Production of high-value chemicals in the button mushroom: a synthetic biology toolkit

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Basidiomycota fungi offer a tractable eukaryotic system for the intracellular manipulation of chemical feedstocks and modified therapeutics. Extensive characterisation of the baker's yeast Saccharomyces cerevisiae genome, comprising 5,400 protein-coding genes, has demonstrated some close similarities to human biochemical processes. The increased complexity of Basidiomycota fungi, with 13,000-16,000 protein-coding genes, provides a biotechnological resource of greater bioprocessing potential with similar underlying biochemistry as yeast. Basidiomycota fungi produce a wealth of valuable chemicals that can be used as antibiotics and agrochemicals, as well as enzymes that can be used as biocatalysts. A Basidiomycota strain that serves as a secondary host to express genes from other higher fungi has not yet been developed, and this is hindering the discovery, study and production of high-value chemicals and enzymes from this prolific group of fungi. A model tractable Basidiomycota fungus such as the button mushroom is ideally placed to fill this gap since it has more complexity and bioprocessing potential than other secondary hosts, such as S. cerevisiae and Aspergillus spp.The aim of this programme is to establish a synthetic biology toolkit for use in the button mushroom. As well as being a model organism and the most widely cultivated mushroom in the world, the button mushroom is able to digest lignocellulose. Therefore, in this work, the growth of the button mushroom will be optimised on different carbon sources that may derive from lignocellulosic waste. Fundamental knowledge will be generated of gene expression on the various carbon sources. Constitutive and inducible promoters will be developed, the latter being activated by lignocellulose derivatives. Temporal control of the expression of the genes of interest will be achieved by switching carbon sources between seed and production culture. This is similar to what is being done for the yeast galactose induction system, in which an initial growth phase in glucose is followed by an expression phase in galactose. The toolkit will facilitate novel uses of cultivated fungi in the discovery, study and production of high-value chemicals, such as terpenoid antimicrobials and other therapeutics, and valuable enzymes, such as lipid-processing and lignocellulose-degrading enzymes. The inducible expression elements developed during this programme will enable researchers to use the button mushroom to produce high-value chemicals in stationary phase, such as antibiotics and antifungals, which may be otherwise toxic if produced during the exponential growth phase. The ability of the button mushroom to grow on lignocellulose will facilitate the use of renewable carbon sources and the implementation of sustainable routes to bioproducts.