Expansion of the ω‐oxidation system AlkBGTL of Pseudomonas putida GPo1 with AlkJ and AlkH results in exclusive mono‐esterified dicarboxylic acid production in E. coli

Abstract

SummaryThe AlkBGTL proteins coded on the alk operon from Pseudomonas putida GPo1 can selectively ω‐oxidize ethyl esters of C6 to C10 fatty acids in whole‐cell conversions with Escherichia coli. The major product in these conversions is the ω‐alcohol. However, AlkB also has the capacity to overoxidize the substrate to the ω‐aldehyde and ω‐acid. In this study, we show that alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH are able to oxidize ω‐alcohols and ω‐aldehydes of esterified fatty acids respectively. Resting E. coli expressing AlkBGTHJL enabled exclusive mono‐ethyl azelate production from ethyl nonanoate, with an initial specific activity of 61 U gcdw −1. Within 2 h, this strain produced 3.53 mM mono‐ethyl azelate, with a yield of 0.68 mol mol−1. This strain also produced mono‐ethyl dicarboxylic acids from ethyl esters of C6 to C10 fatty acids and mono‐methyl azelate from methyl nonanoate. Adding ethyl nonanoate dissolved in carrier solvent bis‐(2‐ethylhexyl) phthalate enabled an increase in product titres to 15.55 mM in two‐liquid phase conversions. These findings indicate that E. coli expressing AlkBGTHJL is an effective producer of mono‐esterified dicarboxylic acids from fatty acid esters.

Biocatalytic, one-pot diterminal oxidation and esterification of n-alkanes for production of α,ω-diol and α,ω-dicarboxylic acid esters

“…The dehydrogenation module consisted of the alcohol dehydrogenase AlkJ and the aldehyde dehydrogenase AlkH, both from Pseudomonas putida GPo1. Like AlkB from module M, it oxidizes alcohols to acids, but is more efficient with respect to cofactor utilization and oxygen consumption (38). We designed E2 based on acyl-CoA ligase AlkK (P. putida GPo1) and the AAT AtfA (from Acinetobacter baylyi).…”

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

“…These ethyl esters were ω-oxidized, resulting in production of 0.36 mM of mono-ethyl azelate after 19 h. This shows that further oxidation to the carboxylic acid is more efficient with ω-hydroxy fatty acid ethyl esters than with ω-hydroxy fatty acids. This is in line with earlier findings, where ethyl esters served as substrate 119 . In contrast to nonanoic acid, hexanoic and octanoic acid were not efficiently converted to MEDA.…”

“…The ω-oxidation module system has been successfully tested before 119 . This system was expanded with AlkK and AtfA to realize the formation of esters.…”

“…The dehydrogenation module consisted of the alcohol dehydrogenase AlkJ and the aldehyde dehydrogenase AlkH, both from Pseudomonas putida GPo1. Like AlkB from module M, it oxidizes alcohols to acids, but is more efficient with respect to cofactor utilization and oxygen consumption 119 . We designed E2 and E3, based on acyl-CoA ligase AlkK (P. putida GPo1) and the AATs AtfA (Acinetobacter baylyi) and Eeb1 (Saccharomyces cerevisiae).…”

“…Its main product is the ω-alcohol, and the ω-aldehyde and carboxylic acid are formed to a limited extent. Recently, we have expanded the AlkBGTL system with alcohol dehydrogenase AlkJ and aldehyde dehydrogenase AlkH in E. coli, resulting in the exclusive and efficient production of MEDAs from esterified fatty acids 119 . However, the AlkBGTLHJ system is not efficient in ω-oxidizing non-esterified medium-chain fatty acids.…”