Integrated Strategies for Enhancing the Expression of Chitosanase AqCoA in the Pichia Pastoris by A Novel High-Copy Plasmid PMC-GAP

In our previous study, the chitosanase AqCoA and its products chitooligosaccharides exhibited signicant application in fungal disease protection. In this study, to enhance the expression of AqCoA, we obtained various strains with multi-copy by a novel plasmid pMC-GAP with stable transformation ability in Pichia pastoris and built an integrated model combining the gene copy number, the chaperones and protein production of AqCoA. In terms of gene dosage, the highest enzyme activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that in strain with only one copy (0.18 U/ml). In addition, we found the chaperone like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased by 61%, 31% and 42% in expression levels of the strain GAP-1AqCoA. Finally, we investigated the effect co-expression of gene copy and chaperones on protein expression. The maximum activity reached 2.319 U/ml by the strain with six chaperones intergrant plus sixteen copies, which was 13-fold higher than that by the control strain with only one copy (GAP-1AqCoA). Co-expression of gene dosage and chaperones signicantly enhanced expression levels of AqCoA, which presented a powerful tool to improve foreign protein expression.

In the past few decades, the yeast Pichia pastoris has been widely used as host organism for heterologous protein production (8) with the advantages of rapid growth and carrying out the posttranslational modi cations to most of proteins (42). However, yeast has limited ability to secrete proteins in its natural state. At present, many genetic methods utilized manipulated Pichia pastoris to obtain high yields of secreted proteins. In general, increasing the gene dosage to construct multi-copy strains is a common method (26). Furthermore, several high-throughput methods were established to screen many transformants with multi-copy integration. Scorer et al. (1994) found that a tight correlation was showed between the copy number and drug-resistance, and then proposed to utilize the high concentration of antibiotics to screen multi-copy strains (38). Moreover, multi-copy plasmids were constructed by the isocaudamer in vitro, like that Li et al (2009) created multiple Pichia clones carrying different copies of the gene expression cassette (23), however, the transformation into P. pastoris is ine cient as result of the constructed plasmids are very large by this way. In addition, Sunga et al. (2008) proposed the method of post-transformational vector amplifcation (PTVA), which mentioning that using the antibiotic to generate the strains with different copies number (41). Strains that survived at higher antibiotic concentrations contained a higher number of intact copies of the gene of interest. In addition, the PTVA was widely employed in many studies, like that Zhu et al. (2009) adopted the method to obtained porcine insulin precursor with 52 copies (58), Rochelle et al. (2016) developed the liquid PTVA on the base of the PTVA (2). Except that, the PTVA also combined with other ways, such as Marx et al. (2009) established the method that combining the vector integration into the rDNA locus with the PTVA to produce strains with different gene copy number (27).
In the most cases, the copy number is determinant in promoting productivity of the recombinant protein; however, the improvement usually reaches a plateau with increasing gene dosage. Overexpression of heterologous proteins may bring about accumulation of misfolded protein (13), which triggering the endoplasmic reticulum (ER) stress and further activating the unfolded protein response (UPR) (49). Thus, overcoming the secretion block that caused by the ER stress may be crucial in enhancing foreign protein production. Recently, some measures, like overexpression of chaperones and foldase, were taken to overrule the obstacle of secretion. To improve the folding and secretion of proteins, the HAC1, as the unfolded-protein response (UPR) pathway regulator (57), was overexpression in the P. pastoris. For example, Li et al (2015) increased the yield of the phytase by 412% by the overexpressing HAC1 (21).
However, the effect of Hac1p to heterologous protein expression needed to be evaluated on a case by case basis, like that Valkonen et al (2003) found that the HAC1 overexpression increased the α-amylase production by 70% but had no in uence on the endoglucanase secretion (43). Furthermore, the chaperone PDI, involved in the disul de bond formation in the ER, increased the capacity for processing proteins of ER in P. pastoris (11). Sha et al (2013) detected that the lipase r27RCL production in the strain with one copy chaperone gene PDI plus ve copies was 2.74-fold higher than that in the control strain with only one copy (39). In addition, the Sso1 and Sso2, as the t-SNAREs protein that working at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane, were demonstrated to modulate the protein expression level (32). Ruohonen et al (1997) found the multiple copies of the SSO2 gene increased the secretion of invertase to the cell surface by 1.5 fold, which suggesting that the SSO gene exert the secretion-enhancing function in the process of the protein secretion (34).
In our previous work, the chitosanase AqCoA from Aquabacterium sp. A7-Y was cloned and successfully expressed in Escherichia coli BL21 (DE3). Furthermore, the AqCoA exhibited higher enzyme activity toward the chitosan and sodium carboxymethylcellulose (CMC) (47). However, the expression level of the AqCoA is limited in E.coli, which cann't meet the application in the industrial production. Thus, in this study, we adopted expression system of Pichia pastoris to improve production of the AqCoA. Furthermore, to elevate the yield of the AqCoA, a novel high-copy yeast expression vector was constructed, which was based on the method for unmarked gene deletions proposed by Pan et al (2011) (30). In addition, the connection among gene copy number, the chaperones and production of AqCoA was investigated.

