Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes

Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C-7-C-16 n-alkanes. Without alkL expression, native E. coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C-7-C-16 n-alkanes and in the activation of a whole-cell alkane biosensor by C-10 and C-11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C-12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 mu mol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C-10-C-16 alkanes and represents the highest reported gain in product yields resulting from its use.