2 research outputs found
Predictive Model for Epoxide Hydrolase-Generated Stereochemistry in the Biosynthesis of Nine-Membered Enediyne Antitumor Antibiotics
Nine-membered enediyne antitumor
antibiotics C-1027, neocarzinostatin
(NCS), and kedarcidin (KED) possess enediyne cores to which activity-modulating
peripheral moieties are attached via (<i>R</i>)- or (<i>S</i>)-vicinal diols. We have previously shown that this stereochemical
difference arises from hydrolysis of epoxide precursors by epoxide
hydrolases (EHs) with different regioselectivities. The inverting
EHs, such as SgcF, hydrolyze an (<i>S</i>)-epoxide substrate
to yield an (<i>R</i>)-diol in C-1027 biosynthesis, whereas
the retaining EHs, such as NcsF2 and KedF, hydrolyze an (<i>S</i>)-epoxide substrate to yield an (<i>S</i>)-diol in NCS
and KED biosynthesis. We now report the characterization of a series
of EH mutants and provide a predictive model for EH regioselectivity
in the biosynthesis of the nine-membered enediyne antitumor antibiotics.
A W236Y mutation in SgcF increased the retaining activity toward (<i>S</i>)-styrene oxide by 3-fold, and a W236Y/Q237M double mutation
in SgcF, mimicking NcsF2 and KedF, resulted in a 20-fold increase
in the retaining activity. To test the predictive utility of these
mutations, two putative enediyne biosynthesis-associated EHs were
identified by genome mining and confirmed as inverting enzymes, SpoF
from Salinospora tropica CNB-440 and
SgrF (SGR_625) from Streptomyces griseus IFO 13350. Finally, phylogenetic analysis of EHs revealed a familial
classification according to inverting versus retaining activity. Taken
together, these results provide a predictive model for vicinal diol
stereochemistry in enediyne biosynthesis and set the stage for further
elucidating the origins of EH regioselectivity
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Engineered Production of Short-Chain Acyl-Coenzyme A Esters in <i>Saccharomyces cerevisiae</i>
Short-chain acyl-coenzyme A esters
serve as intermediate compounds
in fatty acid biosynthesis, and the production of polyketides, biopolymers
and other value-added chemicals. <i>S. cerevisiae</i> is a model organism that has been utilized for the biosynthesis
of such biologically and economically valuable compounds. However,
its limited repertoire of short-chain acyl-CoAs effectively prevents
its application as a production host for a plethora of natural products.
Therefore, we introduced biosynthetic metabolic pathways to five different
acyl-CoA esters into <i>S. cerevisiae</i>. Our engineered
strains provide the following acyl-CoAs: propionyl-CoA, methylmalonyl-CoA, <i>n</i>-butyryl-CoA, isovaleryl-CoA and <i>n</i>-hexanoyl-CoA.
We established a yeast-specific metabolite extraction protocol to
determine the intracellular acyl-CoA concentrations in the engineered
strains. Propionyl-CoA was produced at 4–9 μM; methylmalonyl-CoA
at 0.5 μM; and isovaleryl-CoA, <i>n</i>-butyryl-CoA,
and <i>n</i>-hexanoyl-CoA at 6 μM each. The acyl-CoAs
produced in this study are common building blocks of secondary metabolites
and will enable the engineered production of a variety of natural
products in <i>S. cerevisiae</i>. By providing this
toolbox of acyl-CoA producing strains, we have laid the foundation
to explore <i>S. cerevisiae</i> as a heterologous production
host for novel secondary metabolites