To improve the efficiency of acyl-ACP conversion through the two-step pathway to alkane production, we are performing optimization through localization of the two catalytic proteins. Decrease of the distance the intermediates must travel between Acyl-ACP Reductase (AAR) and Aldehyde Decarbonylase (ADC) is expected to improve overall alkane synthesis.
Initially, we attempted a direct fusion of the two enzymes with a linker. Our main approach is to optimize alkane production by re-use of Biobrick parts from a previous iGEM team. For this, we are re-adapting Biobrick parts from 2010 Slovenia's zinc finger violacein biosysnthesis project. By colocalization of the individual violacein producing enzymes in it's five-step pathway, they were able to increase their pigment production by ~6-fold. Our goal for alkane production optimization is to use the zinc finger localization method to significantly increase yields.
Zinc Finger Fusion
Contrary to what was expected, our GCMS data demonstrated that localization has a negative effect on alkane production. Our attempt at zinc finger localization resulted in an alkane production of approximately 7 mg/L, which is significantly less than our delocalized PetroBrick standard, which produced around 36 mg alkanes per liter. Direct protein fusion produced a yield of 0.408 mg/L, which is essentially negligible. Because our alkane yields were low, we refrained from comprehensive testing of our localization constructs, so future groups are encouraged to refine our methods, as localization holds promise to theoretically increase the efficiency of this system.