Directed evolution of Arabidopsis thaliana carotenoid cleavage dioxygenase 1

Arabidopsis thaliana Carotenoid Cleavage Dioxygenase 1 (AtCCD1) is the role model for CCDs, a class of enzymes that cleave highly specific C=C double bonds in carotenoids and apo-carotenoids. CCD1s cleave carotenoids symmetrically and regio-selective at the 9-10 and 9’-10’ position. The active site of CCDs contains a central, heme-free Fe2+, which is coordinated by four histidines. The enzymatic oxidation utilizes molecular oxygen and does not require further cofactors. This unique cleavage step is used in nature to create a huge variety of colorants, flavors and fragrances. It occurs in all branches of life from protozoa, metazoa to plants and fungi. In vitro, organic solvents show an activation effect towards CCD1 cleavage. The understanding of this mechanism has not been resolved up till now. Vast numbers of cleaved carotenoids show a complex chemistry and cannot be synthetized by chemical methods. Utilization of carotenoids as substrates to produce apo-carotenoids is a promising and novel approach. However, recombinant expression of AtCCD1 occurs predominantly as inclusion bodies and thus low enzymatic activity is achieved. CCD1 can be refolded from inclusion bodies, or purified by N- and C-terminal tags. Both methods yield a modified or tagged enzyme, and are limited in their application to characterize CCDs. A tag-free purification protocol was developed and permits investigation of the native AtCCD1 in vitro. High throughput screening (HTS) of AtCCD1 was possible by establishing the expression in a 96 well micro-titer-plate (MTP) format. The activity and stability of AtCCD1 with organic co-solvents was of special interest because it aided solubility characteristics of the hydrophobic carotenoids. In addition, the conversion of different carotenoid substrates was of key interest because of the regio-specificity of the cleavage reaction. Four complimentary mutagenesis methods were used to generate libraries of AtCCD1 in iterative rounds of directed evolution. Development of recombinant MTP expression in combination with novel HTS methods allowed; (I) screening conversion of carotenoids by substrate depletion, (II) fluorescence of the central cleavage product of carotenoids, (III) the conversion of apo-8’-retinal in the presence of organic co-solvents. The methods were applied to evolve AtCCD1 towards; (a) stability in the presence of organic solvents and (b) conversion of carotenoids. Several key-residues for AtCCD1 were determined, including serine 363, which is involved in substrate determination