Novel strain development for the mycoprotein fungus Fusarium venenatum

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There is ever growing demand for meat replacement protein sources on environmental and health grounds. The fungus Fusarium venenatum is used in the production of mycoprotein, a high-protein, low cholesterol meat substitute sold under the trade name 'Quorn', which has been very commercially successful. A particular strain A3/5 was identified in the 1960s, being chosen for mycoprotein production as it yields a high protein textured product with negligible toxins under industrial growth conditions. However, there are inefficiencies and problems. For example after prolonged growth the fungus is prone to excess branching unsuitable for Quorn production, has low levels of certain vitamins, and there remains a risk of mycotoxin production. Perhaps surprisingly the same strain A3/5 has been used since the 1970s with no strain development. Therefore there is now commercial and environmental interest in obtaining novel strains with improved characteristics for mycoprotein production. In parallel, scientific advances have recently been made in fungal genomics and genetic manipulation meaning that F. venenatum is an attractive model organism for biotechnological exploitation, with research having the potential for significant fundamental scientific and real world applied impact. Experimental Approaches, Methodology and Techniques We plan to use leading genomic, molecular biology, biochemical and classical culturing techniques to explore strain improvement in F. venenatum. A genome sequence of A3/5 is already available to facilitate investigations. (1) Sexual biology of F. venenatum. The species is only known to reproduce asexually, which has impeded strain improvement. However, we have recently developed a molecular sex-test diagnostic, which has identified F. venenatum isolates of different MAT1-1 and MAT1-2 mating type needed for sexual reproduction. We plan to screen isolates by qRT-PCR for expression of genes required for sexual development and then mate chosen isolates under conditions favourable for sexual reproduction, using a range of microscopy to monitor development. If sex can be induced, there is the exciting prospect that strains with desirable characteristics (e.g. higher growth rate and vitamin content, absence of mycotoxins) can be crossed and offspring identified with combinations of favourable traits; the sexual cycle may also generate completely novel, favourable genotypes. This approach is attractive as sexual breeding is a natural, rather than GM, method of strain development allowing exploitation of strains. The discovery of a sexual cycle in F. venenatum would be a world first and provide general insights into fungal sexuality and asexuality. (2) Manipulation of F. venenatum - classical genetics. Pilot studies suggest that diploid strains are more stable under prolonged fermenter growth than the haploid strain A3/5 in current use. We will generate diploid strains to see if more stable production strains with less branching can be obtained, drawing on cutting-edge microscopy and proteomic methods developed at Marlow Foods to monitor hyphal branching and protein composition. We will also develop parasexual crossing to generate novel combinations of traits in haploid strains. (3) Manipulation of F. venenatum - genome editing. GM and CRISPR genome-editing tools will be used to try and alter traits of interest e.g. mycotoxin synthesis and vitamin production. Electronic nose GC-MS will be used to identify undesirable flavour volatiles that also might be removed. Although arising strains could not yet be used commercially, they will provide key insights which might be exploited by (1+2) above.