Two Novel Alkane Hydroxylase-Rubredoxin Fusion Genes Isolated from a Dietzia Bacterium and the Functions of Fused Rubredoxin Domains in Long-Chainn-Alkane Degradation

Abstract

ABSTRACTTwo alkane hydroxylase-rubredoxin fusion gene homologs (alkW1andalkW2) were cloned from aDietziastrain, designated DQ12-45-1b, which can grow on crude oil andn-alkanes ranging in length from 6 to 40 carbon atoms as sole carbon sources. Both AlkW1 and AlkW2 have an integral-membrane alkane monooxygenase (AlkB) conserved domain and a rubredoxin (Rd) conserved domain which are fused together. Phylogenetic analysis showed that these two AlkB-fused Rd domains formed a novel third cluster with all the Rds from the alkane hydroxylase-rubredoxin fusion gene clusters in Gram-positive bacteria and that this third cluster was distant from the known AlkG1- and AlkG2-type Rds. Expression of thealkW1gene in DQ12-45-1b was induced when cells were grown on C8to C32n-alkanes as sole carbon sources, but expression of thealkW2gene was not detected. Functional heterologous expression in analkBdeletion mutant ofPseudomonas fluorescensKOB2Δ1 suggested thealkW1could restore the growth of KOB2Δ1 on C14and C16n-alkanes and induce faster growth on C18to C32n-alkanes thanalkW1ΔRd, the Rd domain deletion mutant gene ofalkW1, which also caused faster growth than KOB2Δ1 itself. In addition, the artificial fusion of AlkB from the Gram-negativeP. fluorescensCHA0 and the Rds from both Gram-negativeP. fluorescensCHA0 and Gram-positiveDietziasp. DQ12-45-1b significantly increased the degradation of C32alkane compared to that seen with AlkB itself. In conclusion, thealkW1gene cloned fromDietziaspecies encoded an alkane hydroxylase which increased growth on and degradation ofn-alkanes up to C32in length, with its fused rubredoxin domain being necessary to maintain the functions. In addition, the fusion of alkane hydroxylase and rubredoxin genes from both Gram-positive and -negative bacteria can increase the degradation of long-chainn-alkanes (such as C32) in the Gram-negative bacterium.