Strategies for Coexpression of Bilin-Producing Enzymes in Escherichia coli.

Protein cofactors vastly expand the chemical space of proteins and thus allow for otherwise inaccessible functionalities. For instance, heme for oxygen transport, flavins that are key to redox reactions, or copper ions facilitating electron transport are only some examples among a plethora of other protein cofactors. In the protein family of phytochromes, bilin compounds act as cofactors and govern their core functionality-the sensing of red/far-red light. This light-sensing capability regulates not only central metabolic pathways in plants but also influences other light-regulated organisms; thus, understanding the molecular mechanisms of phytochromes is of great interest. For biochemical characterization, the phytochrome proteins are most commonly overexpressed in heterologous expression hosts. However, many hosts, like Escherichia coli, lack the machinery to produce sufficient levels of bilins. For the generation of biliverdin, this can be alleviated by the coexpression of a heme oxygenase and the phytochrome, most commonly in a two-plasmid (helper plasmid/expression plasmid) setup. In this chapter, we introduce two alternative strategies, bi-cistronic coexpression and genomic integration of the heme oxygenase. The generation of these systems is shown in detail, and their advantages/disadvantages are discussed compared to the well-established two-plasmid systems. We anticipate that these approaches are also feasible for other bilins, for example, phytochromobilin and/or phycocyanobilin, which will expand the toolbox for phytochrome expression and provide alternatives for challenging study designs.