Data underlying the manuscript 'Evolution-Guided Engineering of Trans-Acyltransferase Polyketide Synthases' by Mabesoone, Leopold-Messer and Minas et al. The repository contains: Sequencing data - Genbanks files of construct designs, ab1 and fasta files for Sanger sequencing. For whole plasmid sequencing, fasta, annotated GenBank files, overviews of sequencing statistics and fastq files for selected plasmids, for which fastq files were provided by the sequencing service, are provided. NMR data - raw data and MestReNova files. Also includes HPLC-MS traces of isolated compounds. HPLC-MS data - Raw data collected on Thermo-Fisher instruments. This data can be analyzed with the Xcalibur software suite. The mzXML data can be analyzed with the Python scripts provided in the scripts folder to generate the images shown in the SI. The data collected for Bacillus and Serratia is MS1 data. The data collected for Gynuella also contains MS-MS data. SCA data - Python scripts, GenBank files and produced data underlying the SCA. Bioactivity data - Data underlying the toxicity assays in Figure S122.