title: Application of AlkBGT and AlkL from Pseudomonas putida GPo1 for Selective Alkyl Ester ω-Oxyfunctionalization in Escherichia coli.
abstract: The enzyme system AlkBGT from Pseudomonas putida GPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (gcdw), 67% of the initial activity on methyl nonanoate. In time-lapse conversions with 5 mM NAEE the main product was 9-hydroxy NAEE (3.6 mM), but also 9-oxo NAEE (0.1 mM) and 9-carboxy NAEE (0.6 mM) were formed. AlkBGT also ω-oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C6 to C10 Increasing the length of the alkyl chain improved the ω-oxidation activity of AlkBGT on esters of C6 and C7 fatty acids. From these esters, application of butyl hexanoate resulted in the highest ω-oxidation activity, 82 U/gcdw Coexpression of AlkL only had a positive effect on ω-functionalization of substrates with a total length of C11 or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for ω-functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids.
  
  
  
  Fatty acid esters are promising renewable starting materials for the production of ω-hydroxy fatty acid esters (ω-HFAEs). ω-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to ω-HFAEs is challenging, as it generates by-products and needs harsh reaction conditions. Biocatalytic production is a promising alternative. In this study, biocatalytic conversion of fatty acid esters toward ω-HFAEs was investigated using whole cells. This was achieved with recombinant Escherichia coli cells that produce the AlkBGT enzymes. These enzymes can produce ω-HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C6 to C10) esterified with ethanol, propanol, or butanol were applied. This is a promising production platform for polymer building blocks that uses renewable substrates and mild reaction conditions.
authors.affiliation: Bioprocess Engineering, Wageningen University, Wageningen, Netherlands youri.vannuland@wur.nl.
authors.forename: Youri M
authors.initials: YM
authors.lastname: van Nuland
authors.affiliation: Biobased Products, Wageningen University and Research Centre, Wageningen, Netherlands.
authors.forename: Gerrit
authors.initials: G
authors.lastname: Eggink
authors.affiliation: Bioprocess Engineering, Wageningen University, Wageningen, Netherlands.
authors.forename: Ruud A
authors.initials: RA
authors.lastname: Weusthuis
mesh_terms: D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D004952:Esters / Q000378:metabolism*; D005227:Fatty Acids / Q000378:metabolism*; D060847:Metabolic Engineering; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D004952:Esters; D005227:Fatty Acids; D011994:Recombinant Proteins; D042926:Cytochrome P-450 CYP4A