Programmed Methylation and Editing in Fungal Polyketide Biosynthesis

biosynthesis;polyketide;methylation;not listed

Storm, Philip AlanTownsend, Craig A ;

Johns Hopkins University

关键词: biosynthesis;    polyketide;    methylation;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/60237/STORM-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Fungal polyketides are natural products assembled by polyketide synthases (PKSs), and among this rich class of secondary metabolites are potent toxins, pigments, and best-selling pharmaceuticals. Such diverse bioactivities all originate from the same core biosynthetic reactions. PKSs catalyze the condensation and modification of small acyl substrates to generate complex scaffolds in an iterative and programmed fashion. Substrates and intermediates are tethered to the PKS and delivered to client domains that act repeatedly during the synthesis of a single product molecule. Non-reducing PKSs (NR-PKSs) generate linear poly-β-ketone intermediates of a specific chain length that are subsequently cyclized in a regiospecific fashion and released to give aromatic mono- and polycyclic products, many of which are further modified by downstream enzymes. In the last decade, significant effort in the Townsend lab has been devoted to understanding NR-PKS programming, a challenging task given the size of these megasynthases and the lack of isolable intermediates in the native producers. Previous work pioneered a domain deconstruction approach, whereby NR-PKSs are dissected at the genetic level to generate mono- and multidomain fragments through heterologous expression. Reconstituting these fragments in vitro enables examination of specific domain combinations, shedding light on the mechanisms and programming of individual catalytic domains. Domain deconstruction has also provided opportunities to obtain atomic-resolution structures of NR-PKS domains. Some NR-PKSs contain an embedded C-methyltransferase (CMeT) that installs one or more methyl groups onto the polyketide scaffold, but little was known about the mechanism or programming of this enzymatic alkylation. In this work, we extend the domain deconstruction approach to explore NR-PKS C-methylation, particularly in the biosynthesis of citrinin, a fungal toxin. In addition to biochemical experiments designed to tease apart how different CMeTs install different methylation patterns, we solved the first crystal structure of a NR-PKS CMeT and found characteristic features that are consistent across all known PKS CMeTs, from marine bacteria to pathogenic fungi. Finally, we identified an editing hydrolase in gene clusters with CMeT-containing NR-PKSs and propose that it serves to maintain efficient biosynthesis by removing off-path intermediates from the PKS in trans. The work in this dissertation will serve as a foundation to rationally engineer methylation and editing of polyketide biosynthesis.