Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme

Robin, Aélig; Roberts, Gareth A.; Kisch, Johannes; Sabbadin, Federico; Grogan, Gideon; Bruce, Neil C.; Turner, Nicholas J.; Flitsch, Sabine L.

doi:10.1039/b901716j

Cited by 61 publications

(70 citation statements)

References 18 publications

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“…Optimization of the flux of electrons is crucial to utilize the full capacity of the biocatalyst to the targeted reaction, and the importance has also been emphasized in the review by Bernhardt and Urlacher (2014). Approaches with fusion constructs have been published, and besides constructs with the reductase domain of BM3, the reductase domain of CYP116B2 (P450RhF) is a promising alternative (Bordeaux et al 2011;Robin et al 2009). This field with a lot of potential for the application of P450s has recently been reviewed (Sadeghi and Gilardi 2013).…”

Section: Electron Transport and Coupling Efficiencymentioning

confidence: 97%

“…This field with a lot of potential for the application of P450s has recently been reviewed (Sadeghi and Gilardi 2013). The length and also structure of the linker within the fusion constructs have been shown to be important for electron transport (Munro et al 2007b;Robin et al 2009). Activity can also be reconstituted with more component systems, especially for eukaryotic systems with unknown redox partners, even though the redox partners are commonly known for eukaryotic systems in contrast to prokaryotic ones.…”

Section: Electron Transport and Coupling Efficiencymentioning

confidence: 99%

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Guidelines for development and implementation of biocatalytic P450 processes

Lundemo

Woodley

2015

Appl Microbiol Biotechnol

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Biocatalytic reactions performed by cytochrome P450 monooxygenases are interesting in pharmaceutical research since they are involved in human drug metabolism. Furthermore, they are potentially interesting as biocatalysts for synthetic chemistry because of the exquisite selectivity of the chemistry they undertake. For example, selective hydroxylation can be undertaken on a highly functionalized molecule without the need for functional group protection. Recent progress in the discovery of novel P450s as well as protein engineering of these enzymes strongly encourages further development of their application, including use in synthetic processes. The biological characteristics of P450s (e.g., cofactor dependence) motivate the use of whole-cell systems for synthetic processes, and those processes implemented in industry are so far dominated by growing cells and native host systems. However, for an economically feasible process, the expression of P450 systems in a heterologous host with sufficient biocatalyst yield (g/g cdw) for non-growing systems or space-time yield (g/L/h) for growing systems remains a major challenge. This review summarizes the opportunities to improve P450 whole-cell processes and strategies in order to apply and implement them in industrial processes, both from a biological and process perspective. Indeed, a combined approach of host selection and cell engineering, integrated with process engineering, is suggested as the most effective route to implementation.

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Section: Electron Transport and Coupling Efficiencymentioning

confidence: 97%

Section: Electron Transport and Coupling Efficiencymentioning

confidence: 99%

Guidelines for development and implementation of biocatalytic P450 processes

Lundemo

Woodley

2015

Appl Microbiol Biotechnol

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“…Another promising application for chimeragenesis is the production of chimeric oxygenases in which the P450 domain is fused to the reductase domain of a self-sufficient P450. A good example for this is the fusion of CYP101A1 to the reductase domain of P450RhF, comprising flavin mononucleotide-and NADH-binding motifs and a [2Fe-2S] ferredoxin-like center [41]. The resulting catalytically self-sufficient chimera can achieve high conversion rates and has a high potential for applications in biocatalysis, especially of the natural terpene substrates.…”

Section: Engineering By Chimeragenesismentioning

confidence: 98%

Terpene Hydroxylation with Microbial Cytochrome P450 Monooxygenases

Janocha

Schmitz

Bernhardt

2015

Biotechnology of Isoprenoids

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Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes.

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“…The stability improvement of these backbones can result in prolonged catalyst lifetimes and enable selective oxidation of a wide range of nonnative substrates (Lewis and Arnold, 2009). Another strategy towards fast CYP variants is e.g., the creation of chimeras in which an interested heme-domain is fused to a reductase domain (Li et al, 2007;Robin et al, 2009). Still, only few examples of preparative scale metabolite synthesis have been published e.g., the use of E. coli cells bicistronically expressing truncated human cytochrome P450 and NADPH-P450 reductase (Vail et al, 2005) and similarly Schizosaccharomyces pombe as a whole cell catalyst expressing a human CYP (Peters et al, 2009).…”

Section: Heme-dependent Monooxygenasesmentioning

confidence: 99%