The construction of recombinant plasmid
First, seven multiple cloning sites (MCS) synthesized was integrated to the plasmid pUC52-kan. Next, some insertion elements, like His4 site, the cassette containing the AOX tanscription termination (AOX TT), the Zeo r selectable marker (ZeocinR cassette) and lox66 sites, the gene cre (CRE) and promoter AOX1 (pAOX1), were ampli cated by the primes in the table S2, and then PCR products were puri ed using the AxyPrep TM DNA Gel Extraction Kit (Axygen). At last, a new expression plasmid was constructed by inserting different insertion elements into the parent plasmid pUC52-Kan in turn. Detailed steps of plasmid construction were presented in the gure S1-S3.
Then, the PCR products of the chitosanase AqCoA and the plasmid pMC-GAP were digested respectively with Xho I and EcoR I and then ligated to generate the plasmid pMC-GAP-AqCoA by the T4 ligase. The transformation of P. pastoris with pMC-GAP-AqCoA and the selection of transformants with pMC-GAP-AqCoA were performed as described by Niu et al (29).
The transformants obtained were cultured overnight on the YPGZ plate at 30℃. Then single transformant selected was transferred to liquid mudium containing 15ml YPD and cultured at 30℃, 200rpm for 24 hours. After centrifugation, the cells harvested were transferred to 100 ml of BMGY medium and cultured at 30˚C, 220 rpm for four days. Lastly, crude enzyme was obtained by centrifugation at 12,000×g at 4˚C for 10 min to remove yeast cells. The puri cation of recombinant protein and determination of content were executed as described by Li et al (24).

The enzymatic activity of the chitosanase AqCoA
The activity of chitosanase AqCoA toward 95% deacetylated chitosan was detected as presented by Wang et al (47). In addition, the activity of chitosanase AqCoA against CMC was tested in this study. In brief, the transformants obtained were cultured overnight on the YPDZ and YPDZ-CMC plate, respectively. Then, YPGZ-CMC plates were stained with 0.1% Congo red solution at room temperature for 30 min, and decolorized with 1M NaCl for 10 min. The enzyme activity was determined by the size of the hydrolysis circle.

Immunodetection of protein expression levels
For western blotting, the crude enzymes from different strains with multi-copy were prepared. The expression level of strains with multi-copy was estimated by the methods described by Zhang et al (2006) (56). Brie y, the same volume supernatants were rst separated by SDS-PAGE. Then, protein was transferred to nitrocellulose membranes, and nonspeci c binding was blocked by incubating the membrane in solution A (10 mM Tris-HCl pH 8.0, 0.9% NaCl, 5% skim milk powder) overnight at 4 °C. The blocked membrane was then incubated with His antibody (10 mM Tris-HCl pH 7.4, 0.9% NaCl, 2% skim milk powder, 0.05% Tween-20, antibody 1:1000) for 2-3 h at room temperature. Goat anti-rabbit secondary antibody conjugated to horseradish peroxidase was used for the AqCoA detection. Antibody-antigen complexes were visualized with ECL Advance Western Blotting Detection Kit. (Amersham Biosciences,

