Abstract
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 C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 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 μmol/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 C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.
Highlights
Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes
We demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes
Our findings are complemented by a recent report showing that coexpression of AlkL significantly increased oxygenation of nonane and dodecanoic acid methyl esters
Summary
Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes. The alkane degradation pathway native to Pseudomonas putida GPo1 has been the focus of extensive research over the past decades and much is understood about the components of the pathway, the substrate range[1,2], enzyme mechanism[3], electron transport coenzymes[4,5,6,7], regulatory system[8,9,10], effect on host cell physiology[11,12,13,14], performance in recombinant hosts[15,16,17,18] and potential applications[19] It has previously been hypothesised[20,21,22] from the presence of alkL in the alk operon and its position in the outer membrane[23] that it plays an important role in transport of alkanes into the cell. Recent work by others has identified that AlkL can enhance oxidation of nonane, methyl dodecanoate[25] and limonene[26] in resting cells which further supports the hypothesis for the role of alkL in the uptake of medium-chain alkanes
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