76 research outputs found
Engineered protein degradation of farnesyl pyrophosphate synthase is an effective regulatory mechanism to increase monoterpene production in Saccharomyces cerevisiae
Monoterpene production in Saccharomyces cerevisae requires the introduction of heterologous monoterpene synthases (MTSs). The endogenous farnesyl pyrosphosphate synthase (FPPS; Erg20p) competes with MTSs for the precursor geranyl pyrophosphate (GPP), which limits the production of monoterpenes. ERG20 is an essential gene that cannot be deleted and transcriptional down-regulation of ERG20 has failed to improve monoterpene production. Here, we investigated an N-degron-dependent protein degradation strategy to down-regulate Erg20p activity. Degron tagging decreased GFP protein half-life drastically to 1 h (degron K3K15) or 15 min (degrons KN113 and KN119). Degron tagging of ERG20 was therefore paired with a sterol responsive promoter to ensure sufficient metabolic flux to essential downstream sterols despite the severe destabilisation effect of degron tagging. A dual monoterpene/sesquiterpene (linalool/nerolidol) synthase, AcNES1, was used as a reporter of intracellular GPP and FPP production. Transcription of the synthetic pathway was controlled by either constitutive or diauxie-inducible promoters. A combination of degron K3K15 and the ERG1 promoter increased linalool titre by 27-fold to 11 mg L−1 in the strain with constitutive promoter constructs, and by 17-fold to 18 mg L−1 in the strain with diauxie-inducible promoter constructs. The sesquiterpene nerolidol remained the major product in both strains. The same strategies were applied to construct a limonene-producing strain, which produced 76 mg L−1 in batch cultivation. The FPPS regulation method developed here successfully redirected metabolic flux toward monoterpene production. Examination of growth defects in various strains suggested that the intracellular FPP concentration had a significant effect on growth rate. Further strategies are required to balance intracellular production of FPP and GPP so as to maximise monoterpene production without impacting on cellular growth.</p
Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids
Metabolic engineering of cucurbitacins in Cucurbita pepo hairy roots
In this paper we show that metabolic engineering in Cucurbita pepo hairy roots can be used to both effectively increase and modify cucurbitacins. Cucurbitacins are highly-oxygenated triterpenoids originally described in the Cucurbitaceae family, but have since been found in 15 taxonomically distant plant families. Cucurbitacin B, D, E and I are the most widespread amongst the Cucurbitaceae and they have both important biological and pharmacological activities. In this study C. pepo hairy roots were used as a platform to boost production and alter the structures of the afore mentioned cucurbitacins by metabolic engineering to potentially provide new or more desirable bioactivities. We report that the ability to induce cucurbitacin biosynthesis by basic Helix-Loop-Helix transcription factors is partially conserved within the Cucurbitaceae and therefore can potentially be used as a biotechnological tool to increase cucurbitacins in several genera of this family. Additionally, overexpression of a novel acyltransferase from cucurbitacin producing Iberis amara generates a hitherto undescribed acetylation at the C3-hydroxyl group of the cucurbitadienol backbone. While overexpression of the cytochromes P450 CsCYP88L2 and McCYP88L7 from Cucumis sativus and Momordica charantia (respectively), results in accumulation of new spectral feature as revealed by High resolution liquid chromatography mass spectroscopy analysis; the m/z of the new peak supports it might be a cucurbitacin hydroxylated at the C19 position in C. pepo hairy roots. Finally, this paper is a case study of how hairy roots can be used to metabolically engineer and introduce novel modifications in metabolic pathways that have not been fully elucidated
Combined metabolome and transcriptome profiling provides new insights into diterpene biosynthesis in S. pomifera glandular trichomes
Background: Salvia diterpenes have been found to have health promoting
properties. Among them, carnosic acid and carnosol, tanshinones and
sclareol are well known for their cardiovascular, antitumor,
antiinflammatory and antioxidant activities. However, many of these
compounds are not available at a constant supply and developing
biotechnological methods for their production could provide a
sustainable alternative. The transcriptome of S. pomifera glandular
trichomes was analysed aiming to identify genes that could be used in
the engineering of synthetic microbial systems.
Results: In the present study, a thorough metabolite analysis of S.
pomifera leaves led to the isolation and structure elucidation of
carnosic acid-family metabolites including one new natural product.
These labdane diterpenes seem to be synthesized through miltiradiene and
ferruginol. Transcriptomic analysis of the glandular trichomes from the
S. pomifera leaves revealed two genes likely involved in miltiradiene
synthesis. Their products were identified and the corresponding enzymes
were characterized as copalyl diphosphate synthase (SpCDS) and
miltiradiene synthase (SpMilS). In addition, several CYP-encoding
transcripts were identified providing a valuable resource for the
identification of the biosynthetic mechanism responsible for the
production of carnosic acid-family metabolites in S. pomifera.
Conclusions: Our work has uncovered the key enzymes involved in
miltiradiene biosynthesis in S. pomifera leaf glandular trichomes. The
transcriptomic dataset obtained provides a valuable tool for the
identification of the CYPs involved in the synthesis of carnosic
acid-family metabolites.General Secretariat of Research and Technology (GSRT) {[}09-SYN-23-879];
grant SEE-ERA. NET PLUS {[}ERA 64/01]; grant KRIPIS {[}MIS 448840
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 SfCPSand 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
DNA gyrase : mechanism of supercoiling and interaction with quinolones
DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. Deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that behaves like a conventional type II topoisomerase, suggesting that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits. However, these results do not unveil the detailed mechanism by which the transported DNA segment is captured and directed through the DNA gate. This mechanism was addressed by probing the topology of the bound DNA segment at distinct steps of the catalytic cycle. A model is proposed in which gyrase captures a contiguous DNA segment with high probability, irrespective of the superhelical density of the DNA, while the efficiency of strand passage depends on the superhelical free-energy. This mechanism is concerted, in that capture of the transported segment induces opening of the DNA gate, which in turn, stimulates ATP hydrolysis.;Mutation of Glu42 to Ala in the B subunit of DNA gyrase abolishes ATP hydrolysis but not nucleotide binding. Gyrase complexes that contain one wild-type and one Ala42 mutant B protein were formed and the ability of such complexes to hydrolyse ATP was investigated. It was found that ATP hydrolysis was able to proceed only in the wild-type subunit, albeit at a lower rate. With only one ATP molecule hydrolysed at a time, gyrase could still perform supercoiling but the limit of this reaction was lower than that observed when both subunits can hydrolyse the nucleotide.;Limited proteolysis was used to identify conformational changes in DNA gyrase and the proteolytic signatures observed were interpreted in terms of four complexes of gyrase, each representing a particular conformational state. Quinolone binding to the gyrase-DNA complex induces a conformational change that results in the blocking of supercoiling. Under these conditions gyrase is still capable of ATP hydrolysis. The kinetics of this reaction have been studied and found to differ from those of the reaction of the drug-free enzyme. By observing the conversion of the ATPase rate to the quinolone-characteristic rate, the formation and dissociation of the gyrase-DNA-quinolone complex can be monitored. Comparison of the time dependence of the conversion of the gyrase ATPase with that of DNA cleavage reveals that formation of the gyrase-DNA-quinolone complex does not correspond to the formation of cleaved DNA. Quinolone binding and drug-induced DNA cleavage are separate processes constituting two sequential steps in the mechanism of action of quinolones on DNA gyrase
Conversion of DNA gyrase into a conventional type II topoisomerase
DNA gyrase is unique among topoisomerases in its ability to introduce negative supercoils into closed-circular DNA. We have demonstrated that deletion of the C-terminal DNA-binding domain of the A subunit of gyrase gives rise to an enzyme that cannot supercoil DNA but relaxes DNA in an ATP-dependent manner. Novobiocin, a competitive inhibitor of ATP binding by gyrase, inhibits this reaction. The truncated enzyme, unlike gyrase, does not introduce a right-handed wrap when bound to DNA and stabilizes DNA crossovers; characteristics reminiscent of conventional type II topoisomerases. This new enzyme form can decatenate DNA circles with increased efficiency compared with intact gyrase and, as a result, can complement the temperature-sensitive phenotype of a parC(ts) mutant. Thus these results suggest that the unique properties of DNA gyrase are attributable to the wrapping of DNA around the C-terminal DNA-binding domains of the A subunits and provide an insight into the mechanism of type II topoisomerases
DEVELOPING A YEAST CELL FACTORY FOR THE PRODUCTION OF TERPENOIDS
AbstractTechnological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with –omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds
Developing a yeast cell factory for the production of terpenoids
Technological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with –omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds
- …