Buchinghamshire, England)
Determination of the Aqcoa copy number by quantitative PCR The copy number of the Aqcoa was studied according the method described by Li et al (2015) (21). In short, the GAPDH (48) gene with the forward primer GCTATCACTGTCGGTATTAACGG and reverse primer TGGGTAGAGTCGTACTTGAACAT was used as the reference sequence, the Aqcoa was detected with the forward primer CACTTCTACGCGGCCTCTCA and reverse primer TGTTGAGCAGGTCGATGTAGG. The gene copy number of the Aqcoa in the P. pastoris was the ratio of Aqcoa gene copy number calculated by standard curve to the GPADH fragment copy number calculated by standard curve.

Result
The construction of the high-copy plasmid with stable transformation stability According to the steps in the gure S1-S3, the recombined plasmids P1 and P2 were constructed successfully. However, in the process of verifying whether the plasmid P3 ( gure S3) is successfully constructed, the target bands (3572 bp) between the two sites lox71 and lox66 in the plasmid P3 were not obtained ( gure S4 (a)). Subsequently, the plasmid of the transformant 2 in the gure S4(a) was extracted and sequenced. The result showed that a new site lox72 replaced the sequence between the two sites lox71 and lox66 in the transformant 2 ( gure S4 (b)), which suggested that leaky expression of AOX promoters in the E. coli drove the translation of the cre gene, resulting the recombination of sequence between lox71 and lox66 sites in the plasmid P3.
To avoid the leaky expression of AOX promoters in E. coli cell, the plasmid P4 was generated by inserting the operon lactose (lacO) gene between pAOX and CRE of the plasmid P3 (Fig. 1). Moreover, as shown in the Fig. 2a, the target bands (3572 bp) were ampli ed by PCR in the transformants 1 and 4. Meanwhile, the circular plasmid (8741 bp) extracted in the transformant 1 was consistent with the theoretical size (5827 bp) in nucleic acid gel (Fig. 2b), which suggested that the operon lacO controlled rigorously the expression of cre in E. coli.
To further verify the function of the plasmid P4, the plasmid P4 linearized by Sal I was transferred to P. pastoris. Then, ve transformants selected were cultured and induced by methanol. After 24h, each strain was separately inoculated on two plates YPG and YPGZ for overnight culture at 30℃. As shown in the Fig. 2c, the colonies treated by methanol grew only on YPD plates, while they did not grow on the YPDZ containing bleomycin, which indicating that the resistance of the strain to bleomycin was eliminated after the inducement of methanol, the plasmid P4 was successfully constructed. In addition, to promote the expression of target genes, promoter GAP and secretory factor α-factor were inserted into the plasmid P4, meanwhile, the restriction sites Xho I and Not I between CRE cassette and ZeocinR cassette were deleted by overlapping extended PCR. At last, the plasmid pMC-GAP was constructed successfully based on the plasmid 4 (Fig. 3).
Heterologous expression of the chitosanase AqCoA in P. pastoris In our previous study, the chitosanase AqCoA was heterologously expressed in E. coli. To improve the expression level of the AqCoA and reduce the cost of postprocessing, the chitosanase AqCoA was heterologously expressed in P. pastoris. Our previous study found the chitosanase AqCoA was able to hydrolyze sodium carboxymethylcellulose (CMC), thus, the strain GAP-1AqCoA was cultured on YPDZ plate containing CMC. As shown in Fig. 4(a), the hydrolytic circle was generated on the YPDZ plate containing CMC. Then, the activity of ten transformants selected in the liquid culture to 95% deacetylated chitosan was detected. As shown in the Fig. 4(b), compared to others, the transformant 3 showed the highest enzyme activity that reached to 0.18 U/ml (Fig. 4(b)).

