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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
Reproducibility of fluorescent expression from engineered biological constructs in E. coli
We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe