Fungi are microorganisms which are found throughout the world and are responsible for many natural processes including the decomposition of plant material and subsequent release of carbon, the preparation of food and beverages, as the source of pharmaceuticals, as well as the cause of a variety of diseases. It is estimated there are 1.5 million species of fungi in the world and the largest organism alive in the world is a fungus colony. Fungi have the ability to make compounds (metabolites) which affect other organisms. A typical example is the antibiotic penicillin. Fungi also produce metabolites that affect weeds (herbicides) and insects (insecticides). Although such herbicides have been reported in the literature useful leads for wide scale use are very rare. Also a natural compound can be evaluated for use in organic farming where there are currently few/no herbicides available. Mevalocidin, the target of this proposal is such an herbicide. The two fungi which have been shown to produce mevalocidin are unique to the Mycosynthetix collection (although they have been deposited in the American Type Culture Collection). By growing these fungi herbicidal activity caused by mevalocidin is produced and this can be detected by it's effect on test plants. Mevalocidin has been subsequently purified and tested against a variety of plants and shows an interesting profile of activity. The yield of mevalocidin production is however low, which limits its use for developing it as a commercial herbicide, and this project attempts to address this problem. After developing a method of accurately measuring mevalocidin, a variety of traditional approaches will be used to improve the yield. Our goal is to be able to make 1 gram per liter of growth medium - at this point we will have a viable product to be used as a new. natural and organic herbicide. OBJECTIVES: This project is focused on a new fungally-derived herbicide, Mevalocidin which has been patented for use in organic farming. Mevalocidin is a novel herbicide produced by filamentous fungi Fusarium sp. DA056446 and Nodulisporum sp. DA092917 from the Mycosynthetix collection. Discovered and patented by DowAgrosciences (DAS) (Gerwick et al, 2008) from the two strains owned by Mycosynthetix, Inc., it was subsequently licensed back to Mycosynthetix. There are few if any other herbicides which could be used for organic farming and therefore Mevalocidin is of high interest. Mycosynthetix owns the rights to develop and market Mevalocidin. By increasing the productivity of the fungi, Fusarium sp. DA056446 and Nodulisporum sp. DA092917, which make this herbicide, we will be in a position to develop the first specific organic herbicide that we are aware of. Moreover, while Mycosynthetix Inc scientists core strengths are fermentation development, we will collaborate with consultants, and/or a third party more familiar with herbicide research and development to take this product to the market as either our own, or as a licensed, product. The following technical goals will be addressed during the project; Develop a physical assay method for measuring Mevalocidin concentration in fermentation extracts; this will involve chromatography, and we shall evaluate using an HPLC-based method. Media investigation using both Fusarium sp. DA056446 and Nodulisporum sp. DA092917 Strain development The effect of co-culture on yield The use of membrane-supports on yield The effect of gross gene regulation using so-called epigenetic effectors Substrate (precursor) feeding and its effect on yield Our anticipated overall goal is to develop a strain of fungus which will produce at least 1 gm per liter of culture and thus provide a starting point for a viable herbicidal material with a minimum amount of further processing. APPROACH: Mevalocidin is produced by culturing either of two fungi in nutrient medium. The following experiments will be conducted; during the optimization phase of the work both fungi will be subjected to the same protocols; if one fungus continues to outperform the other, then at some point during this work our focus will be on the former. 1. Develop a physical assay method for measuring mevalocidin concentration in fermentation extracts; this will involve chromatography, and we shall evaluate using an HPLC-based method. We will initially investigate methods based on those developed by DAS, i.e., using HPLC. 2, Media investigation using both Fusarium sp. DA056446 and Nodulisporum sp. DA092917. This series of experiments will constitute the main effort of the project. In the initial screening experiments DA056446 produced maximum herbicidal activity on malt extract agar and strain DA092917 produced maximum activity on oatmeal agar. Using these observations as a starting point, we will culture both strains using a wide variety of standard fungal media including those above, and including a variety of complex and defined media. 3. Strain development (Elander,1989). The fungi being studied are both filamentous and success of the strain development will to an extent depend on our ability to generate spores which can be subsequently germinated to a clonal filamentous growth. We have a lot of experience in growing and sporulating fungi, and typically will vary the media, temperature of cultivation, light/dark cycles, and incubation period. The initial development of penicillin fermentation followed this approach and the current yield of penicillin G from Penicillium chrysogenum is 14000 times that which was initially observed. 4. The effect of co-culture on yield. It has been reported that culturing fungi together with other fungi or bacteria can improve the yield of the desired product(for example see Zuck et al, 2011). We plan on initiating experiments to investigate this phenomena. 5. The use of membrane-supports on yield. Some fungi respond to being grow on a physical support, and produce higher levels of metabolites that when grown in liquid media (for example see Bigelis et al, 2006). In our research we have used a small bioreactor (Reacsyn, Biodiversity, UK., shown below) which provides a membrane which allows nutrients to be supplied to the growing fungus, which in turn colonizes the membrane. 6. The effect of gross gene regulation using so-called epigenetic effectors. A fungus needs to be able to turn on biosynthetic genes for a given pathway before that product can be made, and up to a point (limitations of substrates) the more enzymes for a given pathway then there will be more product biosynthesized. Research into the expression of pathways has shown from a practical perspective that either genetic modification to deregulate a specific enzymes(s), or by adding certain chemicals to a culture are ways of enhancing this process. 7. Substrate feeding and its effect on yield. The use of feeding exogenous material to support yield improvement is a traditional method and has been employed for a number of fungal products including penicillins
