Ketosynthase engineering enhances activity and shifts specificity towards non-native extender units in type I linear polyketide synthase

Abstract

Engineering modular type I polyketide synthases (PKS) for the targeted incorporation of non-natural 15 substrates to create variations in the polyketide backbone is a long-standing goal of PKS research. Thus far, 16 most approaches focused on engineering the acyltransferase domain (AT) of PKS, whereas the effects of 17 other ubiquitous domains such as the ketosynthase domain (KS) have received much less attention. In this 18 work, we investigated the effects of thirteen active site substitutions in the module 3 KS (KS3) of the 6- 19 deoxyerythronolide B synthase (DEBS) on incorporation of non-natural extender units in vitro. Using a 20 truncated and a complete DEBS assembly line, we show that substitutions of F263 in KS3 invert specificity 21 up to 1,250-fold towards incorporation of non-natural extender units in the terminal position. In contrast, 22 substitutions of I444 in KS3 show up to 8-fold increased production of 6-deoxyerythonolide B (6-dEB) 23 analogues with non-natural extender units at internal positions.