Identification of RoCYP01 (CYP716A155) enables construction of engineered yeast for high-yield production of betulinic acid.

Betulinic acid (BA) and its derivatives possess potent pharmacological activity against cancer and HIV. As with many phytochemicals, access to BA is limited by the requirement for laborious extraction from plant biomass where it is found in low amounts. This might be alleviated by metabolically engineering production of BA into an industrially relevant microbe such as Saccharomyces cerevisiae (yeast), which requires complete elucidation of the corresponding biosynthetic pathway. However, while cytochrome P450 enzymes (CYPs) that can oxidize lupeol into BA have been previously identified from the CYP716A subfamily, these generally do not seem to be specific to such biosynthesis and, in any case, have not been shown to enable high-yielding metabolic engineering. Here RoCYP01 (CYP716A155) was identified from the BA-producing plant Rosmarinus officinalis (rosemary) and demonstrated to effectively convert lupeol into BA, with strong correlation of its expression and BA accumulation. This was further utilized to construct a yeast strain that yields > 1 g/L of BA, providing a viable route for biotechnological production of this valuable triterpenoid.

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.

Isolation, purification and characterisation of transglutaminase from rosemary (Rosmarinus officinalis L.) leaves.

BACKGROUND: Rosemary (Rosmarinus officinalis L.) is a spice and medicinal herb widely used around the world of the natural antioxidants, and it has been widely accepted as one of the spices with the highest antioxidant activity. Transglutaminase (EC 2.3.2.13: TGase) is an enzyme capable of catalysing acyl transfer reactions by introducing covalent cross-links between proteins, as well as peptides and various primary amines. TGase activity in plants was first observed in pea seedlings, and subsequently found in organs of both lower and higher plants. Recently. TGase has captured researchers' interest due to its attractive potential application in food industries. Therefore, the objectives of this study are isolation and purification of TGase from new plant source rosemary (Rosmarinus officinalis L.) leaves at the laboratory scale. Moreover, investigation of the biochemical properties of the purified TGase to provide a suitable TGase enzyme for food industry applications are in focus. MATERIAL AND METHODS: Rosemary (Rosmarinus officinalis L.) leaves was used as a new plant source to TGase. The biochemical characteristics of the crude and purified enzyme were determined. RESULTS: Rosemary (Rosmarinus officinalis L.) TGase was purified to homogeneity by successive three purification steps including ammonium sulfate precipitatation, ion exchange chromatography on DEAE-Sephadex A-50 column and Size exclusion column chromatography on Sephadex G-100 column. Under experimental conditions. 20-30% of ammonium sulfate saturation in the enzyme solution had a high yield of enzyme activity could be obtained. The purified enzyme from the Sephadex G-100 column had 21.35% yield with increased about 7.31 in purification fold. Rosemary TGase exhibited optimum activity at pH 7.0 and 55°C for the catalytic reaction of hydroxylarnine and Z-Gln-Gly. The purified TGase almost maintained full activity after incubation for 15 ruin up to 60°C and it was completely inactivated at 85°C. The rosemary TGase was stimulated at 2-6 rnM CaCl2 concentrations while it lost about 5-20% from its activity by increasing CaCl2 concentration. Sodium chloride (2-14%) shows no noticeable inhibition of the purified TGase activity. Mg+2, Ba+2 were acivited by the purified TGase while it was str ongly inhibited by Fe+2, moderately by Cir2 and Mn+2. CONCLUSION: This paper reports on the purification and characterisation of TGase from newly isolated plant, rosemary (Rosmarinus officinalis L.) leaves. Finding results of the TGase properties make this enzyme a good candidate for application in the food industry. However, additional work is required to increase activity yield during extraction and purification for commercial scale of TGase from this plant.

Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps. Thus, a copalyl diphosphate synthase (RoCPS1) and two kaurene synthase-like (RoKSL1 and RoKSL2) encoding genes were identified and characterized. Expression in yeast (Saccharomyces cerevisiae) and Nicotiana benthamiana demonstrate that RoCPS1 converts geranylgeranyl diphosphate (GGDP) to copalyl diphosphate (CDP) of normal stereochemistry and that both RoKSL1 and RoKSL2 use normal CDP to produce an abietane diterpene. Comparison to the already characterized diterpene synthase from Salvia miltiorrhiza (SmKSL) demonstrates that the product of RoKSL1 and RoKSL2 is miltiradiene. Expression analysis supports a major contributing role for RoKSL2. Like SmKSL and the sclareol synthase from Salvia sclarea, RoKSL1/2 are diterpene synthases of the TPS-e group which have lost the internal gamma-domain. Furthermore, phylogenetic analysis indicates that RoKSL1 and RoKSL2 belong to a distinct group of KSL enzymes involved in specialized metabolism which most likely emerged before the dicot-monocot split.