Repurposing a megasynthase biocatalyst towards the production of industrial surfactants

Abstract

In Nature, thousands of diverse, bioactive polyketides are assembled by a family of multifunctional “assembly line” enzyme complexes called polyketide synthases (PKS). Since the discovery of 6-deoxyerythronolide B synthase (DEBS) in the late 20th century, many attempts have been made to decode, re-arrange and “re-programme” the PKS assembly line to generate value chemicals such as biofuels and platform chemicals. Here, the first module from the MtPKS12 (hereby termed [M₁]) was repurposed towards the production of 2-methyltetradecanol (2-MTDOL), a valuable lubricant with wide applications in industry. Following a bottom-up, deconstructive study of the 440 kDa fully-reducing megasynthase, MtPKS12 [M1] was shown to condense 2(S)-methylmalonyl-CoA with C₁₂-CoA in a single-turnover assay to produce a 2-methyltetradecyl (2-MTD) scaffold, an important intermediate towards 2- MTDOL. The synthetic utility of MtPKS12 was further demonstrated with the successful production of C₁₃-C₂₁ methyl-branched scaffolds using C₁₀-C₁₈-CoA substrates. Following this, three trans-acting auxiliary enzymes (MtPapA5, UcFatB and the Mycobacterial protein Rv2047c) were investigated for the enzymatic release of polyketide products from MtPKS12. All three candidates presented significant barriers ranging from a lack of crosstalk to insolubility. The investigation concluded with the successful utilisation of ketosynthase (KS) – sourced from MtPKS12 module 2 ([M₂]) – to untether intermediates from MtPKS12 [M₁]. By exploiting the mutual recognition between the two MtPKS12 modules, the 2-MTD scaffold was hydrolysed to produce 2-methyltetradecanoic acid (2-MTDA). The challenge remains to improve substrate and intermediate flux through the PKS to benefit product titres, which presently remains very low. The study herein demonstrates the feasibility of repurposing a fully-reducing PKS module towards the manufacture of useful 2,2-dialkyl chemicals.