Cholesterol oxidase is the predominant enzyme engaged in the pathway of cholesterol degradation in a number of soil bacteria, and this FAD-containing flavoprotein assembles the oxidation of cholesterol to 4-cholesten-3-one in the presence of molecular oxygen (O2) and yields hydrogen peroxide (H2O2) as a by-product. Heterologous expression suggests the expression of a gene or part of a gene in a host organism which does not naturally have this gene or gene fragment. It is the prevail technique to produce interested enzyme for function studies and industrial applications, where Escherichia coli is widely-applied heterologous host due to its fast growth, economical cultivation, and easily introducing exogenous gene.