Effect of different gene doses on expression of the AqCoA
To construct the plasmid pMC-GAP-2AqCoA, the pMC-GAP-1AqCoA was divided into two parts. One part was digested with two restriction enzymes Spe I and Xba I to produce a cassette with single copy (1 AqCoA). The other part was cut with single enzyme Xba I and dephosphorized with alkaline phosphatase to prevent the vector from self-linking. The pMC-GAP-2AqCoA was generated by linking the dephosphorization product with 1 AqCoA cassette. The method was also applied in the construction of the plasmid pMC-GAP-4AqCoA and pMC-GAP-8AqCoA. The recombinant plasmids (pMC-GAP-2AqCoA, pMC-GAP-4AqCoA and pMC-GAP-8AqCoA) were linearized and transferred into the P. pastoris. The transformants were selected according to the size of the hydrolysis circle to culture in vitro and activity detection. Similarly, the transformants containing pMC-GAP-3AqCoA, pMC-GAP-5AqCoA and pMC-GAP-5AqCoA were obtained and named as GAP-3AqCoA, GAP-5AqCoA, GAP-7AqCoA, respectively. As shown in the Fig. 4(c), activity of the AqCoA against 95% deacetylated chitosan was gradually enhanced with increase of the AqCoA cope number. Among them, the GAP-4AqCoA exhibited the highest enzyme activity (0.32 U/ml). However, the enzyme activity began to decline as the copy number of the AqCoA exceeded four.
To improve more e ciently the expression of the AqCoA, the plasmid pMC-GAP-PDI-ERO1-SSA4-SSE1-YDJ1-SSO2 was constructed and transferred to the strain GAP-1AqCoA or GAP-4AqCoA or GAP-8AqCoA, respectively. The transformants obtained were named as GAP-1AqCoA − 6chaperone GAP-4AqCoA − 6chaperone and GAP-8AqCoA − 6chaperone, respectively. Then, based on the strain GAP-8AqCoA-6chaperone, the transformants GAP-12AqCoA-6chaperone, GAP-16AqCoA-6chaperone, GAP-20AqCoA-6chaperone and GAP-24AqCoA-6chaperone were separately constructed. The result showed the synergistic effect between gene copy number and molecular chaperones in enhancing expression of the AqCoA. As indicated in the Fig. 6(c), when the co-expression of six chaperones, the enzyme activity of AqCoA enhanced gradually with the increase of the copy number of the AqCoA, suggesting that combination of six chaperones synergistically enhanced the secretion levels of the recombinant protein. Moreover, the maximum enzyme activity reached 2.319 U/ml by the recombinant strain with six chaperones combination plus sixteen copies chitosanase gene Aqcoa, which was 13-fold higher than that for the control strain GAP-1AqCoA with only one copy (Table 1). However, the activity gradually decreased as the copy number exceeded 16, indicating that molecular chaperones had certain limitation in relieving secretion bottlenecks of protein. Besides, the expression level of the AqCoA was detected by western blot. The result exhibited that the expression level of the AqCoA increased gradually with the increase of the copy number of the AqCoA in the Fig. 6(d), the highest amount of protein attained 125.20 mg/l by the recombinant strain with six chaperones combination plus sixteen copies chitosanase gene Aqcoa (Table  1), which was consistent with the result in the Fig. 6(c). The copy number of target genes had a crucial effect on the production of recombinant protein, thus, the Aqcoa gene copy number in the genomes of the integrants was determined by the qPCR assays. As shown in Table 2, the Aqcoa gene copy numbers of strains GAP-1AqCoA-6chaperone, GAP-4AqCoA-6chaperone, GAP-8AqCoA-6chaperone, GAP-12AqCoA-6chaperone, GAP-16AqCoA-6chaperone, GAP-20AqCoA-6chaperone and GAP-24AqCoA-6chaperone were 0.98, 2.77, 6.02, 9.79, 12.36, 15.47 and 18.02, respectively, which was not consistent with theoretical copy number. It was likely that the recombinant plasmid was not fully integrated into the yeast genome, which caused that the copy number of the gene had difference between the theoretical and measured values.

