Fasan, Rudi and Crook, Nathan C. and Peters, Matthew W. and Meinhold, Peter and Buelter, Thomas and Landwehr, Marco and Cirino, Patrick C. and Arnold, Frances H. (2011) Improved Product-Per-Glucose Yields in P450-Dependent Propane Biotransformations Using Engineered Escherichia coli. Biotechnology and Bioengineering, 108 (3). pp. 500-510. ISSN 0006-3592 http://resolver.caltech.edu/CaltechAUTHORS:20110301-113228764
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P450-dependent biotransformations in Escherichia coli are attractive for the selective oxidation of organic molecules using mild and sustainable procedures. The overall efficiency of these processes, however, relies on how effectively the NAD(P)H cofactors derived from oxidation of the carbon source are utilized inside the cell to support the heterologous P450-catalyzed reaction. In this work, we investigate the use of metabolic and protein engineering to enhance the product-per-glucose yield (Y_(PPG)) in wholecell reactions involving a proficient NADPH-dependent P450 propane monooxygenase prepared by directed evolution [P450_(PMO)R2; Fasan et al. (2007); Angew Chem Int Ed 46:8414–8418]. Our studies revealed that the metabolism of E. coli (W3110) is able to support only a modest propanol: glucose molar ratio (Y_(PPG)~0.5) under aerobic, nongrowing conditions. By altering key processes involved in NAD(P)H metabolism of the host, considerable improvements of this ratio could be achieved. A metabolically engineered E. coli strain featuring partial inactivation of the endogenous respiratory chain (Δndh) combined with removal of two fermentation pathways (ΔadhE, Δldh) provided the highest YPPG (1.71) among the strains investigated, enabling a 230% more efficient utilization of the energy source (glucose) in the propane biotransformation compared to the native E. coli strain. Using an engineered P450_(PMO)R2 variant which can utilize NADPH and NADH with equal efficiency, we also established that dual cofactor specificity of the P450 enzyme can provide an appreciable improvement in Y_(PPG). Kinetic analyses suggest, however, that much more favorable parameters (K_M, k_(cat)) for the NADH-driven reaction are required to effectively compete with the host’s endogenous NADH-utilizing enzymes. Overall, the metabolic/protein engineering strategies described here can be of general value for improving the performance of NAD(P)H-dependent whole-cell biotransformations in E. coli.
|Additional Information:||© 2010 Wiley Periodicals, Inc. Received 11 July 2010; revision received 10 September 2010; accepted 18 October 2010. Published online 28 October 2010 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bit.22984. This work was supported by USDA grant number 2006-35505-16660 (F.H.A.) and by the NSF grant number BES-0519516 (P.C.C.). This material is also based upon work supported by the U.S. Department of Defence Contract No. DAAD19-02-D-0004.|
|Subject Keywords:||cytochrome P450 monooxygenase; alkane oxidation; BM-3; whole-cell biotransformations; protein engineering; Escherichia coli|
|Official Citation:||Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli Rudi Fasan1, Nathan C. Crook1, Matthew W. Peters2, Peter Meinhold2, Thomas Buelter2, Marco Landwehr2, Patrick C. Cirino3, Frances H. Arnold1,*Article first published online: 10 NOV 2010 DOI: 10.1002/bit.22984|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Ruth Sustaita|
|Deposited On:||03 Mar 2011 16:00|
|Last Modified:||03 Mar 2011 16:00|
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