Abstract
BackgroundThe engineering of metabolism holds tremendous promise for the production of desirable metabolites, particularly alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. Iron sulfur proteins therefore require mechanisms to ensure interaction between proper partners, analogous to many signal transduction proteins. While there has been progress in the isolation of engineered metabolic pathways in recent years, the design of insulated electron metabolism circuits in vivo has not been pursued.ResultsHere we show that a synthetic hydrogen-producing electron transfer circuit in Escherichia coli can be insulated from existing cellular metabolism via multiple approaches, in many cases improving the function of the pathway. Our circuit is composed of heterologously expressed [Fe-Fe]-hydrogenase, ferredoxin, and pyruvate-ferredoxin oxidoreductase (PFOR), allowing the production of hydrogen gas to be coupled to the breakdown of glucose. We show that this synthetic pathway can be insulated through the deletion of competing reactions, rational engineering of protein interaction surfaces, direct protein fusion of interacting partners, and co-localization of pathway components on heterologous protein scaffolds.ConclusionsThrough the construction and characterization of a synthetic metabolic circuit in vivo, we demonstrate a novel system that allows for predictable engineering of an insulated electron transfer pathway. The development of this system demonstrates working principles for the optimization of engineered pathways for alternative energy production, as well as for understanding how electron transfer between proteins is controlled.
Highlights
The engineering of metabolism holds tremendous promise for the production of desirable metabolites, alternative fuels and other highly reduced molecules
Hydrogenase genes from Chlamydomonas reinhardtii and the ferredoxin I gene from Spinacia olearcea were commercially synthesized by Codon Devices (Cambridge, MA), codon optimized for expression in Saccharomyces cerevisiae and acceptable for use in E. coli for wide applicability
pyruvate-ferredoxin oxidoreductase (PFOR) from Desulfovibrio africanus was isolated from plasmid pLP1 [28] provided by Laetitia Pieulle (Centre National de la Recherche Scientifique, Marseille, France) and ydbK was obtained through colony PCR of E. coli BL21
Summary
The engineering of metabolism holds tremendous promise for the production of desirable metabolites, alternative fuels and other highly reduced molecules. Engineering approaches must redirect the transfer of chemical reducing equivalents, preventing these electrons from being lost to general cellular metabolism. This is especially the case for high energy electrons stored in iron-sulfur clusters within proteins, which are readily transferred when two such clusters are brought in close proximity. There are many appealing applications for such engineered electron transfer systems in vitro, such as miniaturized biofuel cells, biocatalysts, and biosensors [7]. These approaches, do not take advantage of the self-assembly and self-regenerating abilities of live cells. Engineered cellular pathways in vivo have the potential to impact our understanding of cellular electron transfer systems in live cells and may provide self-renewing platforms for the continuous production of fuels and other useful molecules [9]
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