Discovery of new bioactive molecules for use in crop protection

Secondary Supervisor(s): Dr Lijiang Song (Chemistry)

University of Registration: University of Warwick

BBSRC Research Themes:

• Sustainable Agriculture and Food (Plant and Crop Science)
• Understanding the Rules of Life (Microbiology)

Project Outline

General background

Many of the bioactive molecules used in agriculture are made by microorganisms or inspired by natural products. For instance, abamectin derives from actinomycete bacteria and is one of the most widely used insecticides in crop protection. The Alberti lab aims to discover new bioactive natural products using a combination of genomics, genetic engineering and analytical chemistry, with the aim to improve the repertoire of molecules available for crop protection.^1–3

Project background

We previously isolated and characterised fungal strains of the genus Trichoderma that show inhibition of the growth of plant pathogenic bacteria.⁴ We have identified candidate biosynthetic gene clusters within their genomes that we believe to be involved in the biosynthesis of novel bioactive molecules.

Aim and Objectives

Aim: to discover and characterise antimicrobial natural products from fungi that can be used in crop protection to fight plant diseases.

Objectives:

• To clone and heterologously express the biosynthetic gene clusters of interest (Fig. 1A);
• To isolate and characterise the corresponding natural products, including polyketides and non-ribosomal peptides (Fig. 1B);
• To assess the bioactivity of the isolated compounds against a panel of plant pathogenic bacteria, including on plants, and to evaluate their cytotoxicity on human cells (Fig. 1C).

Methodology

• Gene cloning, such as Golden Gate assembly, Gibson assembly and yeast-based homologous recombination will be used to clone the genes of interest and assemble them into suitable expression vectors for heterologous expression in Aspergillus oryzae;
• Metabolite analyses will be performed through LC-MS and NMR spectroscopy (previous experience in these techniques is not essential) in order to characterise the pathway products and define the catalytic function of the relevant enzymes;
• Antimicrobial bioassays will be performed against a panel of plant pathogenic bacteria, including on plants, to assess the bioactivity of the isolated compounds;
• Genetic engineering will be performed (e.g. through CRISPR/Cas9) with the aim to study and improve the production of the molecules of interest.

Impact

This PhD project will lead to the development of biological control agents for plant disease, and will have long term impact on agricultural practices.

References

(1) Al-salihi, S. A. A.; Alberti, F. Genomic Based Analysis of the Biocontrol Species Trichoderma Harzianum: A Model Resource of Structurally Diverse Pharmaceuticals and Biopesticides. Journal of Fungi 2023, 9, 895.

(2) Brooks, S.; Weaver, J. A.; Klomchit, A.; Alharthi, S. A.; Onlamun, T.; Nurani, R.; Vong, T. K.; Alberti, F.; Greco, C. Unveiling the Potential of Daldinia Eschscholtzii MFLUCC 19-0629 through Bioactivity and Bioinformatics Studies for Enhanced Sustainable Agriculture Production. Frontiers in Chemical Biology 2024, 3, 1362147. https://doi.org/10.3389/fchbi.2024.1362147.

(3) Klomchit, A.; Calabon, M. S.; Worabandit, S.; Weaver, J. A.; Karima, E. M.; Alberti, F.; Greco, C.; Mahanil, S. Unveiling Novel Neocosmospora Species from Thai Mangroves as Potent Biocontrol Agents against Colletotrichum Species. J Appl Microbiol 2024, 135 (5), lxae114. https://doi.org/10.1093/jambio/lxae114.

(4) Tamizi, A.; Mat-amin, N.; Weaver, J. A.; Olumakaiye, R. T.; Akbar, M. A.; Jin, S.; Bunawan, H.; Alberti, F. Genome Sequencing and Analysis of Trichoderma (Hypocreaceae) Isolates Exhibiting Antagonistic Activity against the Papaya Dieback Pathogen, Erwinia Mallotivora. Journal of fungi 2022, 8, 246. https://doi.org/https://doi.org/10.3390/jof8030246.

Understanding the biosynthesis and mode of action of the anticancer antibiotic pleurotin for production of new analogues

Secondary Supervisor(s): Dr Manuela Tosin, Dr Lona Alkhalaf

University of Registration: University of Warwick

BBSRC Research Themes: Understanding the Rules of Life (Microbiology, Structural Biology)

Project Outline

General background

Fungi are prolific producers of bioactive molecules used in medicine. For example, penicillin and cephalosporin antibiotics represent together 47% of the global antibiotic market. The Alberti lab aims to understand the biosynthesis of bioactive molecules, with the goal to direct it towards the production of new analogues.^1,2

Project background

Pleurotin is an anticancer antibiotic made by fungi that has potential to be an anticancer lead against breast and colon cancer. The Alberti lab recently used a combination of genome sequencing, comparative transcriptomics and isotope-labelled precursor feeding to study the early steps of pleurotin biosynthesis (Fig. 1).³

Aim and Objectives

Aim: to fully elucidate the biosynthesis of pleurotin and study its interaction with its target in human cells, in order to further develop new and improved pleurotin analogues.

