title: Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes.
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.
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Chris
authors: C
authors: Grant
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Dawid
authors: D
authors: Deszcz
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Yu-Chia
authors: YC
authors: Wei
authors: Dept. of Structural and Molecular Biology, ISMB, University College London, Gower Street, London WC1E 6BT, U.K.
authors: Rubéns Julio
authors: RJ
authors: Martínez-Torres
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Phattaraporn
authors: P
authors: Morris
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Thomas
authors: T
authors: Folliard
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Rakesh
authors: R
authors: Sreenivasan
authors: 1] Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K [2] Dept. of Structural and Molecular Biology, ISMB, University College London, Gower Street, London WC1E 6BT, U.K.
authors: John
authors: J
authors: Ward
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Paul
authors: P
authors: Dalby
authors: Department of Chemical and Biochemical Engineering, Technical University of Denmark, DK 2800 Lyngby, Denmark.
authors: John M
authors: JM
authors: Woodley
authors: Dept. of Biochemical Engineering, Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, U.K.
authors: Frank
authors: F
authors: Baganz
mesh_terms: D000473:Alkanes / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D001692:Biological Transport; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis; D005233:Fatty Alcohols / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D060847:Metabolic Engineering; D008958:Models, Molecular; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000737:chemistry* / Q000378:metabolism; D019076:Transgenes
chemical_list: D000473:Alkanes; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005227:Fatty Acids; D005233:Fatty Alcohols