A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis
<p>Abstract</p> <p>Background</p> <p>Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
BMC
2012-06-01
|
| Series: | BMC Biotechnology |
| Subjects: | |
| Online Access: | http://www.biomedcentral.com/1472-6750/12/31 |
| _version_ | 1811280933838389248 |
|---|---|
| author | van Bloois Edwin Dudek Hanna M Duetz Wouter A Fraaije Marco W |
| author_facet | van Bloois Edwin Dudek Hanna M Duetz Wouter A Fraaije Marco W |
| author_sort | van Bloois Edwin |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered.</p> <p>Results</p> <p>Here, we have used phenylacetone monooxygenase (PAMO) from <it>Thermobifida fusca</it>, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant <it>E. coli</it> expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening.</p> <p>Conclusions</p> <p>Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well.</p> |
| first_indexed | 2024-04-13T01:23:28Z |
| format | Article |
| id | doaj.art-b2cafa8dd3d347fb9910b4e1994f902f |
| institution | Directory Open Access Journal |
| issn | 1472-6750 |
| language | English |
| last_indexed | 2024-04-13T01:23:28Z |
| publishDate | 2012-06-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Biotechnology |
| spelling | doaj.art-b2cafa8dd3d347fb9910b4e1994f902f2022-12-22T03:08:41ZengBMCBMC Biotechnology1472-67502012-06-011213110.1186/1472-6750-12-31A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysisvan Bloois EdwinDudek Hanna MDuetz Wouter AFraaije Marco W<p>Abstract</p> <p>Background</p> <p>Baeyer-Villiger monooxygenases (BVMOs) represent a group of enzymes of considerable biotechnological relevance as illustrated by their growing use as biocatalyst in a variety of synthetic applications. However, due to their increased use the reproducible expression of BVMOs and other biotechnologically relevant enzymes has become a pressing matter while knowledge about the factors governing their reproducible expression is scattered.</p> <p>Results</p> <p>Here, we have used phenylacetone monooxygenase (PAMO) from <it>Thermobifida fusca</it>, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant <it>E. coli</it> expressing PAMO in 96-well microtiter plates in a reproducible fashion. Using this system, the best expression conditions of PAMO were investigated first, including different host strains, temperature as well as time and induction period for PAMO expression. This optimized system was used next to improve biotransformation conditions, the PAMO-catalyzed conversion of phenylacetone, by evaluating the best electron donor, substrate concentration, and the temperature and length of biotransformation. Combining all optimized parameters resulted in a more than four-fold enhancement of the biocatalytic performance and, importantly, this was highly reproducible as indicated by the relative standard deviation of 1% for non-washed cells and 3% for washed cells. Furthermore, the optimized procedure was successfully adapted for activity-based mutant screening.</p> <p>Conclusions</p> <p>Our optimized procedure, which provides a comprehensive overview of the key factors influencing the reproducible expression and performance of a biocatalyst, is expected to form a rational basis for the optimization of miniaturized biotransformations and for the design of novel activity-based screening procedures suitable for BVMOs and other NAD(P)H-dependent enzymes as well.</p>http://www.biomedcentral.com/1472-6750/12/31Baeyer-Villiger monoxygenase<it>Escherichia coli</it>BiocatalysisSquare deep-well microtiter platesScreening |
| spellingShingle | van Bloois Edwin Dudek Hanna M Duetz Wouter A Fraaije Marco W A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis BMC Biotechnology Baeyer-Villiger monoxygenase <it>Escherichia coli</it> Biocatalysis Square deep-well microtiter plates Screening |
| title | A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis |
| title_full | A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis |
| title_fullStr | A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis |
| title_full_unstemmed | A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis |
| title_short | A stepwise approach for the reproducible optimization of PAMO expression in <it>Escherichia coli</it> for whole-cell biocatalysis |
| title_sort | stepwise approach for the reproducible optimization of pamo expression in it escherichia coli it for whole cell biocatalysis |
| topic | Baeyer-Villiger monoxygenase <it>Escherichia coli</it> Biocatalysis Square deep-well microtiter plates Screening |
| url | http://www.biomedcentral.com/1472-6750/12/31 |
| work_keys_str_mv | AT vanblooisedwin astepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT dudekhannam astepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT duetzwoutera astepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT fraaijemarcow astepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT vanblooisedwin stepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT dudekhannam stepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT duetzwoutera stepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis AT fraaijemarcow stepwiseapproachforthereproducibleoptimizationofpamoexpressioninitescherichiacoliitforwholecellbiocatalysis |