Difference between revisions of "Team:Heidelberg/Cytochrome Engineering"

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Revision as of 13:24, 1 November 2017


Cytochrome Engineering
Modulating CYP1A2 product specifity
Cytochromes are heme-dependent enzymes of immense importance across all kingdoms of life. Due to their often highly promiscuous nature, the cytochrome P450 superfamily is of particular interest in context of enzyme engineering. In this subproject, we aimed at applying our PREDCEL toolbox for re-directing the catalytic activity of cytochromes towards desired products. Employing the caffeine-metabolizing human Cytochrome P450 1A2 (CYP1A2) as example, our team implemented a PREDCEL enzyme evolution workflow coupling phage survival to the production of a naturally unfavored catalytic product: theophylline. We created M13 phages encoding CYP1A2 as well as a corresponding accessory plasmid linking the intracellular theophylline levels to geneIII production via a theophylline riboswitch. After iterative propagation of the CYP1A2-encoding phages on mutagenic selection cells transformed with our accessory plasmid, we observed numerous, partially recurrent point mutations in CYP1A2, indicative of a successful evolution. Taken together, our work lays the foundation for the future engineering of enzymes by means of in vivo directed evolution with PREDCEL.

Introduction

Enzymes, i.e. proteins mediating specific, catalytic functions, are amongst the most powerful molecular machines invented by nature. Since decades, humans utilize naturally occurring enzymes as bio detergents (e.g. in washing powder kirk2002industrial), in the paper industry bajpai1999application and for food processing alkorta1998industrial.
The engineering of novel enzymes catalyzing reactions that do not or only inefficiently occur in nature holds great promise for biotechnological production of regenerative fuel, biomaterials and novel pharmaceuticals, e.g. based on Organosilicons. However, so far, enzyme engineering has typically been a time-consuming, elaborate, expensive and inefficient process, usually requiring laborious, iterative trial-and-error optimization of engineered candidates packer2015methods .
To accelerate the development of novel enzymes, our team harnessed the engineering strategy nature uses: Evolution.
Figue 1: title
Figure on with description of theophylline PACE

Background

The human Cytochrome CYP1A2 is an example of a heme-dependent-thiolate monooxygenase, member of the P450 superfamily and plays an important role in the metabolism of many structurally unrelated substrates. Primarily the CYP1A2 is located in the endoplasmic reticulum of liver cells. It is of great interest as it is involved in the oxidative metabolism of many commonly used therapeutics and xenobiotics ahn2004high, such as caffeine. Caffeine undergoes degradation by CYP1A2 through an initial N3- demethylation into three xanthine derivates, 81.5% paraxanthine, 10.8% theobromine and 5.4% theophylline perera2010caffeine. The chemical structure of these three primary metabolites of caffeine only differ in their methylation pattern (Fig.2).
Figue 2: Chemical structure of caffeine
Caffeine is a substrate of CYP1A2.
Figue 2: Chemical structures of Paraxanthine, theobromine and theophylline
By N3 demethylation caffeine is metabolized into these three xanthine derivates by CYP1A2.
Figue 1: title
gigure x with explanation of plaque assays

References