Engineering NADPH Production in E. coli with Modified Lipoamide Dehydrogenase

Background:The use of fossil fuels as the primary source of energy concerns environmentally aware citizens and nations. Concerned citizens reduce their carbon footprint by carpooling and other means of transportation, composting or recycling as a means of waste reduction, and using wind turbines or solar panels as sources of renewable energy. Biofuels are another source of renewable energy. Our research group is using modified Escherichia coli as a potential candidate in biofuel research.  In this study, we examined growth of transformed E. coli growth activity on various carbon sources.  Our hypothesis is that E. colican be genetically modified to produce fatty alcohols. Methods: We obtained mutant strains that produce the enzyme lipoamide dehydrogenase (Lpd) and redesigned its active site. These E. coli strains were transformed with the Maqu_2220 plasmid containing an alcohol reductase. We used the Gibson method to replace the gene producing fatty acids for a gene of similar structure that produced fatty alcohols. We grew the transformed bacteria in a series of M-9 solutions with different carbon sources. Optical density readings from a Tecan F200 were used to produce growth curves to better understand how well our different mutant strains would grow on these carbon sources. Gas-chromotagraph-mass-spectrometry (GC-MS) was used to detect the presence of fatty alcohols. Results: We successfully transformed the mutant E. coli and produced three new transformed strains with the Maqu_2220 plasmid. Growth curve data indicate that these mutant strains grown on glucose have similar growth curves to wild type strains; however, these strains grew poorly on gluconate. Growth on other carbon sources was intermediate between these two extremes. GC-MS results indicate that one strain produced the target fatty alcohols. Conclusion: Our preliminary data demonstrate fatty alcohol production in E. coli. Such fatty alcohol production is an important step for developing advanced biofuels.