Funding Information We gratefully acknowledge financial support from the European Union's 7th Framework Programme FP7/2007–2013 under grant agreement no. 266025.
Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct
Article first published online: 14 AUG 2013
© 2013 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Thematic Issue on Biomaterials
Volume 6, Issue 6, pages 694–707, November 2013
How to Cite
Scheps, D., Honda Malca, S., Richter, S. M., Marisch, K., Nestl, B. M. and Hauer, B. (2013), Synthesis of ω-hydroxy dodecanoic acid based on an engineered CYP153A fusion construct. Microbial Biotechnology, 6: 694–707. doi: 10.1111/1751-7915.12073
- Issue published online: 11 OCT 2013
- Article first published online: 14 AUG 2013
- Manuscript Accepted: 8 JUN 2013
- Manuscript Revised: 7 JUN 2013
- Manuscript Received: 28 FEB 2013
- European Union's 7th Framework Programme FP7/2007–2013. Grant Number: 266025
A bacterial P450 monooxygenase-based whole cell biocatalyst using Escherichia coli has been applied in the production of ω-hydroxy dodecanoic acid from dodecanoic acid (C12-FA) or the corresponding methyl ester. We have constructed and purified a chimeric protein where the fusion of the monooxygenase CYP153A from Marinobacter aquaeloei to the reductase domain of P450 BM3 from Bacillus megaterium ensures optimal protein expression and efficient electron coupling. The chimera was demonstrated to be functional and three times more efficient than other sets of redox components evaluated. The established fusion protein (CYP153AM. aq.-CPR) was used for the hydroxylation of C12-FA in in vivo studies. These experiments yielded 1.2 g l–1 ω-hydroxy dodecanoic from 10 g l–1 C12-FA with high regioselectivity (> 95%) for the terminal position. As a second strategy, we utilized C12-FA methyl ester as substrate in a two-phase system (5:1 aqueous/organic phase) configuration to overcome low substrate solubility and product toxicity by continuous extraction. The biocatalytic system was further improved with the coexpression of an additional outer membrane transport system (AlkL) to increase the substrate transfer into the cell, resulting in the production of 4 g l–1 ω-hydroxy dodecanoic acid. We further summarized the most important aspects of the whole-cell process and thereupon discuss the limits of the applied oxygenation reactions referring to hydrogen peroxide, acetate and P450 concentrations that impact the efficiency of the production host negatively.