Because the system is used widely, a number of strategies for enhancing protein production have been developed. However, most strategies do not target the protein itself, but instead modify other aspects of the process, such as the host, promoter, signal peptide, chaperone, fusion proteins, protease, fermentation conditions, codon optimization, or gene copy number. Although these methods can effectively enhance the Forsythin expression of some proteins, it is challenging to enhance the secretion of certain proteins that are retained in the cell. Previous studies have shown that the rational design of the internal regions of a protein can enhance its secretion by P. pastoris. Therefore, some secretion signals that affect protein secretion must exist in the internal regions of proteins. It may therefore be necessary to modify the sequence or structure of a protein to enhance its secretion. In this study, we used methyl parathion hydrolase from Ochrobactrum sp. M231 to investigate the effect of modification of its N-terminus on secretion. Organophosphorus hydrolases play important roles in the decontamination and bioremediation of environments polluted by organophosphate pesticides. MPH, isolated by our lab from Ochrobactrum sp. M231 in 2008, can efficiently and specifically Evodiamine degrade methyl parathion, but it cannot be secreted from P. pastoris. However, another OPH, OPHC2, isolated from Pseudomonas pseudoalcaligenes, also by our laboratory, has a similar three-dimensional structure to MPH. It has been over-expressed and efficiently secreted at concentrations of up to 5.5 g/L in P. pastoris. These two proteins have the same function and share a sequence identity of 47.7%. However, they have different secretion pa erns when expressed in the same P. pastoris expression system with an identical promoter and signal peptide. Although it has been reported that integrating 12 copies of the MPH expression casse e into the P. pastoris system can significantly improve the secretion of MPH and that MPH can be expressed and secreted by Bacillus subtilis WB800, secretion levels with these two methods are still close to 100-fold lower than the levels of OPHC2 secreted by P. pastoris. We a empted to identify the protein sequence factor that affected the secretion of MPH by P. pastoris. Sequence and structural analyses revealed that MPH and OPHC2 have different Nterminal regions. Therefore, the aim of this study was to improve the secretion of MPH by P. pastoris through the modification of its N-terminal structure. In this study, MPH from 
Ochrobactrum sp. M231 was selected and the importance of its N-terminus in its secretion by P. pastoris was determined. We swapped the corresponding block of sequence from OPHC2 based the Schema software analysis and removed the N-terminal block of MPH according to the 3D protein structures. Our results revealed that the N-terminal region plays an important role in secretion. In addition, the improved secretion of the MPH mutants was not due to differences in growth rate, mRNA expression, gene copy number, or stability. Although the mutant proteins had reduced catalytic efficiency, the secretion of both D10-MPH and N9-MPH was improved significantly compared to wild-type MPH, as demonstrated by SDS-PAGE.