The article introduces the updated version 4 of antiSMASH, a comprehensive open-source platform for mining and analyzing biosynthetic gene clusters (BGCs) from bacterial, fungal, and plant genomes. Key improvements in antiSMASH 4.0 include: 1. **Improved Gene Cluster Boundary Prediction**: New methods, such as the Cluster Assignment by Islands of Sites (CASSIS) algorithm for fungal genomes and the ClusterFinder algorithm for both bacterial and fungal genomes, are added to predict gene cluster boundaries more accurately. 2. **Enhanced Chemistry Prediction**: The SANDPUMA algorithm, which integrates multiple prediction methods, is used to predict substrate specificities for non-ribosomal peptide synthetase adenylation domains. Additionally, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides (RiPPs) are provided. 3. **Comparative Analysis Tools**: A domain-level alignment tool for trans-AT polyketide synthase (PKS) assembly line architectures is introduced, allowing for rapid assessment of biochemical relationships between assembly lines. 4. **User Interface and Usability Improvements**: The user interface has been updated to improve usability, and several other efficiency improvements have been made, including an updated ClusterBlast database and support for GFF3-formatted files. 5. **Additional Features**: The platform now supports TTA codon annotation, which is crucial for understanding the regulation of secondary metabolism in certain bacteria. These enhancements make antiSMASH a more powerful and user-friendly tool for the discovery of novel bioactive molecules through genome mining.The article introduces the updated version 4 of antiSMASH, a comprehensive open-source platform for mining and analyzing biosynthetic gene clusters (BGCs) from bacterial, fungal, and plant genomes. Key improvements in antiSMASH 4.0 include: 1. **Improved Gene Cluster Boundary Prediction**: New methods, such as the Cluster Assignment by Islands of Sites (CASSIS) algorithm for fungal genomes and the ClusterFinder algorithm for both bacterial and fungal genomes, are added to predict gene cluster boundaries more accurately. 2. **Enhanced Chemistry Prediction**: The SANDPUMA algorithm, which integrates multiple prediction methods, is used to predict substrate specificities for non-ribosomal peptide synthetase adenylation domains. Additionally, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides (RiPPs) are provided. 3. **Comparative Analysis Tools**: A domain-level alignment tool for trans-AT polyketide synthase (PKS) assembly line architectures is introduced, allowing for rapid assessment of biochemical relationships between assembly lines. 4. **User Interface and Usability Improvements**: The user interface has been updated to improve usability, and several other efficiency improvements have been made, including an updated ClusterBlast database and support for GFF3-formatted files. 5. **Additional Features**: The platform now supports TTA codon annotation, which is crucial for understanding the regulation of secondary metabolism in certain bacteria. These enhancements make antiSMASH a more powerful and user-friendly tool for the discovery of novel bioactive molecules through genome mining.