Overexpression And Secretion Of The Lipase From Rhizopus Chinensis In Heterologous Hosts

Escherichia coli and Pichia pastoris are two most widely used expression systems during expressing proteins in heterologous hosts. E. coli is usually utilized to express proteins, which has the advantages of short cultivation cycles, clear genetic background and overexpression. However, the heterologous proteins, especially for the eukaryotic proteins, often exist as the inclusion body(IB), and are extremely difficult to secret into the medium due to lacking of perfect secretion mechnism in E. coli. While P. pastoris could be cultivated in high-density fermentation and modify the heterologous proteins. But high-level expression in P. patoris is usually limited to gene dosage, protein translation and translocation et al.. Therefore, in this study, lipase pro RCL was used as a model enzyme, and high-level expression and secretion of this lipase in two hosts were investigated by technology of molecular biology. The main contents are as follows:(1) Recombinant plasmid p ET22b-pel B-pro RCLC was constructed to express lipase pro RCL in E. coli. However, the solubility of the lipase was not improved by optimization of the environmental factor. Four soluble tag genes(Trx A, Dsb C, Nus A, and Skp) were cloned from the genome of E. coli. After fusion expression, only fusion protein Trx A-pro RCL was soluble. Then the lipase gene was cloned into the vector MBP3. The fusion protein MBP-pro RCL was also soluble. Accroding to the analysis of cellular localization, both the fusion protein Trx Apro RCL and MBP-pro RCL existed in the cytoplasm of E. coli. Then the specific activity of the lipase MBP-pro RCL was vastly increased by optimization of the expression hosts BL21 trx B(DE3) and Origami2(DE3), the ability of which for synthesising disulfide bonds was much higher. For further enhancing the lipase secretion, three membrane proteins(lpp, Omp X, Osm Y) were fused to N terminal of the lipase. More than 90% fusion proteins(Osm Y-pro RCL) were soluble in E. coli at 17 oC, and they also existed in the cytoplasm, rather than in the periplasm or in the medium.(2) Five kinds of signal peptides from type II secretion pathway were cloned into the vector MBP, and a series of secretion vectors SP-MBP were constructed. Then the lipase gene was cloned into SP-MBP and vectors SP-MBP-pro RCL were also constructed. After induction, only three fusion proteins(Omp A-MBP-pro RCL, Dsb A-MBP-pro RCL and Ycd O-MBP-pro RCL) were secreted into the periplasm by SDS-PAGE. Then the periplasm secretion levels of fusion proteins Omp A-MBP-pro RCL and Dsb A-MBP-pro RCL were enhanced by optimization of temperature. But more fusion proteins Ycd O-MBP-pro RCL were secreted into the medium. By analysis of flow cytometry(FCM) and transmission electron microscopy(TEM), the secretion of fusion protein Ycd O-MBP-pro RCL was probably due to the leakage of the strain.(3) Overexpression in P. pastoris might triger UPR, which could be solved by optimization of the hetorologous gene dosage. One method for quantifing the gene copy number of foreign gene in P. pastoris was established by RT-q PCR. A range of recombinant strains with different copies of lipase gene in muts phenotype were screened by RT-q PCR. And the highest enzyme activity reached 280 U?m L-1 by SRCL-3.(4) The chaperone gene PDI was cloned from the genome of P. pastoris and the relationship of gene copy numbers between chaperone gene and lipase gene was systematically explored. It was suggested that the enzyme activity of all the recombinant strains with different copies lipase gene increased by a certain degree. And the highest lipase enzyme activity reached 355 U?m L-1 by the strain PDI-1-SRCL-5. Then we investigated the effects of co-expression with multi-copy PDI gene and multi-copy lipase gene. It showed that the expression level of recombinant strains with high copies lipase gene was not always positively correlated well with the copy number of chaperone gene PDI, which may be due to the characteristic of the protein itself.(5) A series of recombinant strains with one, three, five and six copies of lipase gene pro RCL was screened to evaluate the effects of pro RCL gene dosage on the lipase expression level. Different from the muts phenotype, it showed that the maximum enzyme activity reached 260 U?m L-1 by XY RCL-5, but both the enzyme activity and extracellular protein concentration decreased in XY RCL-6. Moreover, the transcription level of lipase gene in XY RCL-6 reached the maximum, and then decreased gradually after 24 h. The transcription levels of two chaperone genes ERO1 and PDI involved in protein folding in the Endoplasmic Reticulum(ER) were further investigated, which showed that Unfolded Protein Response(UPR) might be trigered in high copy strain. One gene of thiol oxidase for oxidative protein folding in the ER was cloned from the genome of P. pastoris. Two key catalytic disulide bonds(outside and inside) existed in the protein sequence, which was the same with that from S. cerevisiae. The increase of foreign proteins in P. pastoris could not be solely achieved by co-expression with chaperone PDI or ERO1 p in mut+ phenotype. Thus we constructed a recombinant strain with one copy PDI gene and one copy ERO1 gene to enhance the lipase production. The enzyme activity reached 370 U?m L-1 by Co XY RCL-5 in shaking flasks. It supplied a potential strategy for overexpression of other heterologous proteins in P. pastoris by co-expression with these two chaperone genes.