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 -functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this -functionalization. In this report, it is shown that whole cells of Escherichia coli expressing the AlkBGT system can also -oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher -oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (g cdw ), 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 -oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C 6 to C 10 . Increasing the length of the alkyl chain improved the -oxidation activity of AlkBGT on esters of C 6 and C 7 fatty acids. From these esters, application of butyl hexanoate resulted in the highest -oxidation activity, 82 U/g cdw . Coexpression of AlkL only had a positive effect on -functionalization of substrates with a total length of C 11 or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for -functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids. IMPORTANCEFatty acid esters are promising renewable starting materials for the production of -hydroxy fatty acid esters (-HFAEs).-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to -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 -HFAEs was investigated using whole cells. This was achieved with recombinant Escherichia coli cells that produce the AlkBGT enzymes. These enzymes can produce -HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C 6 to C 10 ) 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. T he global demand for polymers is expected to grow in the coming years (1) and thus also the need for sustainable polymer production processes. -Hydroxy fatty acids (-HFAs) and dicarboxylic acids (DCAs) are building blocks of polymers such as polyesters and polyamides (1-3). These compounds can be produced from medium-chain-length fatty acids (MCFAs) by oxidation of the terminal methyl group, a reaction called -oxidation (4).A recent development is the production of fatty acids by processes using microbial chain elongation from organic waste material, yielding both odd-and even-chain-length fatty acids ranging from C 4 to C 9 (5, 6).Chemical -oxidation of nonactivated terminal methyl groups remains challenging due to the inert nature of these bonds. This results in poor selectivity (7,8). Biocatalytic -oxidation can be a solution for the terminal activation of fatty acid (est...

Production of medium-chain, a, omega-bifunctional monomers from fatty acids and n-alkanes

“…Pathway AlkBGTHJL facilitated exclusive mono-ethyl DCA production from ethyl nonanoate and methyl nonanoate. This exclusive production was a clear improvement compared to earlier studies, wherein AlkBGTL or AlkBGTJL enzymes were applied 58,65,78,116 . Furthermore, mono-ethyl dicarboxylic acid production using AlkBGTHJL also requires less energy and oxygen input than when only AlkBGTL is applied.…”

“…This could be due to the fact that a methyl ester was the substrate. AlkB activities are higher on methyl nonanoate then on ethyl nonanoate 58,116 ; this might apply to the ω-aldehydes as well. Still, this would not explain the large differences between the rates of alcohol and aldehyde oxidation.…”

“…Ethyl heptanoate does not seem to be a good substrate, as can be concluded from the low product titres. Methyl/ethyl heptanoate were tested before as substrates for the AlkBGT system and were also not ω-oxidized efficiently 65,116 . The reason for this remains unknown.…”

“…Recently, we have demonstrated that E. coli expressing AlkBGT(L) can also ω-oxidize esters with an alkyl chain >1 116 . By increasing the alkyl chain length, the ω-oxidation activity on shorter fatty acid esters improved greatly.…”

“…AlkB overoxidizes ethyl nonanoate to 9-oxo ethyl nonanoate and mono-ethyl azelate 116 . We determined this overoxidation activity of whole cells expressing AlkBGTL from the pBTL10 plasmid by the addition of the intermediates 9-hydroxy ethyl nonanoate and 9-oxo methyl nonanoate and measuring the initial activities (Table 3.1).…”