Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAA -> ncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes.