The programming of widely distributed iterative fungal hr-PKS is mysterious, yet it is central for generating polyketide natural product diversity by controlling the chain length, β-processing level and methylation patterns of fungal polyketides. For the iterative hr-PKS TENS, responsible for producing the pentaketide-tyrosine hybrid pretenellin A 1, the chain length programming is known to be determined by the KR domain. Structure prediction of the KR domain enabled the identification of a relevant substrate binding helix, which was the focus of swap experiments with corresponding sequences from the related hr-PKS DMBS and MILS that produce similar hexa- and heptaketides (2, 3). The investigations of chimeric TENS variants expressed in vivo in the host Aspergillus oryzae NSAR1 revealed the substrate binding helix as a promising target for further investigations, evidenced by observed increase of the chain length during swap experiments. Building on these findings, rational engineering of TENS was applied based on structural analysis and sequence alignment. A minimal set of four simultaneous amino acid mutations achieved the re-programming of TENS by producing hexaketides in minor amounts. To refine our understanding and minimize the number of mutations impacting polyketide chain length, we conducted an alanine scan, pinpointing crucial amino acid positions. Our findings give indications on the intrinsic programming of hr-PKS domains by minimal changes in the amino acid sequence as one influence factor for programming.