Mass Directed Auto Purification System with DAD and ELSD detectors
Final Report Abstract
The mass-directed purification system has underpinned almost all of the research output of the Cox group in Hannover over the past 4 years and has supported collaborations within Hannover, with other German groups and with international collaborators. The role of the instrument is to give high level analytical information about complex mixtures of natural products, obtained either from natural sources such as fungi, or from bio-engineered sources. The instrument allows rapid and accurate purification of compounds of interest which is a crucial first step in their identification by NMR and other MS methods. At least 13 scientific papers have arisen from its use. Here I summarise some of the main outputs. 1. Investigations of silent fungal gene clusters involved in avirulence signalling. Here we used the instrument to probe the production of secondary metabolites potentially involved in signalling between fungal pathogens and plants during disease processes. We transferred genes known to be highly expressed in fungi during this process to a heterologous host and activated them. This produced new compounds which were identified and isolated using the instrument and this led in-turn to the award of DFG funding to further investigate the biosynthesis of cytochalasins and related compounds. Further work with Bayer is ongoing. 2. Discovery of a new class of fungal metabolites: The Maleidrides. Here the instrument was used during the analysis of gene expression in B. fulva and A. oryzae and again used for the purification of new metabolites and intermediates in the pathways to byssochlamic acid and cornexistin. The work was in collaboration with Syngenta and led to further funding (in the UK) to investigate microorganisms for the production of useful agrochemical compounds. 3. Determination of the True Citrinin Biosynthetic Pathway. Citrinin is a model compound which has been much studied over the years as a key fungal polyketide. We dissected the gene cluster by expression genes one-by-one in A. oryzae. The instrument was used for the purification of unstable intermediates which allowed the complete and correct citrinin pathway to be determined. In-turn this led to a collaboration with a Chinese group which elucidated the biosynthesis of important fungal pigments known as azaphilones used in the food industry. Further work with related sorbicillins is ongoing (DFG). 4. Identification of the Squalestatin Biosynthetic Pathway and its Engineering. Squalestatins are very potent anti-cholesterol compounds obtained from fungi. We identified the fungal gene clusters involved in their biosynthesis and expressed key genes in E. coli allowing in vitro investigation of key acyl-transferase steps. We were able to engineer these in vitro to produce novel squalestatin analogs. The instrument was used in the identification and purification of key intermediateds and products. More recently we have dissected complex oxidation steps involved in the biosynthesis of the multicyclic core of the squlestatins, again by using the instrument to purify highly unstable intermediates for further characterisation. 5. Structural Revision of the Phyllostictines. These metabolites were reported with very unusual structures, but we isolated them using the instrument and we were able to obtain highly pure sampels which allowed reassignment of the structures and correct NMR identification. This in-turn allowed us to find the gene cluster and make targetted knockouts for the production of key intermediates. This allowed the biosynthetic pathway to be fully understood. 6. Biosynthersis of Xenovulenes. These complex molecules possess interesting neuropharmacological properties and are biosyntehsised by fungi. We discivered the biosynthetic genes and engineered the pathway by inserting the genes in A. oryzae. Use of the mass-directed purification system allowed us to isolate key intermediates and determine the presence of a new type of terpene cylase; the first example of a true hetero-Diels Alder enzyme; and unique oxidative ring-contraction enzymes. Current work involves the discovery and exploitation of parallel pathways in fungi.
Publications
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2015. Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae. Chem Sci 6, 4837–4845
Song, Z., Bakeer, W., Marshall, J., Yakasai, A., Khalid, R., Collemare, J., Skellam, E., Tharreau, D., Lebrun, M.-H., Lazarus, C., Bailey, A., Simpson, T., Cox, R.
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2015. Novel nonadride, heptadride and maleic acid metabolites from the byssochlamic acid producer Byssochlamys fulva IMI 40021 – an insight into the biosynthesis of maleidrides. Chem Commun 51, 17088–17091
Szwalbe, A., Williams, K., O’Flynn, D., Bailey, A., Mulholland, N., Vincent, J., Willis, C., Cox, R., Simpson, T.
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2015. The molecular steps of citrinin biosynthesis in fungi. Chem Sci 7, 2119–2127
He, Y., Cox, R.
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2016. Heterologous Production of Fungal Maleidrides Reveals the Cryptic Cyclization Involved in their Biosynthesis. Angewandte Chemie Int Ed 55, 6784–6788
Williams, K., Szwalbe, A., Mulholland, N., Vincent, J., Bailey, A., Willis, C., Simpson, T., Cox, R.
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2017. Genetic and chemical characterisation of the cornexistin pathway provides further insight into maleidride biosynthesis. Chem Commun 53, 7965–7968
Williams, K., Szwalbe, A., Dickson, C., Desson, T., Mulholland, N., Vincent, J., Clough, J., Bailey, A., Butts, C., Willis, C., Simpson, T., Cox, R.
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2017. Orange, red, yellow: biosynthesis of azaphilone pigments in Monascus fungi. Chem Sci 8, 4917– 4925
Chen, W., Chen, R., Liu, Q., He, Y., He, K., Ding, X., Kang, L., Guo, X., Xie, N., Zhou, Y., Lu, Y., Cox, R., Molnár, I., Li, M., Shao, Y., Chen, F.
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2017. Structural Revision and Biosynthesis of the Fungal Phytotoxins Phyllostictines A and B. Journal of Natural Products 80, 1235-1240
Trenti, F., Cox, R.
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2017. Welwitschianalol A and B, two cyclohexene derivatives and other insecticidal constituents of Caesalpinia welwitschiana (Oliv.) Brenan. Phytochem Lett 22, 81–86
Essoung, F., Mba’ning, B., Mohamed, S., Lwande, W., Ngouela, S., Tsamo, E., Chhabra, S., Hassanali, A., Cox, R.
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2018. The cycloaspeptides: uncovering a new model for methylated nonribosomal peptide biosynthesis. Chem Sci 9, 4109–4117
Mattos-Shipley, K., Greco, C., Heard, D., Hough, G., Mulholland, N., Vincent, J., Micklefield, J., Simpson, T., Willis, C., Cox, R., Bailey, A.
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2018. Three previously unrecognised classes of biosynthetic enzymes revealed during the production of xenovulene A. Nature Communications 9, 1963
Schor, R., Schotte, C., Wibberg, D., Kalinowski, J., Cox, R.
