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 substrates to create variations in the polyketide backbone is a long-standing goal of PKS research. Thus far, most approaches focused on engineering the acyltransferase domain (AT) of PKS, whereas the effects of other ubiquitous domains such as the ketosynthase domain (KS) have received much less attention. In this work, we investigated the effects of thirteen active site substitutions in the module 3 KS (KS3) of the 6-deoxyerythronolide B synthase (DEBS) on incorporation of non-natural extender units in vitro. Using a truncated and a complete DEBS assembly line, we show that substitutions of F263 in KS3 invert specificity up to 1,250-fold towards incorporation of non-natural extender units in the terminal position. In contrast, substitutions of I444 in KS3 show up to 8-fold increased production of 6-deoxyerythonolide B (6-dEB) analogues with non-natural extender units at internal positions. The latter notably without compromising overall productivity of the assembly line. Our study further elucidates the underlying mechanisms for these different behaviors, highlighting the potential of KS engineering for the production of designer polyketides in the future.

From waste to health-supporting molecules: biosynthesis of natural products from lignin-, plastic- and seaweed-based monomers using metabolically engineered Streptomyces lividans

“…The optimized instrumental settings applied for the analysis of 3-oxoadipyl-CoA were operation in positive ion mode [M + H] + and specific values for the curtain gas (35 psi), collision gas flow rate (medium), ion spray voltage (4.5 kV), temperature (400 °C), ion source gas (60 psi), entrance potential (10 V), mass of the parent ion ( m/z 910.3) and the daughter ion ( m/z 403.1), declustering potential (173 V), collision energy (43.2 V) and cell exit potential (24.3 V). The settings for the other CoA esters were taken from previous work [ 46 , 93 , 95 , 96 ].…”