Objective:Peptidyl alkaloids,a series of important natural products can be assembled by fungal non-ribosomal peptide synthetases(NRPSs).However,many of the NRPSs associated gene clusters are silent under laboratory conditions,and the traditional chemical separation yields are low.In this study,we aim to discovery and efficiently prepare fungal peptidyl alkaloids assembled by fungal NRPSs.Methods:Bioinformatics analysis of gene cluster containing NRPSs from the genome of Penicillium thymi-cola,and heterologous expression of the putative gene cluster in Aspergillus nidulans were performed.Isolation,structural identification,and biological evaluation of the product from heterologous expression were carried out.Results:The putative tri-modular NRPS AncA was heterologous-expressed in A nidulans to give anacine(1)with high yield,which showed moderate and selective cytotoxic activity against A549 cell line.Conclusion:Heterologous expression in A nidulans is an efficient strategy for mining fungal peptidyl alkaloids.