Discussion
The pMC-GAP is a novel plasmid with rapid and convenient in construction of high-copy strain. At present, the method to build multi-copy strain mainly depended on the different concentration of antibiotics, which led to the increase of the cost in the experiment. Pan et al (2011) proposed the effective strategy to delete gene from genome, in which referred to that resistance genes on foreign plasmids integrated into P. pastoris genome via homologous recombination were at last deleted by the Cre recombinase (30). However, the application of this system in yeast expression has not been reported. Therefore, we rebuilt the plasmid P3 ( gure S1-S3) and found the promoter AOX1 leaked expressing in the E. coli activated the translation of the Cre enzyme that led to recombination between the two lox sites ( gure S4), which hindered the application of this system in yeast expression. Therefore, some strategies took to regulate the expression of the Cre protein. Some investigators mentioned that the promoter lac (P lac ) (45) of E.coli was admittedly a rather simple system in gene regulation (20) and strictly modulated by the inducer isopropyl β-D-thiogalactopyranoside (IPTG) (1). To prevent the leakage of the gene expression, the plasmid P3 was added the promoter lac (P lac ) and generated the plasmid P4 (Fig. 1). In addition, different promoters were successfully employed for the expression of recombinant proteins in P. pastoris (7). The alcohol oxidase (AOX1) promoter, strongly induced by addition of methanol, was extensively used for recombinant protein production in P. pastoris (9,15). However, the methanol needed to be added continuously during the growth of the P. pastoris, which increased the risk of bacterial contamination. Nevertheless, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (pGAP) has been turn into a wide alternative (4,55), the promoter utilized other carbon sources like glucose, glycerol as a substrate (48,16) and avoid the use of methanol in the industry, which possessed important advantageous in industrial production. Thus, to facilitate industrial production of the recombinant proteins in P. pastoris, we employed the pGAP and constructed the novel plasmid pMC-GAP (Fig. 3). Moreover, in this study, some strains containing different gene copy were obtained, indicating that the plasmid pMC-GAP is more e cient in generating multi-copy strains.
Increase the copy number of the AqCoA could signi cantly enhanced the protein yield (Fig. 4c). However, it gradually reached the plateau in the increase of the protein production once gene dosage exceeded a certain limit, suggesting that it was not the linear relation between gene dosage and protein yield. Previous studies showed that over-expression of heterologous proteins in yeasts increased the burden of the folding capacity in the ER and activated the unfolded protein response (17), which limiting the secretory pathway and further reducing the production of the foreign protein.
To alleviate the stress on the ER, a few chaperone genes were overexpressed. In the study, ten chaperone genes were employed to augment the recombinant protein expression Different chaperones exhibited different e ciencies towards the foreign protein expression. In among of them, up-regulating of PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 enhanced the expression of the AqCoA (Fig. 5). Pdip, as the protein disul de isomerase, assisted in rearrangement of incorrect disul de pairings by isomerase activity (50). Overexpression of PDI increased recombined protein production in the P. pastoris (40). Similarly, the overexpression of the avoenzyme Ero1p that producing disul de bonds for oxidative protein folding in the ER (10) improved Fab fragment secretion in P. pastoris (46). As the most prominent components regulating the UPR (3), the overexpression of the transcription factor Hac1p increased translation of the chaperones and further enhanced expression of heterologous protein (14). Ydj1p and Ssa1p were the cytoplasmic chaperones. Ssa1p directly interacted with Ydj1p, that facilitating to assist in the transportation of intermediate proteins to the ER (28). Increasing the expression of chaperone Ydj1p and Ssa1p improved production of foreign proteins (37). Ssa4 was also the cytosolic chaperone and responsible for the transport of target nascent proteins to the ER membrane (5,12). Overexpression of Ssa4 increased protein expression level in P. pastoris (53). As the secretion helper factor, Sso2 acted as one of the soluble N-ethylmaleimidesensitive factor attachment protein receptors and aided the fusion of secretory vesicles at the plasma membrane (34,51). Overexpression of Sso2 also achieved the purpose of enhancing protein production (18). Consistent with the above reports, in our work, enhancing the expression level of PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 had a positive impact on the expression of the AqCoA.
At last, we investigated the effects of co-expression between gene dosages and different chaperones on the expression of the AqCoA. In the strain containing one copy, the yield of the protein produced by the coexpression of six chaperones increased by 2.17 times than that of the control strain with single copy (Table 1) and was higher than that of the strain with the individual overexpression of one chaperone, indicating the potential synergistic effects of combined chaperones. Based on the strain with one copy plus six chaperones, the strains with various copy numbers were generated. Furthermore, the highest yield of the protein reached 125.20 mg/l by the strain containing six chaperones and 16 copies, which increased by 13 times than that of the control strain with one copy (Table 1), suggesting co-expression of six chaperones markedly improve secretion pathway of the multi-copy strains to facilitate the expression of the AqCoA. Zhang et al (2006) proposed that some pairs of chaperones could enhance the secretion levels of the protein. However, overexpression of the chaperone pairs exclusively located in the cytoplasm or ER had a negative effect to the protein expression, in which secretion was lower than that mediated by single chaperone (56). In this study, to avoid the above case, the chaperones from the ER (PDI and ERO1) and the cytoplasm (YDJ1, SSA1, SSO2 and SSE4) were co-expressed, which prominently increased the yield of recombinant protein. Therefore, integrated expression of chaperones and gene dosages presented a powerful tool to enhance foreign protein expression.

