| Pichia pastoris has been developed into a highly-successful protein expression system in recent years. It combines the advantages of low cost, high expression level, stable heredity and high secretion efficiency and has been widely used in laboratories and factories all over the word. The successful development of P. pastoris expression system could be partly contributed to the strong and easily-manipulated AOX1 promoter. However, researchers have to use methanol, which is a toxic, flammable and explosive carbon source, to induce the expression from AOX1 promoter. The transportation, storage and use of methanol are undoubtedly potential dangers in industrial production. In recent years, researchers paid more and more attention to the promoter of formaldehyde dehydrogenase (FLD1 promoter) of P. pastoris. On one hand, FLD1 promoter is very strong and stringent, comparable to the AOX1 promoter. On the other hand, FLD1 promoter can be strongly induced by methylamine salts. Methylamine salts are much less toxic, dangerous and volatile than methanol. Therefore, it is more convenient and safe to use methylamine salts than methanol in laboratories or factories. To improve the MPH production, we selected the strong FLD1 promoter to drive the secreted expression of MPH protein.Firstly, we fused FLD1 promoter region and a-mating factor signal sequence by overlap extension PCR and constructed P. pastoris expression vector pEFaA. Then we ligated the previously-resynthesized MPH coding gene smpd (according to the P. pastoris preferred codons) into the pEFaA vector to construct the MPH expression vector pEFaA-smpd. After electroporation, the pEFaA-smpd vector was integrated into the genome of P. pastoris X33 strain. We achieved durative and secreted expression of MPH protein in P. pastoris recombinant strains. After incubation for 72 h, the accumulative MPH enzyme activity in culture supernatant reached 5.4 U/mL.During our study, we found that there were two different bands of secreted MPH protein in SDS-PAGE Gel. Except the anticipated MPH band, there was still a smaller protein band. After analyses, we found that the Kex2 protease of P. pastoris could recognize the KR site (-Lys-Arg-) in MPH sequence and cleave 37 amino acids residues from the N terminal of MPH, thus forming the truncated MPH protein. After analyzing the crystal structure of MPH protein, we thought it was probable that one or more amino acids in the cleaved 37 amino acid residues played an important role in methyl parathion hydrolyzation. So the catalyzing activity of truncated MPH protein might undergo a big decrease. At the same time, the truncated MPH protein accounted for more than 30% of all MPH protein in medium supernatant. Therefore, we deduced that the truncated MPH protein may greatly affected the total MPH enzyme activity in culture supernatant. Besides, the KR site in MPH protein may compete for the limited Kex2 protease with the KR site in a-mating factor signal peptide, thus resulting in the decrease of MPH secretion efficiency. Based on these facts, we decided to remove the KR site in MPH sequence by site-directed mutagenesis. The mutated MPH coding sequence smpdK (KR→KK) could greatly increase the total enzyme activity in medium supernatant. The average MPH enzyme activity increased by 59%, from 5.4 U/mL to 8.6 U/mL.It is commonly accepted that increasing the copy number of heterologous protein expression cassettes usually could greatly increase the expression level of foreign proteins. In subsequent experiments, we ligated the FLD1-drived MPH expression cassette into the pPIC9K plasmid (which could be used for screening high-copy transformants) and replaced the AOX1 promoter region to construct MPH expression plasmid p9KFLD-smpdK. Then we firstly got lots of transformants by electrotransformation and selected multiple-copy transformants by using high concentration G418 antibiotics. Because yeast cells with low-copy expression cassettes could spontaneously generate high-copy recombinant strains at a rare possibility. Subsequently, we increased G418 concentrations step by step to screen P. pastoris recombinant strains with higher copy numbers. Finally, the extracellular MPH enzyme activity of those recombinant strains, which can resist 8000 mg/L or 12000 mg/L G418, reached 45 U/mL. In other words, increasing copy number of MPH expression cassette improved the total MPH enzyme activity by 5 times.Another part of our work was focused on establishment of genetic manipulation systems in P. pastoris. We optimized an unmarked gene deletion method in P. pastoris. We could use this method repeatedly to disrupt P. pastoris genes while without introducing selectable markers. This method combined the advantages of fusion-PCR, Cre/lox system and stringent AOX1 promoter. The entire gene deletion procedure could be finished within 5 days. It is much faster and more convenient than other currently-available methods for unmarked gene deletions in P. pastoris.Currently, almost all P. pastoris expression plasmids are integration plasmids without eukaryotic replication origin. These expression plasmids could integrate into yeast chromosomes by homologous recombination and replicate as the yeast chromosomes replicate. Before electroporation, researchers usually have to linearize expression vectors in homologous recombination region by restriction endonucleases, then electroporate P. pastoris competent cells with linear expression plasmids and select positive transformants. During our study, we found that we could get enough correct transformants when electroporating P. pastoris with circular plasmids. This helps solve the following two possible problems in plasmid linearization:(1) heterologous protein coding genes and homologous recombination regions might have the same restriction sites, and it is difficult to find a proper linearization site; (2) when one expression plasmid has multiple protein expression cassettes, linearization at promoter (which is usually used as homologous recombination region) will cleave the circular plasmid into several fragments. So the whole expression plasmid could not be integrated into P. pastoris chromosome. In our study, we observed that circular expression plasmid, using AOX1ã€FLD1 or GAP promoter regions as homologous recombination sites, could integrate into P. pastoris genome at an acceptable efficiency. These findings provided more convenience and flexibility to researchers when using P. pastoris expression system. |
