ArticleBMC biotechnology2012
A stepwise approach for the reproducible optimization of PAMO expression in Escherichia coli for whole-cell biocatalysis.
Article in BMC biotechnology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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3 citing papers in PubMed, 11 citations in OpenAlex.
- Spray-Dried Engineered Escherichia coli Whole-Cell Biocatalysts Enable Cell Recycling and In Vivo α-Ketoglutarate and NADPH Regeneration for Efficient Codeine Manufacture.Biotechnology and bioengineering · 2026Article
- Efficient Synthesis of Methyl 3-Acetoxypropionate by a Newly Identified Baeyer-Villiger Monooxygenase.Applied and environmental microbiology · 2019Article
- Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications.Frontiers in microbiology · 2014Review
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Authors and funding
4 authors at 1 institution in 1 country.
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Abstract
backgroundBaeyer-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.
resultsHere, we have used phenylacetone monooxygenase (PAMO) from Thermobifida fusca, a prototype Type I BVMO, as a model enzyme to develop a stepwise strategy to optimize the biotransformation performance of recombinant E. coli 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.
conclusionsOur 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.
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