دورية أكاديمية
Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli.
| العنوان: | Improved product-per-glucose yields in P450-dependent propane biotransformations using engineered Escherichia coli. |
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| المؤلفون: | Fasan R; Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA., Crook NC, Peters MW, Meinhold P, Buelter T, Landwehr M, Cirino PC, Arnold FH |
| المصدر: | Biotechnology and bioengineering [Biotechnol Bioeng] 2011 Mar; Vol. 108 (3), pp. 500-10. Date of Electronic Publication: 2010 Nov 10. |
| نوع المنشور: | Journal Article; Research Support, U.S. Gov't, Non-P.H.S. |
| اللغة: | English |
| بيانات الدورية: | Publisher: Wiley Country of Publication: United States NLM ID: 7502021 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1097-0290 (Electronic) Linking ISSN: 00063592 NLM ISO Abbreviation: Biotechnol Bioeng Subsets: MEDLINE |
| أسماء مطبوعة: | Publication: <2005->: Hoboken, NJ : Wiley Original Publication: New York, Wiley. |
| مواضيع طبية MeSH: | Cytochrome P-450 Enzyme System/*metabolism , Escherichia coli/*genetics , Escherichia coli/*metabolism , Glucose/*metabolism , Metabolic Networks and Pathways/*genetics , Propane/*metabolism, Biotransformation ; Genetic Engineering ; NADP/metabolism ; Organisms, Genetically Modified ; Oxidation-Reduction ; Recombinant Proteins/genetics |
| مستخلص: | 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 whole-cell 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 (YPPG ~ 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 Y(PPG) (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. (Copyright © 2010 Wiley Periodicals, Inc.) |
| المشرفين على المادة: | 0 (Recombinant Proteins) 53-59-8 (NADP) 9035-51-2 (Cytochrome P-450 Enzyme System) IY9XDZ35W2 (Glucose) T75W9911L6 (Propane) |
| تواريخ الأحداث: | Date Created: 20110120 Date Completed: 20110420 Latest Revision: 20131121 |
| رمز التحديث: | 20221213 |
| DOI: | 10.1002/bit.22984 |
| PMID: | 21246504 |
| قاعدة البيانات: | MEDLINE |
Full Text Finder | تدمد: | 1097-0290 |
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| DOI: | 10.1002/bit.22984 |