Discovery of fungal depsipeptide based on a synthetic biology- based approach Teigo Asai, Yuto Homma, Akihiro Sugawara, Yohei Morishita, Kento Tsukada, Taro Ozaki Tohoku University, Japan A synthetic biology approach by combination use of genome mining and heterologous expression enables us to rationally access new natural products hidden in the fungal genomic information. Fungal depsipeptides and lipopeptides are one of the most attractive sources for natural product-based drug discovery, 1 and their structural frameworks are usually constructed by both highly-reducing polyketide synthase (HR-PKS) and non-ribosomal peptide synthetase (NRPS). Thus, to mine new fungal depsipeptides and lipopeptides, we carried outgenome mining to find biosynthetic gene clusters (BGCs) containing both an HR-PKS gene and an NRPS gene using our draft genome database. During our genome mining effort, we found a candidate cluster, cmlp cluster, composed of an HR-PKS gene ( cmlpA ), an AMP-binding domain-containing enzyme ( cmlpB ), an acyltransferase (AT) gene ( cmlpC ), and an NRPS gene ( cmlpD ) (Figure. 1A ) in Chaetomium mollipilium genomic information. Comparing cmlp cluster with that of emericellamide A suggested this cluster is related to a cyclic depsipeptide. 2 Bioinformatics analyses and reported natural depsipeptides from fungi indicated the possibility of cmlp cluster producing a new cyclic depsipeptide. 2, 3 Therefore, we conducted a heterologous expression of the cmlp cluster to obtain a natural product derived from this BGC.
Figure 1 (A) cmlp cluster (B) putative biosynthetic pathways of 2a and 2b . Based on previously reported biosynthetic studies on fungal cyclic depsipeptide and lipopeptide, 2, 4 we introduced cmlpA, cmlpAC, and cmlpABCD , into Aspergillus oryzae to construct AO-cmlpA, AO-cmlpAC, and AO-cmlpABCD . AO-cmlpAC produced (2 R ,3 R ,4 S )-3-hydroxy-2,4-dimethylhexanoic acid 1 , whereas AO-cmlpA didn't produce any metabolites. Interestingly, this result suggested that CmlpC (AT) is responsible for hydrolytic chain release of 1 from CmlpA (HR-PKS). The metabolite analysis of AO-cmlpABCD showed the production of two equilibrium compounds 2a and 2b , and structure elucidation based on spectral analyses and chemical derivatization revealed their structures to be a new cyclic depsipeptide 2a and its an unprecedented cyclol derivative 2b . We hypothesize the biosynthetic pathway for 2a / 2b based on the results of heterologous expression experiments and the structures of the metabolites (Figure 1B). We also investigated inactive domains in CmlpD (NRPS) by mutagenesis experiments (Figure 1A). We thus predictably obtained new cyclic depsipeptides 2a / 2b based on genome mining and heterologous expression. In our poster, we will discuss the detailed genome mining of a candidate BGC, cmlp cluster, and structure elucidation and biosynthetic mechanisms of new cyclic depsipeptides 2a / 2b . References 1. (a) Wang, X. et al. Molecules 2018 , 23 , 169. (b) Zhao, P. et al. Peptides 2019 , 113 , 52–65. 2. Chiang, Y.–M. et al. Chem. Biol. 2008 , 15 , 527–532. 3. (a) Wang, X. et al. J. Biotech. 2020 , 309 , 85–91. (b) Sinha,S. et al. BMC Genom. 2019 , 20 , 374. (c) Matsui, M. et al. J. Biosci. Bioeng . 2017 , 123 , 147–153. 4. Cacho, R. et al. J. Am. Chem. Soc. 2012 , 134 , 16781–16790.
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