The development of a heterologous expression system is often a principal step in biochemical and biotechnological studies on cytochromes P450 (P450s). However, heterologous expression of eukaryotic membrane-bound P450s in Escherichia con is still a trial-and-error process because theoretical and systematical experimental procedures have not yet been established. In this study, we generated a series of chimeric variants of fungal P450s by replacing their N-terminal domains with the N-terminal domains of other P450s and explored their potentials for heterologous expression in E. con. Large-scale screening demonstrated that a number of fungal P450s could be expressed when their N-terminal amino acid sequences were replaced with the corresponding domain of CYP5144C1, even when the expression of the non-chimeric sequence was unpromising. Furthermore, a comprehensive screening resulted in the identification of 64 different types of chimeric partners whose N-terminal domains could potentially be used to increase the expression levels of various P450s. These findings will help to elaborate experimental strategies for high-level heterologous expression of a variety of eukaryotic membrane-bound P450s in E. coli. (c) 2015, The Society for Biotechnology, Japan. All rights reserved.