Stereochemistry in Biology and Synthetic Biology
thesisposted on 2020-12-01, 00:00 authored by Taylor Kornfuehrer
As molecular asymmetry is a defining feature of living systems, stereochemistry is of crucial importance in biological studies. Herein, stereochemistry is explored at the level of both primary and secondary metabolism. First, we describe a novel cellular mechanism for maintenance of cofactor homochirality in bacteria. S-adenosyl-L-methionine (SAM) is a biochemical cofactor that is ubiquitous across all domains of life. It is the primary methyl donor in various methylation reactions involving DNA, RNA, and proteins. Under physiological conditions, the active form (S,S)-SAM spontaneously racemizes about its sulfonium center to (R,S)-SAM. The (R,S) form is inactive as a methyl donor and has been suggested to inhibit methyltransferases, which may lead to cellular damage over time. Previous studies identified a bacterial domain of unknown function, DUF62, that can hydrolyze SAM. However, these studies did not examine the differential hydrolysis of the (S,S) and (R,S) forms. Herein, we show that a recombinant protein containing DUF62 from the actinomycete bacterium Salinispora tropica functions as a stereoselective (R,S)-SAM hydrolase. Polyketide natural products exhibit remarkable structural diversity and equally variable biological activity. The biosynthetic logic of polyketide synnthases (PKSs) makes polyketides an attractive target for the employment of synthetic biology techniques to generate non-natural analogues with enhanced therapeutic properties. Biosynthesis of polyketides and synthetic biology strategies for effecting structural modifications are reviewed. Using the anticancer pladienolide/FD-895 class of macrolides as a model, we describe preliminary efforts to employ a reductive loop swap for alteration of stereochemistry. We successfully cloning of the third extension module of the PKS and purified recombinant protein for use in in vitro extension assays. Using an evolution-guided approach, we identified a pool of donor reductive loops and performed in silico analyses to determine the best candidates. Additionally, we carried out molecular and microbiological examination of the pladienolide-producing strain, Streptomyces sp. FERM BP-7812.