Reaction and catalyst engineering to exploit kinetically controlled whole-cell multistep biocatalysis for terminal FAME oxyfunctionalization.
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.
Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Emil-Figge-Strasse 66, Dortmund, 44227, Germany.
Manfred
M
Schrewe
Mattijs K
MK
Julsing
Kerstin
K
Lange
Eik
E
Czarnotta
Andreas
A
Schmid
Bruno
B
Bühler
D000438:Alcohols / Q000378:metabolism* / Q000633:toxicity; D000447:Aldehydes / Q000378:metabolism* / Q000633:toxicity; D001709:Biotechnology / Q000379:methods; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D007850:Lauric Acids / Q000378:metabolism* / Q000633:toxicity; D060847:Metabolic Engineering; D010084:Oxidation-Reduction; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D000438:Alcohols; D000447:Aldehydes; D007850:Lauric Acids; D011994:Recombinant Proteins; D042926:Cytochrome P-450 CYP4A
AlkBGT; AlkJ; AlkL; E. coli W3310; in situ product recovery; process integration