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

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         <h1>Introduction</h1>
 
         <h1>Introduction</h1>
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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; [Reference: Kirk O, Borchert TV, Fuglsang CC (August 2002). "Industrial enzyme applications". Current Opinion in Biotechnology. 13 (4): 345–351. PMID 12323357. doi:10.1016/S0958-1669(02)00328-2.]) , in the paper industry [Bajpai P (March 1999). "Application of enzymes in the pulp and paper industry". Biotechnology Progress. 15 (2): 147–157. PMID 10194388. doi:10.1021/bp990013k.] and for food processing [Alkorta I, Garbisu C, Llama MJ, Serra JL (January 1998). "Industrial applications of pectic enzymes: a review". Process Biochemistry. 33 (1): 21–28. doi:10.1016/S0032-9592(97)00046-0.].<br>
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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 <x-ref>kirk2002industrial</x-ref>), in the paper industry <x-ref>bajpai1999application</x-ref> and for food processing <x-ref>alkorta1998industrial</x-ref>.<br>
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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 (LINK zu Organosilicon Projekt). 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 [ref: review directed evolution page 1].<br>
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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 (LINK zu Organosilicon Projekt). 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 <x-ref>packer2015methods</x-ref> .<br>
 
To accelerate the development of novel enzymes, our team harnessed the engineering strategy nature uses: Evolution.  
 
To accelerate the development of novel enzymes, our team harnessed the engineering strategy nature uses: Evolution.  
 
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Revision as of 11:47, 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 (LINK zu Organosilicon Projekt). 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
Figue 1: title
gigure x with explanation of plaque assays

References