Abstract:[Background] The expression of fugal P450s in Escherichia coli system is often in low efficiency. The N-terminal domain (NTD) amino acid sequence modification has been emerged as a potential strategy to achieve high-level expression of these membrane-bound proteins. [Objective] The Au8002 gene in Aspergillus ustus 094102 is predicted to encode a fungal P450. It cannot be expressed successfully in its natural sequence. Three NTD modifications were carried to prove the efficiency of this strategy on the heterologous expression in E. coli system. [Methods] Three NTD-modified Au8002 protein were designed to facilitate the gene expression and 5-aminolevulinic acid (5-ALA), the substrate for biosynthesis of P450s, was added during the heterologous expression to increase the yield of protein. [Results] The identifying results by SDS-PAGE and western blot indicated that the NTD modifications used in this study could improve the fungal gene expression and the addition of 5-ALA improved the P450 protein yield in E. coli. When the NTD was truncated in full length, the NTD-modified Au8002 appeared to be partially soluble, and CO binding test also proved its solubility and proved that it was a P450 protein. [Conclusion] The NTD modification of Au8002 of strain 094102 can help to overcome the low efficiency of its heterologous expression in E. coli and the addition of 5-ALA can also help to increase its expression level. The observation made in this study may provide a useful guideline for improving the expression yield of fungal P450s in E. coli.