Objectives:

• To isolate and characterise pleurotin biosynthetic intermediates from engineered fungal strains;
• To study the interaction of pleurotin with its target in human cells;
• To produce new analogues of pleurotin using precursor-directed biosynthesis.

Methodology

• Metabolite analyses will be performed through LC-MS and NMR spectroscopy in order to characterise the pathway products and define the catalytic function of the relevant enzymes (Fig. 1A);
• Synthetic organic chemistry will be utilised to generate some precursors for feeding experiments.
• X-ray crystallography will be used to investigate the interaction between pleurotin and its human target, thioredoxin reductase (Fig. 1B);
• Precursor-directed biosynthesis, potentially in combination with gene knockouts through CRISPR/Cas9, will be used to produce new analogues of pleurotin with the aim to improve its bioactivity and pharmacokinetic properties (Fig. 1C). These will be purified using HPLC and tested for bioactivity.

Impact

This PhD project will increase our understanding of the biosynthesis of an anticancer lead and of its interaction with its molecular target, which will allow us to design more effective analogues.

References

(1) Alberti, F.; Khairudin, K.; Venegas, E. R.; Davies, J. A.; Hayes, P. M.; Willis, C. L.; Bailey, A. M.; Foster, G. D. Heterologous Expression Reveals the Biosynthesis of the Antibiotic Pleuromutilin and Generates Bioactive Semi-Synthetic Derivatives. Nat Commun 2017, 8 (1), 1831. https://doi.org/10.1038/s41467-017-01659-1.

(2) Alberti, F.; Khairudin, K.; Davies, J. A.; Sangmalee, S.; Willis, C. L.; Foster, G. D.; Bailey, A. M. Biosynthesis of Pleuromutilin Congeners Using an Aspergillus Oryzae Expression Platform. Chem Sci 2023, 14, 3826–3833. https://doi.org/10.1039/d2sc06638f.

(3) Weaver, J. A.; Alkhder, D.; Prasongpholchai, P.; Tadesse, M. D.; Santos, E. L. D. L.; Song, L.; Corre, C.; Alberti, F. Early Steps of the Biosynthesis of the Anticancer Antibiotic Pleurotin. bioRxiv 2024. https://doi.org/https://doi.org/10.1101/2024.08.27.609176.

CASE: Mining biocatalysts from talented microorganisms for industrial biocatalysis applications

University of Registration: University of Warwick

Non-academic partner: Dr Vincent Poon, Hypha Discovery Ltd

Project Outline

This project is a new research collaboration between Dr. Alberti, Prof. Corre (University of Warwick) and Dr. Poon (Hypha Discovery) to elucidate the genes responsible for specific, but currently poor yielding biotransformation reactions and engineering into higher-yielding host systems for enabling industrial biotechnology applications.

Specific xenobiotic metabolites known as dihydrodiols are of high interest; derived from epoxide hydrolase (EPHX) enzymes acting upon epoxides formed by Cytochrome P450 (CYP450) enzymes. These metabolites are formed from epoxides of aromatic as well as non-aromatic alkene moiety-containing parent molecules. These dihydrodiol metabolites are highly challenging to manufacture by chemical syntheses, leading to reliance on animal-derived materials for their biosynthetic production to allow toxicological and drug-drug interaction testing of medicines in development. The improved understanding and expression of these EPHX enzymes with and without coexpression with CYP450 enzymes will enable acceleration of impacted medicines through development. It will also reduce or eliminate the use of animal materials and open further avenues for their application in production of chiral feedstock chemicals.

C-H bond activation for lead compound diversification can be particularly difficult by chemical synthesis as it is challenging, often non-selective, involves costly multi-step procedures and may not fully consider other derivatives. An alternative method, which is currently practiced at Hypha Discovery, is to produce metabolite derivatives through the use of microbial biotransformation. To expand on Hypha’s capability, the aim of the project is to capture the activity of a human-like EPHX and CYP450s from one of Hypha Discovery’s fungal strains for the production of dihydrodiol metabolites.

Objectives

The objectives of the project are:

1. To perform genome sequencing and annotation of one of Hypha Discovery’s selected fungal strains identified for human-like EPHX biocatalytic capability.

2. To perform bioinformatic analyses and genome mining of genes-of-interest (GOIs)

3. To construct vectors to express GOIs in various expression strains (yeast, fungi, bacteria).

4. To express GOIs at Hypha Discovery and to test the efficacy and activity of the recombinant systems against several drug compounds (e.g., carbamazepine) and their intermediate epoxides by UPLC-MS.

5. Optimise and develop methods for the scale-up of selected metabolites for structure elucidation to confirm trans-diol product formation, as seen in human metabolism.

Overall, the recombinant cell factory developed in this project will allow for the efficient expression of fungal biocatalysts and will be the base to generate other recombinant strains by swapping other epoxide-producing cytochrome P450s to cover broader substrate specificity.

Candidates are encouraged to contact Dr Fabrizio Alberti to discuss the project before applying if they wish to.