Screening Of Endogenous Signal Peptides And Secretory Expression Of Cephalosporin C Acylase In Pichia Pastoris

The methylotrophic yeast Pichia pastoris is an important host for the production of secreted heterologous protein, and the secretion of the recombinant protein is usually mediated by an N-terminal signal peptide. Endogenous signal peptides, from natively secreted P. pastoris proteins, can be correctly processed, may exhibitstrong secretion efficiency. Cephalosporin C (CPC) acylase is the key enzyme for one-step enzymatic production of 7-aminocephalosporanic acid acid (7-ACA), which is very attractive at the industrial level because of the prospects of simplifying the process and reducing costs. In order to obtain efficient signal peptides, signal peptides from the endogenously secreted P. pastoris proteins were screened using bioinformatics analysis, and their capability to mediate the secretion of reporter proteins, such as enhanced green fluorescent protein (EGFP) and (3-galactosidase (?-gal), was investigated. In addition, these endogenous signal peptides were used to direct the secretion of recombinant CPC acylase in P. pastoris.Four potential endogenous signal sequences were obtained from P. pastoris secretome using SignalP4.1 software:DAN4, GAS1, MSB2 and FRE2. The widely used signal peptide a-mating factor (a-MF) and a reported endogenous signal peptide DSE4 were employed as controls to investigate secretion efficiency of the four signal peptides. Recombinant strains, each expressing a reporter protein with the tested signal peptide driven by a constitutive strong mutant promoter PG1, were constructed. The CPC acylase gene from Pseudomonas sp. Strain SE83 was codon optimized according to the codon bias in P. pastoris, synthesized, and named SECA. After the preliminary optimization of fermentation conditions in shake flasks, extracellular and intracellular expression levels of EGFP, ?-gal and SECA were detected. The results showed that, secretion levels of EGFP, P-gal and SECA mediated by MSB2 were higher than that of a-MF and DSE4. DAN4 was effective in secreting P-gal and SECA, but was not efficient in the secretion of EGFP. While the highest secretory expression of ?-gal was obtained with the GAS1', it did not direct the secretion of EGFP and SECA comparable to that of a-MF. FRE2 mediated a moderate level of EGFP and ?-gal secretion lower than DSE4, and the lowest secretion of SECA. Taken together, MSB2 can be considered as the most efficient signal peptide for heterologous protein secretion. It mediated a 1.5-,7.97-, and 1.94-fold higher secretion of EGFP, ?-gal and SECA, respectively, in comparison to a-MF. SECA was successfully secreted guided by DAN4 and MSB2, with the highest level of 3.81-and 2.65-fold higher than that of a-MF and DSE4 respectively, mediated by DAN4. To further improve SECA secretory expression mediated by MSB2, a multi-copy strain G/MSBCAm was screened by increasing Zeocin concentration, with a 28.4% and 53.2% higher intracellular and extracellular activity of CPC acylase respectively, than that of the low-resistance strain G/MSBCA.