Publications

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7 Publications visible to you, out of a total of 7
Nicotiana benthamiana as a competitive host for large-scale oxidative biotransformations and an efficient chassis for metabolic engineering of CYP-mediated biosynthetic pathways

Abstract (Expand)

Because of their remarkable catalytic diversity, human cytochrome P450 enzymes (CYPs) are valuable tools for biotransformation. They enable regio- and stereospecific oxidation of non-activated hydrocarbon … bonds, a process often difficult to achieve by conventional chemical methods. Plants serve as versatile host platforms for expressing foreign CYPs, providing access to cofactor flux and a variety of reducing partners and membrane systems suitable for enzyme integration. However, it is essential to adapt heterologous CYPs to the plant metabolic environment to establish this system as a viable option for large-scale biotechnological applications. To achieve this, we tested various approaches aimed at enhancing the functional activity of heterologous CYPs, including co-expression of heterologous redox partners and adapting CYPs for transfer to various subcellular compartments. While compartmentalization did not enhance catalytic function, we found that the transient co-expression of redox partners significantly influenced the performance of human CYPs in a plant production system. While human cytochrome P450 reductase (CPR) typically enhances or maintains enzyme activity, plant-derived CPRs can either enhance or significantly reduce the metabolism of specific drugs. For most CYP/CPR/substrate combinations, we increased product accumulation by 1.8- to 4.3-fold. By swapping the structural domains of human and plant CPR, we created a hybrid CPR variant that demonstrates improved productivity across all tested CYPs. This variant may serve as a potential universal redox partner for human CYPs expressed in plant hosts. Using scaled transient expression of CYPs and the hybrid CPR gene, we performed preparative biotransformation of the pharmaceutically valuable prodrug yohimbine, yielding around 50 mg of 11-hydroxyyohimbine. This represents the highest ever reported CYP-mediated biotransformation of exogenous substrates in a plant system, comparable to that achieved in yeast. To diversify the spectrum of natural products, we integrated human CYP and CPR enzymes into the engineered irregular monoterpene pathways. These secondary metabolites function as pheromones and insecticides, and demonstrate cytotoxic and antibiotic activities. We established a sustainable platform for irregular monoterpene accumulation in Nicotiana benthamiana, achieving the highest level in plant systems. Two CYPs converted branched and cyclopropane monoterpenes into novel hydroxylated derivatives, with human CPR co-expression aiding in product accumulation. Thus, by integrating human CYPs into a network of heterologous genes from different organisms, we initiated new biosynthetic pathways in N. benthamiana and expanded the range of irregular monoterpene structures. Acknowledgements: YVS acknowledges the financial support from the German Research Foundation (DFG), grant number 516587177.

Authors: Yuriy V. Sheludko, Iryna Gerasymenko, Heribert Warzecha

Date Published: 20th Oct 2026

Publication Type: Conference Paper

Co-expression of cytochrome P450 reductases modulates the activity and selectivity of human cytochrome P450 enzymes transiently expressed in Nicotiana benthamiana

Abstract (Expand)

The transient co-expression of various redox partners affects the efficiency of human hepatic cytochrome P450 enzymes (CYP) in a plant-based biotransformation system. Using Nicotiana benthamiana as a … host, we investigated the interaction of CYP1A2, CYP2D6, and CYP3A4 with different cytochrome P450 reductases (CPRs) derived from animal and plant sources. While human CPR typically enhance or maintain CYP activity, plant-derived CPRs can either enhance or significantly inhibit the metabolism of specific drugs. Additionally, the co-expression of an alternative redox partner, cytochrome b5, can enhance, inhibit, or have no effect on human CYP catalysis, depending on the specific CYP involved. For the majority of CYP/CPR/substrate combinations, we selected variants beneficial for CYP-mediated biotransformation, resulting in a 1.8-4.3-fold increase in product accumulation. We also developed chimeric human-plant CPR variants by swapping structural domains, thereby allowing identification of specific regions responsible for different levels of CYP2D6 productivity. This approach led to the creation of an enhanced CPR variant with a modified transmembrane domain and improved characteristics compared to the native versions.

Authors: Yuriy V. Sheludko, Iryna M. Gerasymenko, Heribert Warzecha

Date Published: 2026

Publication Type: Preprint

Biofactoring of Irregular Monoterpenes in Nicotiana benthamiana: From Key Precursors to New Molecules

Abstract

Not specified

Authors: Iryna Gerasymenko, Yuriy V. Sheludko, Heribert Warzecha

Date Published: 7th Oct 2025

Publication Type: Conference Paper

Efficient accumulation of new irregular monoterpene malonyl glucosides in Nicotiana benthamiana achieved by co-expression of isoprenyl diphosphate synthases and substrate-producing enzymes

Abstract (Expand)

Irregular monoterpenes have limited natural sources but possess unique activities applicable in medicine and agriculture. To enable sustainable plant-based production of these compounds, we established … a transient expression procedure to enhance the biosynthetic flux in Nicotiana benthamiana toward dimethylallyl diphosphate (DMAPP), a substrate for isopentenyl diphosphate synthases (IDSs) that generate irregular monoterpene skeletons. Considering the benefits of glycosylation for accumulating and storing monoterpenes in extractable form, we focused on developing a platform for the production of non-volatile glycosylated irregular monoterpenes using three IDS that form branched and cyclic structures. The analysis of methanolic leaf extracts from transiently transformed N. benthamiana plants revealed six major new components, 6-O-malonyl-β-D-glucopyranoside and 6-O-malonyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside derivatives of chrysanthemol, lavandulol and cyclolavandulol, five of which are novel compounds. Alleviating two bottlenecks in the DMAPP formation in plastids by co-expressing 1-deoxyxylulose 5-phosphate synthase and isopentenyl diphosphate isomerases increased the yield of chrysantemyl and lavandulyl glucosides produced by plant-derived IDS to 1.7 ± 0.4 μmol g-1 FW and 1.4 ± 0.3 μmol g-1 FW, respectively. A bacterial cyclolavandulyl diphosphate synthase operated efficiently in chloroplasts and cytoplasm. The highest irregular monoterpene concentrations were achieved in cytoplasm by co-expression of hydroxymethylglutaryl-CoA reductase, the bottleneck enzyme of the mevalonate pathway for DMAPP biosynthesis. The mean level of cyclolavandulyl glucosides reached 3.9 ± 1.5 μmol g-1 FW; the top-performing plants contained 6.6 μmol g-1 FW. This yield represents the highest amount of irregular monoterpenes produced in plant systems.

Authors: Iryna Gerasymenko, Yuriy V. Sheludko, Volker Schmidts, Heribert Warzecha

Date Published: 6th Oct 2025

Publication Type: Journal Article

The race with microbial factories: Metabolic and protein engineering for production small molecules in plants

Abstract

Not specified

Authors: Iryna Gerasymenko, • Sheludko YV, Heribert Warzecha

Date Published: 5th Mar 2025

Publication Type: Conference Proceeding

Selecting the right partner is crucial: Co-expressing CPR effectively modulates the activity of foreign P450s in plant cells

Abstract

Not specified

Authors: Yuriy V. Sheludko, Iryna Gerasymenko, Heribert Warzecha

Date Published: 5th Mar 2025

Publication Type: Conference Paper

Choosing the right partner: Maximizing the efficiency of foreign P450s in plant cells.

Abstract

Not specified

Authors: Yuriy V. Sheludko, Iryna Gerasymenko, Heribert Warzecha

Date Published: 7th Oct 2024

Publication Type: Conference Paper

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