Reaction and catalyst engineering to exploit kinetically controlled whole‐cell multistep biocatalysis for terminal FAME oxyfunctionalization

Abstract

The oxyfunctionalization of unactivated C−H bonds can selectively and efficiently be catalyzed by oxygenase-containing whole-cell biocatalysts. Recombinant Escherichia coli W3110 containing the alkane monooxygenase AlkBGT and the outer membrane protein AlkL from Pseudomonas putida GPo1 have been shown to efficiently catalyze the terminal oxyfunctionalization of renewable fatty acid methyl esters yielding bifunctional products of interest for polymer synthesis. In this study, AlkBGTL-containing E. coli W3110 is shown to catalyze the multistep conversion of dodecanoic acid methyl ester (DAME) via terminal alcohol and aldehyde to the acid, exhibiting Michaelis-Menten-type kinetics for each reaction step. In two-liquid phase biotransformations, the product formation pattern was found to be controlled by DAME availability. Supplying DAME as bulk organic phase led to accumulation of the terminal alcohol as the predominant product. Limiting DAME availability via application of bis(2-ethylhexyl)phthalate (BEHP) as organic carrier solvent enabled almost exclusive acid accumulation. Furthermore, utilization of BEHP enhanced catalyst stability by reducing toxic effects of substrate and products. A further shift towards the overoxidized products was achieved by co-expression of the gene encoding the alcohol dehydrogenase AlkJ, which was shown to catalyze efficient and irreversible alcohol to aldehyde oxidation in vivo. With DAME as organic phase, the aldehyde accumulated as main product using resting cells containing AlkBGT, AlkL, as well as AlkJ. This study highlights the versatility of whole-cell biocatalysis for synthesis of industrially relevant bifunctional building blocks and demonstrates how integrated reaction and catalyst engineering can be implemented to control product formation patterns in biocatalytic multistep reactions.

Application of AlkBGT and AlkL from Pseudomonas putida GPo1 for Selective Alkyl Ester ω-Oxyfunctionalization in Escherichia coli

“…This was comparable with findings in an earlier work (17). A recent publication by the same group reported the apparent K s values for C 12 oxygenation products (19), and the apparent K s for the aldehyde was lower than for the alcohol. This might apply to C 9 as well, resulting in higher activity for aldehyde oxidation and thus 9CNAME accumulation.…”

“…The AlkBGT enzyme system was successfully applied for -oxidation of NAEE, and an activity of 67% compared to when NAME was used as the substrate when AlkL was also expressed. It has been shown before that AlkB overoxidizes the -hydroxy product of methyl esters (17)(18)(19). AlkB also overoxidizes NAEE to the acid product, after conversion of the hydroxy derivative toward the aldehyde.…”

“…Outer membrane protein AlkL was successfully applied to further increase the activity of E. coli expressing AlkBGT on larger, more hydrophobic molecules. The -oxidation activity of cells harboring AlkL on methyl dodecanoate was improved 28-fold in small-scale assays and 62-fold in a two-liquid-phase setup (18,19).…”

Efficient production of the Nylon 12 monomer ω-aminododecanoic acid methyl ester from renewable dodecanoic acid methyl ester with engineered Escherichia coli

“…To support ODAME formation, the 326 heterologous pathway was amended by the NAD(P)H independent alcohol dehydrogenase AlkJ 327 from P. putida GPo1. AlkJ transfers electrons to the electron transport chain (Kirmair and 328 Skerra, 2014) and thus catalyzes irreversible alcohol oxidation (Schrewe et al, 2014). 329…”

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

“…An Escherichia coli strain was created that expressed monooxygenase AlkB, along with rubredoxin AlkG and rubredoxin reductase AlkT that are necessary to deliver electrons. This strain efficiently ω-oxidized methyl esters of C5 to C12 fatty acids 58,65,78 . The activity with methyl nonanoate as substrate was even higher than with the natural substrate, n-nonane.…”