Quantification of N-acetylcysteamine activated methylmalonate incorporation into polyketide biosynthesis

Abstract

SummaryPolyketides are biosynthesized through consecutive decarboxylative Claisen condensations between a carboxylic acid and differently substituted malonic acid thioesters, both tethered to the giant polyketide synthase enzymes. Individual malonic acid derivatives are typically required to be activated as coenzyme A-thioesters prior to their enzyme-catalyzed transfer onto the polyketide synthase. Control over the selection of malonic acid building blocks promises great potential for the experimental alteration of polyketide structure and bioactivity. One requirement for this endeavor is the supplementation of the bacterial polyketide fermentation system with tailored synthetic thioester-activated malonates. The membrane permeable N-acetylcysteamine has been proposed as a coenzyme A-mimic for this purpose. Here, the incorporation efficiency into different polyketides of N-acetylcysteamine activated methylmalonate is studied and quantified, showing a surprisingly high and transferable activity of these polyketide synthase substrate analogues in vivo.

Flexible enzymatic activation of artificial polyketide extender units by Streptomyces cinnamonensis into the monensin biosynthetic pathway

“…Also, analogous nonactivated building blocks were not accepted for incorporation into the related polyketide erythromycin A in our earlier experiments using S. erythraea (Klopries et al . ).…”

“…Several approaches to AT engineering are currently being discussed in the literature, ranging from reductionistic in vitro approaches using isolated enzymes to holistic in vivo experiments on whole assembly lines in natural producer organisms, typically Actinomycetes (Koryakina and Williams ; Klopries et al . ; Koryakina et al . , ; Musiol‐Kroll et al .…”

“…1b) Bravo-Rodriguez et al 2015;Koryakina et al 2017). Several approaches to AT engineering are currently being discussed in the literature, ranging from reductionistic in vitro approaches using isolated enzymes to holistic in vivo experiments on whole assembly lines in natural producer organisms, typically Actinomycetes (Koryakina and Williams 2011;Klopries et al 2013;Koryakina et al 2013Koryakina et al , 2017Musiol-Kroll et al 2017). Important in AT engineering experiments is the activation of artificial extender unit building blocks that are to be incorporated into biosynthetic pathways.…”

Exploring the Promiscuous Enzymatic Activation of Unnatural Polyketide Extender Units in Vitro and in Vivo for Monensin Biosynthesis

“…While direct supplementation of CoA‐activated malonate derivatives is unsuitable due to its limited membrane permeability and synthetic accessibility, N ‐acetylcysteamine esters (SNAC esters), acting as a CoA‐mimic, have been successfully used in feeding experiments . However, significant synthetic effort ahead of the fermentation, toxicity in fermentations and inferior activation compared to CoA‐thioesters have limited their application …”

Engineering the acyltransferase substrate specificity of assembly line polyketide synthases

“…Ketosynthase (KS) domains accept the polyketide chain from the upstream ACP and catalyse a Claisen-like condensation between it and the ACP-bound a-carboxyacyl-CoA extender AT substrate specificity engineering toolbox AT sequence data [4,[40][41][42][43][44] . Trans-AT complementation erythromycin AT1,2,6/ rapamycin AT2,14 [34,[57][58][59][60][61] erythromycin AT4/niddamycin AT5 [62] erythromycin AT6/FK520 AT8 [63] tylosin AT6/erythromycin AT1 [53] erythromycin AT1,2/ putative AT domains [58] erythromycin AT1/epothilone AT2 [65] erythromycin AT1/ epothilone AT3 [65] geldanamycin AT2,5/ rapamycin AT2,14 [64] geldanamycin AT1,3,4,7/ rapamycin AT2,14 [64] substrate specificity of acceptor/donor AT [52][53] erythromycin AT6/ a structurally identified residue [54] erythromycin AT6/saturation mutagenesis at selected positions based on promiscuous epothilone AT3 [54] MMCoA/MCoA…”