Biotransformation of medium-chain alkanes using recombinant P450 monooxygenase from Alcanivorax borkumensis SK2 expressed in Escherichia coli

Abstract

A bioconversion system for medium-chain alkanes was constructed by using a recombinant Escherichia coli whole-cell biocatalyst expressing P450 monooxygenase genes, ferredoxin, and ferredoxin reductase cloned from Alcanivorax borkumensis as an operon. The recombinant E. coli harboring the P450 gene and two related expression component enzymes, ferredoxin and ferredoxin reductase, was constructed in a single vector pET21(a) and successfully expressed in E. coli BL21(DE3) as a soluble form, showing a molecular weight of 53 kDa on 10% SDS-PAGE. When the cell-free extract of E. coli BL21 expressing p450 monooxygenase was subjected to reduced CO difference spectral analysis, a soret band near 450 nm appeared indicating that the cloned P450 was expressed as a functionally active enzyme. The E. coli cells harboring the expressed P450 gene were able to convert n-octane and 1-decene, producing approximately 450 µg/ml of n-octanol and 290 µg/ml of 1,2-epoxydecane, respectively, at pH 7.0 and 30 o C. However, the recombinant E. coli cells were not able to convert the branched alkane, 2,6,10,14-tetramethylpentadecane (C19).

Elucidation of multiple alkane hydroxylase systems in biodegradation of crude oil n ‐alkane pollution by Pseudomonas aeruginosa DN1

“…In many cases, but not always, these alkane hydroxylases have different substrate ranges or different induction patterns. Most of AlkB or AlkB‐like family and cytochromes P450 prefer to degrade medium‐chain n ‐alkanes, which have been found in both Gram‐positive and Gram‐negative bacteria and show a high sequence diversity (Baik ; Xu et al . ).…”

The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6

“…Molecular methods for enzyme engineering such as rational design, directed evolution and site directed mutagenesis have been reported to yield up to 8-fold improvement for CYP whole-cell processes while increments of over 25-fold have been achieved by considering factors such as microbial host selection, reaction and process optimisation [3,9-15]. For the hydroxylation of alkanes using whole cell biocatalysts expressing CYP153s, significant studies have centred on the choice of host organism, vector systems and other molecular tools to improve protein expression and whole cell activity [4,5,8,16-18]. Although this has led to meaningful progress in biocatalyst design, productivity in most in vivo alkane biotransformation studies fall outside the operating window described as essential for fine chemical synthesis, a minimum of 0.1 g -1  l -1  h -1 [12].…”

Corksorb Enhances Alkane Degradation by Hydrocarbonoclastic Bacteria

“…A mixture of n -alkanes, the main components of crude oil, was used as carbon and energy source. Two different bacterial strains were tested: R. opacus B4, a Gram-positive bacterium able to degrade both aromatic and aliphatic hydrocarbons ( Na et al, 2005 ; Castro et al, 2016 ), and A. borkumensis SK2, a marine Gram-negative bacterium isolated from seawater/sediment samples that uses almost exclusively alkanes as carbon and energy source ( Yakimov et al, 1998 ; Baik, 2010 ). Transcriptomics analysis of A. borkumensis SK2 growing on alkanes in the presence and absence of corksorb was also performed.…”