Abstract
BackgroundSub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions. These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Such advantages have been recently harnessed by metabolic engineers to improve the production of various high-value chemicals via compartmentalized metabolic engineering. However, measuring sub-cellular concentrations of key metabolites represents a grand challenge for compartmentalized metabolic engineering.MethodsTo this end, we developed a synthetic biosensor to measure a key metabolite, acetyl-CoA, in a representative compartment of yeast, the peroxisome. This synthetic biosensor uses enzyme re-localization via PTS1 signal peptides to construct a metabolic pathway in the peroxisome which converts acetyl-CoA to polyhydroxybutyrate (PHB) via three enzymes. The PHB is then quantified by HPLC.ResultsThe biosensor demonstrated the difference in relative peroxisomal acetyl-CoA availability under various culture conditions and was also applied to screening a library of single knockout yeast mutants. The screening identified several mutants with drastically reduced peroxisomal acetyl-CoA and one with potentially increased levels. We expect our synthetic biosensors can be widely used to investigate sub-cellular metabolism and facilitate the “design-build-test” cycle of compartmentalized metabolic engineering.
Highlights
Sub-cellular compartmentalization is used by all eukaryotes and some prokaryotes as a means to create favorable micro-environments for various metabolic reactions
To test the viability of the localized synthetic biosensor, a proof-of-concept study was performed in which yeast cells with PHB-producing genes were analyzed to compare acetyl-CoA levels in the cytosol and peroxisome
One limitation of this biosensor is that the PHB production pathway uses NADPH as a cofactor allowing for the possibility that NADPH will become the limiting reagent at sufficiently high acetyl-CoA levels
Summary
Sub-cellular compartmentalization is used by all eukaryotes and some prokaryotes as a means to create favorable micro-environments for various metabolic reactions These compartments concentrate enzymes and substrates (Choi & Montemagno, 2006), increasing the rate of reaction. A synthetic biosensor to detect peroxisomal acetyl-CoA concentration for compartmentalized metabolic engineering. Sub-cellular compartmentalization is used by cells to create favorable microenvironments for various metabolic reactions These compartments concentrate enzymes, separate competing metabolic reactions, and isolate toxic intermediates. Measuring sub-cellular concentrations of key metabolites represents a grand challenge for compartmentalized metabolic engineering. To this end, we developed a synthetic biosensor to measure a key metabolite, acetyl-CoA, in a representative compartment of yeast, the peroxisome. We expect our synthetic biosensors can be widely used to investigate sub-cellular metabolism and facilitate the ‘‘design-build-test’’ cycle of compartmentalized metabolic engineering
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