Conclusion
In this study, a novel plasmid pMC-GAP with stable transformation ability was constructed in P. pastoris. By adopting pMC-GAP, we constructed series of plasmids with different copy number and obtained corresponding transformants. In point of gene dosages, the highest enzyme activity got 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that of strain with a single copy. In addition, the chaperones like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 elevated level of protein expression, and PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased protein expression. Lastly, the maximum enzyme activity reached 2.319 U/ml by the recombinant strain with six chaperones (PDI-ERO1-SSA4-SSE1-YDJ1-SSO2) intergrant plus sixteen copies chitosanase gene Aqcoa, that was 13-fold higher than that for the control strain (GAP-1AqCoA). It presented an effective strategy that co-expression of gene copy and molecular chaperone in improvement of foreign protein expression.

Data Availability
All data generated or analyzed during this study are included in this paper and its supplementary information les.
Ethical Approval Not applicable.
Consent to Participate Not applicable. Supplementary Tables S1-S4 are not available with this version. Figure 1 The addition of lactose operon (lacO) on the plasmid to prevent the leaked expression of AOX in E. coli. The cassette with the lacO and CRE was ampli ed. The cassette and the plasmid P3 were digested by Xho I and Not I and then ligated to generate the recombined plasmid by the T4 ligase. Finally, the plasmid P4 was generated by deleting the restriction site Xho I and Not I on the recombinant plasmid.  Construction of the plasmid pMC-GAP. The AOX tanscription termination codon (AOX TT) and the cassette with the promoter GAP and secretory factor α-factor were respectively ampli ed and ligated to the linearized plasmid P4 in turn to generate recombinant plasmid pMC-GAP. The activity assay of strain GS115 with one-copy or multi-copy. (a) Activity of the strain with single copy to the sodium carboxymethylcellulose (CMC) was detected in the plate YPDZ-CMC by the hydrolysis circle. (b) Activity determination to 95% deacetylated chitosan in ten transformants containing one copy. (c) Activity detection to 95% deacetylated chitosan in the strains with various copies. Effects of expression of different molecular chaperones on the hydrolysis activity toward 95% deacetylated chitosan in the strains with single-copy or four-copy. (a) Effects of expression of different molecular chaperones on the hydrolysis activity to 95% deacetylated chitosan in the strains with single copy. (b) Effects of expression of different molecular chaperones on the hydrolysis activity to 95% deacetylated chitosan in the strains with four copies.