ArticleMicrobial cell factories2018
Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.
Article in Microbial cell factories, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 24 papers.
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24 citing papers in PubMed, 53 citations in OpenAlex.
- Diterpenoids in medicinal plants: structure, distribution, biological activities, biosynthesis, and bioengineering prospects.Frontiers in plant science · 2026Review
- Advances in semi-rational design of terpene synthases and their modifying enzymes.AMB Express · 2025Review
- Heterologous Production of the Structurally Complex Diterpenoid Forskolin inBioengineering (Basel, Switzerland) · 2025Article
- Bacterial biosynthesis of abietane-type diterpene ferruginol from glucose.Microbial cell factories · 2025Article
- Advances in the Structures, Pharmacological Activities, and Biosynthesis of Plant Diterpenoids.Journal of microbiology and biotechnology · 2024Review
- Biotechnological interventions for the production of forskolin, an active compound from the medicinal plant,Physiology and molecular biology of plants : an international journal of functional plant biology · 2024Review
- Functional Study and Efficient Catalytic Element Mining of CYP76AHs inMolecules (Basel, Switzerland) · 2023Article
- A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast.Nature communications · 2023Review
- Genome-wide identification of the AOMT gene family in wax apple and functional characterization ofFrontiers in plant science · 2023Article
- Engineering medicinal plant-derived CYPs: a promising strategy for production of high-valued secondary metabolites.Planta · 2022Review
- A gene cluster in Ginkgo biloba encodes unique multifunctional cytochrome P450s that initiate ginkgolide biosynthesis.Nature communications · 2022Article
- Ancestral sequence reconstruction of the CYP711 family reveals functional divergence in strigolactone biosynthetic enzymes associated with gene duplication events in monocot grasses.The New phytologist · 2022Article
- Tripterygium wilfordii cytochrome P450s catalyze the methyl shift and epoxidations in the biosynthesis of triptonide.Nature communications · 2022Article
- Biodiscoveries within the Australian plant genus Eremophila based on international and interdisciplinary collaboration: results and perspectives on outstanding ethical dilemmas.The Plant journal : for cell and molecular biology · 2022Review
- First purified recombinant CYP75B including transmembrane helix with unexpected high substrate specificity to (2R)-naringenin.Scientific reports · 2022Article
- Biosynthesis of a novel ganoderic acid by expressing CYP genes from Ganoderma lucidum in Saccharomyces cerevisiae.Applied microbiology and biotechnology · 2022Article
- A cytochrome P450 CYP81AM1 from Tripterygium wilfordii catalyses the C-15 hydroxylation of dehydroabietic acid.Planta · 2021Article
- Progress in heterologous biosynthesis of forskolin.Journal of industrial microbiology & biotechnology · 2021Review
- Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9.Microbial cell factories · 2021Article
- Rational design strategies for functional reconstitution of plant cytochrome P450s in microbial systems.Current opinion in plant biology · 2021Review
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
Abstract
backgroundForskolin 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.
resultsAs 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.
conclusionsAs 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.
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