Development of understanding of the genetic elements for biosurfactant production by a basidiomycete yeast – Mark Caddick – The University of Liverpool
Project partners – Doug Cossar – Croda Europe Ltd
Biosurfactants have the potential to transform the specialty chemicals industry. They provide typically higher performance characteristics at lower temperatures than conventional materials. In addition they often demonstrate synergistic behaviour and added benefits such as narrow spectrum antibiosis. However, other than a very limited number of cases, the yield of such products is too low to be other than niche market products. To bring biosurfactants to a broader market requires a significant increase in yield. This is optimally achieved by developing an understanding of the genes involved in biosynthesis of the biosurfactant along with insight into their control and regulation. This project will determine the genome sequence of a strain known to produce a particular biosurfactant with known economic potential. In comparison with related strains and using standard gene function annotation methods, the probable gene cluster encoding this phenotype will be identified. A transcriptomic approach will be applied to determine the relationship of gene transcription and product accumulation. This will validate the identity of the gene cluster and provide further insight into the key components of the biosynthetic apparatus. This work will provide a platform to conduct a programme of directed mutagenesis leading to creation of strains with enhanced yield. Further expansion is envisaged to probe the structure to activity relationship of the molecule and thereafter precisely engineer performance variants for specific applications.
Discovery of potential pyrrolizidine natural products by genomic scanning of actinomycetes from NCIMB culture collection – Hai Deng – University of Aberdeen
Project partners – Carol Philips – NCIMB Ltd
The discovery of natural products, an important source of human medicines, is critical for the development of new therapeutics against health threats, including cancer and multidrug-resistant pathogens. Yet, in recent years, industrial efforts for natural product discovery has been hampered due to a variety of reasons, including the repeated discovery of previously known compounds. Many have suggested that the solution lies in genomics and focusing research efforts on strains that encode genes for the biosynthesis of uncharacterized natural products can dereplicate, streamline, and accelerate the discovery process. This project is designed to maximise the chance of harnessing a group of previously underexploited bacterial pyrrolizidine alkaloids (PAs). Pyrrolizidines constitute an important nucleus in many pharmaceutically important agents. Naturally occurring PAs are mainly discovered from plants and fungi. Only a few PAs have been discovered from bacteria to date. Bacterial PAs display promising antibacterial and antitumour activities and have attracted considerable interest from academic research groups and the pharmaceutical industry. This group of metabolites, however, have been underexploited for many years. Recently, we elucidated the biosynthetic pathway of bacterial PAs, legonmycins, for the first time and characterized the monooxygenase enzyme LgnC (encoded by lgnC). Building on this track record, we used bioinformatics analysis on available genomic data and found that genes for the synthesis of PA-like metabolites may be ubiquitously present in bacterial genomes. We have developed a methodology needed for gene-based discovery of PA biosynthetic loci. This method relies on the fact that the PA biosynthetic pathways contain the key LgnC-like monooxygenases. Thus amplification of an internal fragment of lgnC with degenerate primers is sufficient to identify strains or plasmid clones that encode PA biosynthetic pathways. Our preliminary results are encouraging. NPRONET funding will enable us to employ this targeted gene-based screening method by mining a collection of actinomycete strains in NCIMB Ltd for PA discovery. The success of this short project will allow us to further collaborate with NCIMB Ltd and fully exploit its bacterial resources in a larger scale (8000 strains) of gene-based survey for PA discovery by applying IUK feasibility grant (i.e. industrial biotechnology catalyst-technical feasibility studies) or BBSRC-funded programme.
Biosurfactant production by yeasts belonging to the genus Pseudozyma – Ian Roberts – Institute of Food Research
Project partners – Doug Cossar – Croda Europe Ltd
Yeast biodiversity has enormous potential for innovative natural products bioengineering. Several different yeast species, in addition to the well-known brewing and baking yeasts, have already proved of considerable value to bioindustry. They are valuable both as sources of novel natural products and as industrial-scale production vehicles for known compounds. Since 1948, the UK National Collection of Yeast Cultures (NCYC) has been collecting diverse yeasts from widely different habitats worldwide and is recognised internationally as one of the largest, most biologically diverse and best-characterised yeast collections in the world. More recently, it has begun an ambitious project to genome sequence all 4,000 strains in the collection. Mining yeast genomes computationally generates new knowledge of species diversity and its origins. Linking genomic knowledge to high-throughput robotic screens allows us to uncover and understand novel yeast properties, leading to innovations of high commercial value. In this project we will seek to expand knowledge of yeast variants using the comprehensive biological resources of the NCYC. We will focus on variation in biosurfactant producing yeasts belonging to Pseudozyma. In particular we will investigate what Mannosylerythritol Lipid (MEL) surfactants are produced by a novel species discovered within the NCYC collection, what additional functional variation can be uncovered by looking at a wider range of Pseudozyma species, and how functional variation is related to genetic variation discovered via whole genome sequencing. In the longer term we will utilise this new knowledge in strain improvement programmes aimed at producing MELs with novel, commercially desirable structure/activity relationships. The project is innovative as it brings together yeast taxonomy, genomics and metabolic engineering to yield environmentally-friendly biosurfactants with improved properties.
Genomics-enabled novel biocatalyst identification and characterisation – Gary Black – Northumbria University
Project partner – Ben Bradley – CHAIN Biotech Ltd.
Clostridia are robust bacteria with a track record for commercial solvent production. They have diverse and unique anaerobic metabolic pathways that produce a wide range of reduced chemical products from a variety of different substrates and consequently a large number of interesting enzymes.
CHAIN Biotech has access to a large collection of Clostridia strains (about 300) representing many different species but today, given the complexity of characterising individual strains both at the genetic and biochemical level, the collection remains largely untouched and under-exploited.
The project is designed to unlock the industrial potential of this unique biological resource. The key enabler is the application of advanced and low cost genome sequencing methods that deliver DNA sequence data from a large number of strains in parallel. Once sequence data is available, bioinformatics tools can be used to identify key sequences encoding enzymes of commercial interest. We will target 5 important enzyme classes during this screen and aim to find new enzymes in Clostridia for each one of these classes. Two of the most promising targets will be amplified and cloned into E.coli to exemplify the research strategy. If successful, this approach will be expanded into a more comprehensive discovery platform for new biocatalysts.
Improving the efficiency of natural product expression and purification in bacterial strains of pharmaceutical relevance by biomimetic culturing – Geertje van Keulen – Swansea University
Project partner – Nick Allenby – Demuris Ltd
Natural products are experiencing a revival due to the lack of novel chemical entities and classes entering the pipeline of antimicrobial drug discovery. The actinomycete culture collection at Demuris Ltd represents an excellent resource for novel NP discovery due to its high biodiversity, which also translates to large chemical diversity. Highly interesting NPs have been identified after screening of the Demuris strain collection using proprietary antifungal bioassays. Development of novel antifungals is pertinent due to invasive fungal infections increasingly becoming resistant to first and second-line treatments, which in turn is currently limited to only three classes of chemotherapy. Antifungal drug discovery with these screened strains is however hampered due to the strains’ poor growth and NP production characteristics in standard laboratory culturing conditions, resulting in very low chemical yields.
We have recently shown that biomimetic culturing is an effective addition to the toolbox of methods for (i) eliciting expression of (cryptic) natural products (NPs), and (ii) for efficient chemical extraction at decent to high yields of NPs from an inert solid biomimetic matrix, using the model NP-producing actinomycete Streptomyces coelicolor. In this short project, we aim to characterise NP production by strains of industrial and pharmaceutical interest via biomimetic culturing. We will also evaluate whether biomimetic culturing is an efficient approach to optimise the yield of antifungal compounds from this three-dimensional matrix, thus allowing structural characterisations.
NPRONET BIV funding will enable us to employ our novel biomimetic culturing method for the first time to industrially relevant strains, especially to strains that are difficult to handle and/or produce novel NPs in media which do not scale up. This short project will allow us to set up a new collaboration with Demuris Ltd, which, if successful, will facilitate systematic exploitation of its bacterial resources at a large scale (Demuris strain collection contains >10,000 dereplicated actinomycetes) for NP discovery by applying for further funding, e.g. to the Life Sciences Research Network Wales and programmes within the RCUK Antimicrobial Resistance themes.
Characterization of a novel antifungal peptide from the entomopathogenic fungus Metarhizium anisopliae – Tariq Butt – Swansea University
Project Partner – Robert Nash – Phytoquest Ltd
Invasive fungal infections cause substantial morbidity and mortality and are a costly, common problem in healthcare settings. Of major concern is the increase in resistance of pathogens including Candida and Aspergillus to current antifungal products. Entomopathogenic fungi (EPF) belonging to the genera Metarhizium, Beauveria, Tolypocladium and Isaria produce an array of secondary metabolites many of which show promise for development as therapeutics. The compounds include cyclic peptides (destruxins, cyclosporin), indolizidine alkaloids (swainsonine) and atypical aminoacids (myriocin). At least two compounds, myriocin and cyclosporine, have been developed as medicines: Myriocin and cyclosporine are immune modulators which were isolated from Tolypocladium inflatum and Isaria sinclarii, respectively. Although antimicrobial activity has been reported in culture filtrates of several EPF very few have been isolated and characterised. This proposal focuses on a small peptide (< 1 kDa) secreted by Metarhizium anisopliae which exhibits strong antifungal activity especially against Candida glabrata, a highly opportunistic pathogen of the urogenital tract and bloodstream of immune compromised persons (e.g. elderly, HIV patients). This fungus, like other Candida species, is becoming increasingly resistant to common antifungal drugs such as fluconazole. There is clearly a need for the development of safe alternatives. Although progress has been made in enriching and partially characterising the Metarhizium-derived anti-Candida compound further work is needed to elucidate its properties. NPRONET funding will enable us to work closely with Phytoquest Ltd in determining: (1) the structure of the peptide and (2) in vivo efficacy using an insect model. Larvae of the greater waxmoth (Galleria mellonella) provide a relatively inexpensive testing system without the ethical implications associated with murine or simian models. The assay is high throughput and the results are easy to score. Importantly, Galleria has an innate immune system with many characteristics similar to the mechanisms of human innate immunity. The success of this short project will allow us to further collaborate with Phytoquest Ltd in exploring the development of this novel compound through an Innovate UK feasibility grant (i.e. industrial biotechnology catalyst-technical feasibility studies) or BBSRC-funded programme.
Identification and testing of genes required for the production of natural UV protectants – Mark Caddick – The University of Liverpool
Project partner – Steve Wilson – Unilever UK
UV is highly damaging to skin and potentially harmful, consequently there is a need to develop good stable ingredients that can minimise these harmful effects. Perhaps surprisingly the number of available compounds suitable for this purpose is very limited and they can have their drawbacks. In nature, a range of compounds exist with major potential for skin protection, however the challenge is to be able to produce these in quantities and at a cost that would make them commercially viable. One major source of these compounds are algae. The aim of this project is to identify and then test the genes responsible for production of one such compound. This will involve sequencing the messenger RNA from different samples of algae followed by computational analysis to identify the genes involved in biosynthesis of the compound of interest, allowing us to eventually test these genes in a simple bacterial system. We also aim to develop and test extraction procedures, comparing the bacteria and algae for both the quantity and purity of these compounds. This will represent a first step in the commercial production