Plasmids

The DNA fragments encoding 6803 AAR, 73102 AAR, and 7942 AAR were prepared by overlap extension polymerase chain reaction. The codons were optimized for strong expression in E. coli [28]. The codon-optimized DNA fragments coding for BP-1 AAR, 7336 AAR, 7421 AAR, 9313 AAR, Ma AAR, 51142 AAR, 1986 AAR, 9917 AAR, and 0205 AAR were constructed by Eurofin Operon Biotechnologies. For coexpression of AAR and ADO in E. coli, the pETDuet-1 coexpression vector (Novagen) was used, into which the codon-optimized DNA fragments of AAR and ADO were cloned via the NcoI and EcoRI restriction sites and the NdeI and AvrII restriction sites, respectively [14]. Both the AAR and ADO genes are preceded by a T7 promoter, lac operator, and ribosome-binding site. 73102 ADO was used because it is known to have a high alkane-producing activity [5]. All the AAR and ADO proteins had a C-terminal extension of Gly–Ser–Ser–Gly and a 6× His-tag. All the AAR proteins had an additional Gly between the N terminus (Met) and the second residue in order to eliminate the frame shift due to the NcoI restriction site (CCATGG + GT). The ESPRESSO server, which estimates protein expression (http://cblab.meiyaku.jp/ESPRESSO/) [29], predicted that all the AAR and ADO constructs can be expressed in E. coli.