Rational design of fatty acid photodecarboxylase enables the efficient decarboxylation of medium- and short-chain fatty acids for the production of gasoline bio-alkanes

Fatty acid photodecarboxylase (CvFAP) from Chlorella variabilis NC64A can convert efficiently long-chain fatty acids to hydrocarbons via decarboxylation, and offer a new opportunity for bio-alkanes production. However, the catalytic activity of CvFAP towards medium- and short- fatty acids significantly decreased, leading to a great limitation for the application of CvFAP into the production of gasoline bio-alkanes, which are one of the most important fuels in our modern society. To improve the catalytic efficiency of CvFAP toward these specific fatty acids, a rational design based on the strategy of ‘Focused Rational Iterative Site-specific Mutagenesis’ (FRISM) was performed, where only dozens of mutants needed to be screened. Two mutants (I398L and P460A/G462A) with 29 to 552-fold increasement in decarboxylation catalytic efficiency (kcat/Km) toward different fatty acids (C3-C14) were achieved. The substrate scope of the best mutants is quite broad. Gram-scale reactions were also tested successfully. MD simulations explained the results of improved catalytic activity of mutants for divergent substrates. Based on our experimental results, the best mutants in this study exhibited great potential to generate high value-added gasoline alkanes.