A synthetic biology method based on heterologous biosynthesis coupled with genome mining is a promising approach for increasing the opportunities to rationally access natural product with novel structures and biological activities through total biosynthesis and combinatorial biosynthesis. Here, we demonstrate the advantage of the synthetic biology method to explore biological activity-related chemical space through the comprehensive heterologous biosynthesis of fungal decalin-containing diterpenoid pyrones (DDPs). Genome mining reveals putative DDP biosynthetic gene clusters distributed in five fungal genera. In addition, we design extended DDP pathways by combinatorial biosynthesis. In total, ten DDP pathways, including five native pathways, four extended pathways and one shunt pathway, are heterologously reconstituted in a genetically tractable heterologous host, Aspergillus oryzae, resulting in the production of 22 DDPs, including 15 new analogues. We also demonstrate the advantage of expanding the diversity of DDPs to probe various bioactive molecules through a wide range of biological evaluations.

Multidrug-resistant (MDR) bacteria are widespread throughout the world and pose an increasingly serious threat to human and animal health. Besides implementing strict measures to prevent improper antibiotic use, it remains essential that novel antibiotics must be developed. These antibiotics need to exert their activity via mechanisms different from those employed by currently approved antibiotics. In this study, we used several 5-fluorouracil (5-FU) analogues as chemical probes and investigated the potential of these pyrimidine analogues as antibacterial agents. Several 5-FU derivatives exerted potent activity against strains of Gram-positive cocci (GPC) that are susceptible or resistant toward approved antibiotics, without showing cross-resistance. Furthermore, we have provided evidence that the pyrimidine analogues exerted anti-GPC activity via thymineless death by inhibition of thymidylate synthetase (ThyA) and/or inhibition of RNA synthesis. Interestingly, whole genome resequencing of in vitro-selected, pyrimidine analogue-resistant Staphylococcus aureus mutants indicated that S. aureus strains with pyrimidine-analogue resistance induced an amino acid (AA) substitution, deletion, and/or insertion into thymineless-death related proteins except for ThyA, or enhanced the ThyA transcription level. Thus, S. aureus may avoid altering the ThyA function by introducing an AA substitution, suggesting that the pyrimidine analogues, which directly bind to ThyA without phosphorylation, may be more effective and show a higher genetic barrier than the pyrimidines that depend on phosphorylation for activity. The findings of this study may assist in the future development of a novel class of antibiotics for combating MDR GPC, including methicillin-resistant S. aureus and vancomycin-resistant Enterococci.

The structural diversity of natural products and their derivatives have long contributed to the development of new drugs. However, the difficulty in obtaining compounds bearing skeletally novel structures has recently led to a decline of pharmaceutical research into natural products. This paper reports the construction of a meroterpenoid‐like library containing 25 compounds with diverse molecular scaffolds obtained from diversity‐enhanced extracts. This method constitutes an approach for increasing the chemical diversity of natural‐product‐like compounds by combining natural product chemistry and diversity‐oriented synthesis. Extensive pharmacological screening of the library revealed promising compounds for anti‐osteoporotic and anti‐lymphoma/leukemia drugs. This result indicates that the use of diversity‐enhanced extracts is an effective methodology for producing chemical libraries for the purpose of drug discovery.