosphate (FDP) in E. coli. Based on these results, we conclude that the limiting factor for sesquiterpene synthesis in E. coli is the poor expression of the cyclase enzyme and not supply of the FDP precursor.     

Ubiquinone-10 production using Agrobacterium tumefaciens dps gene in Escherichia coli by coexpression system

Ubiquinone (Coenzyme Q; abbreviation, UQ) acts as a mobile component of the respiratory chain by playing an essential role in the electron transport system, and has been widely used in pharmaceuticals. The biosynthesis of UQ involves 10 sequential reactions brought about by various enzymes. In this study we have cloned, expressed the decaprenyl diphosphate synthase, designated dps gene, from Agrobacterium tumefaciens, and succeeded in detecting UQ-10 in addition to innate UQ-8 in Escherichia coli. Furthermore, the production of UQ-10 was higher than UQ-8. To establish an efficient expression system for UQ-10 production, we used genes, including ubiC, ubiA, and ubiG involved in UQ biosynthesis in E. coli, to construct a better co-expression system. The expression coupled by dps and ubiCA was effective for increasing UQ-10 production by five times than that by expressing single dps gene in the shake flask culture. To study for a large-scale production of UQ-10 in E. coli, fed-batch fermentations were implemented to achieve a high cell density culture. A cell concentration of 85.40 g/L and 94.58 g/L dry cell weight (DCW), and UQ-10 content of 50.29 mg/L and 45.86 mg/L was obtained after 32.5 h and 27.5 h of cultivation, subsequent to isopropyl-β-d-thiogalactopy ranoside and lactose induction, respectively. In addition, plasmid stability was maintained at high level throughout the fermentation.     

Biosynthetic study of conidiation-inducing factor conidiogenone: heterologous production and cyclization mechanism of a key bifunctional diterpene synthase

Conidiogenone, a diterpene with a unique structure, is known to induce the conidiation of Penicillium cyclopium. The biosynthetic pathway of (?)-conidiogenone has been fully elucidated by the heterologous expression of biosynthetic genes in Aspergillus oryzae and by in vitro enzyme assay with ¹³C-labeled substrates. After construction of deoxyconidiogenol by the action of bifunctional terpene synthase, one cytochrome P450 catalyzes two rounds of oxidation to furnish conidiogenone. Notably, similar biosynthetic genes are conserved among more than 10 Penicillium sp., suggesting that conidiogenone is a common conidiation inducer in this genus. The cyclization mechanism catalyzed by terpene synthase, which involves successive 1,2-alkyl shifts, was fully elucidated using ¹³C-labeled geranylgeranyl pyrophosphate (GGPP) as substrate. During the structural analysis of deoxyconidiogenol, we observed broadening of some of the ¹³C signals measured at room temperature, which has not been observed with other structurally related compounds. Careful examination using techniques including ¹³C NMR studies at ?80 °C, conformational analysis and prediction of the ¹³C chemical shifts using density functional theory gave insights into this intriguing phenomenon.     

Isocarnosol,a diterpene from salvia lanigera

Isocarnosol, a novel diterpene from the leaves of Salvia lanigera, has been characterized.     

Antileishmanial activity of diterpene acids in copaiba oil

Leishmaniasis is a neglected tropical disease. According to the World Health Organization, there are approximately 1.5-two million new cases of cutaneous leishmaniasis each year worldwide. Chemotherapy against leishmaniasis is based on pentavalent antimonials, which were developed more than a century ago. The goals of this study were to investigate the antileishmanial activity of diterpene acids in copaiba oil, as well as some possible targets of their action against Leishmania amazonensis. Methyl copalate and agathic, hydroxycopalic, kaurenoic, pinifolic and polyaltic acids isolated from Copaifera officinales oleoresins were utilised. Ultrastructural changes and the specific organelle targets of diterpenes were investigated with electron microscopy and flow cytometry, respectively. All compounds had some level of activity against L. amazonensis. Hydroxycopalic acid and methyl copalate demonstrated the most activity against promastigotes and had 50% inhibitory concentration (IC₅₀) values of 2.5 and 6.0 µg/mL, respectively. However, pinifolic and kaurenoic acid demonstrated the most activity against axenic amastigote and had IC₅₀ values of 3.5 and 4.0 µg/mL, respectively. Agathic, kaurenoic and pinifolic acid caused significant increases in plasma membrane permeability and mitochondrial membrane depolarisation of the protozoan. In conclusion, copaiba oil and its diterpene acids should be explored for the development of new antileishmanial drugs.     

Mutation in dipZ leads to reduced production of active human placental alkaline phosphatase in Escherichia coli

Abstract In Escherichia coli , glucose 6-phosphate is transported via the Uhp system which is inducible by glucose 6-phosphate. We showed that, in a uhp -deficient strain, glucose 6-phosphate was dephosphorylated in the periplasm and that the resulting glucose was subsequently transported into the cells via the phosphotransferase system. The uptake of glucose generated from glucose 6-phosphate allowed the bacteria to produce an increased level cAMP compared to cells grown on non-limiting concentrations of glucose.     

Succinate production in Escherichia coli

Succinate has been recognized as an important platform chemical that can be produced from biomass. While a number of organisms are capable of succinate production naturally, this review focuses on the engineering of Escherichia coli for the production of four-carbon dicarboxylic acid. Important features of a succinate production system are to achieve an optimal balance of reducing equivalents generated by consumption of the feedstock, while maximizing the amount of carbon channeled into the product. Aerobic and anaerobic production strains have been developed and applied to production from glucose and other abundant carbon sources. Metabolic engineering methods and strain evolution have been used and supplemented by the recent application of systems biology and in silico modeling tools to construct optimal production strains. The metabolic capacity of the production strain, the requirement for efficient recovery of succinate, and the reliability of the performance under scaleup are important in the overall process. The costs of the overall biorefinery-compatible process will determine the economic commercialization of succinate and its impact in larger chemical markets.     

Isolation and Identification of Cotylenins F and G

The structures of cotylenins F and G, isolated from the culture filtrate of a fungus (Cladosporium sp.), have been assigned as IV and V, respectively. Cotylenin G was derived from cotylenin F by treatment with a strong base.     

The structure and stereochemistry of phlogantholide-A,a diterpene from Phlogacanthus thyrsiflorus

The structure and stereochemistry of phlogantholide-A, a new diterpene lactone isolated from the leaves of Phlogacanthus thyrsiflorus has been determined as 2β,15,18-trihydroxy-ent-labd-8(17),13-dien-16-oic lactone by chemical and spectroscopic means.     

Neue diterpene aus der subtribus espeletiinae

The investigation of several species from the newly created subtribe Espeletiinae afforded in addition to known compounds seven new diterpenes and two norditerpenes. The structures were elucidated by intensive ¹H-NMR studies. The chemotaxonomic situation is discussed briefly.     

Polyhydroxyalkanoate production in recombinant Escherichia coli

Abstract The bacterial species Escherichia coli has proven to be a powerful tool in the molecular analysis of polyhydroxyalkanoate (PHA) biosynthesis. In addition, E. coli holds promise as a source for economical PHA production. Using this microorganism, clones have been developed in our laboratory which direct the synthesis of poly-β-hydroxybutyrate (PHB) to levels as high as 95% of the cell dry weight. These clones have been further enhanced by the addition of a genetically mediated lysis system that allows the PHB granules to be released gently and efficiently. This paper describes these developments, as well as the use of an E. coli strain to produce the copolymer poly-(3-hydroxybutyrate- co -3-hydroxyvalerate (PHB- co -3-).