克氏原螯虾i-型溶菌酶在巴斯德毕赤酵母中的高效胞外表达及其抑菌活性(英文).浙江大学学报（农业与生命科学版）,2019,45(5):526-532

水燕，管政兵，叶俊贤，史永红，刘国锋，徐增洪（.中国水产科学研究院淡水渔业研究中心，农业农村部淡水渔业和种质资源利用重点实验室，江苏 无锡408；.江南大学生物工程学院，工业生物技术教育部重点实验室，江苏 无锡4；.中国农业科学院上海兽医研究所，上海004）

Lysozymes are antibacterial enzymes that are widely distributed in organisms[1-2]. They are important immune effectors in resisting bacterial pathogens,particularly for aquatic animals[3]. Animal lysozymes are categorized into four types as follows: chickentype (c-type), goose-type (g-type), invertebrate-type(i-type), and chalaropsis-type[4]. The i-type lysozymes only existed in invertebrates, and they are considered as essential immune effectors for resisting bacterial pathogen attack in the innate immune system[3-4].

Red swamp crayfish,Procambarus clarkii, is an important aquaculture species in China, and the expansion ofP. clarkiiaquaculture is severely impaired by pathogens[5]. Two i-type lysozymes, pc-iLys1 and pc-iLys2,have been identified in this crayfish in 2010[6].However, due to possessing toxic effects to Escherichia coli, the recombinant pc-iLys1 and pc-iLys2 proteins expressed in theE. colisystem were mainly as insoluble inclusion bodies with low soluble-expression efficiency. To characterize theP. clarkiii-type lysozyme and develop a crayfish-sourced i-type lysozyme for the applications of potential feed additive in the future, a recombinant expression method must be established for the efficient production of pc-iLys.

TheP. pastorisis a good host for the yields of various heterologous proteins in the industry and the academy[7-8]. The advantages of this system include easy genetic manipulation, post-translational modification, and high cell density with a cheap medium.Furthermore, extracellularly expressed proteins can be collected directly from the culture medium, thus simplifying the purification steps of downstream[7,9].The present study intended to establish an efficient strategy for the heterologous expression of pc-iLys1 in P.pastoris.

1 Materials and methods

1.1 Strains,plasmids,and reagents

E. coli TOP10, P. pastoris SMD1168, and plasmid pPIC9K were purchased from Invitrogen(Carlsbad, USA). Micrococcus lysodeikticus cells(Shanghai Rebiosci Biotechnology Co., Ltd. in China) were employed for the lytic activity assay of lysozymes. DNA polymerases, restriction endonucleases,and ligases were acquired from TaKaRa (Dalian,China). All chemicals were of the biological reagent grade.

1.2 Synthesis of the codon-optimized pc-iLys1 cDNA

The usage bias of codons differed for P. clarkii and P. pastoris. The open reading frame (ORF) of pc-iLys1 cDNA (471 bp) (GenBank accession No.GQ301200) was optimized in favor of P. pastoris expression by using the online program DNAorks 3.2.4 (https://hpcwebapps.cit.nih.gov/dnaworks/) (Fig.1). The optimized pc-iLys1 cDNA was synthesized(Sangon, China), and a His6-tag was added at the Cterminus of pc-iLys1. The pc-iLys1 cDNA was ligated into the pPIC9K between the EcoRⅠand NotⅠsites, which was controlled by the alcohol oxidase 1(AOX1) promoter. The recombinant plasmid pPIC9Kpc-iLys1 was transformed into E. coli TOP10 for amplification.

Fig.1 Alignment of nucleotide sequences between the optimized and the original cDNA of pc-iLys1

1.3 Transformation of P. pastoris and screening of transformants

The SalⅠ-linearized pPIC9K-pc-iLys1 was transformed with the electrocompetent cells of P. pastoris SMD1168. A multi-copy selection of resistant P.pastoris transformants was performed by successively replicating the His+transformants onto a series of YPD(1% yeast extract, 2% peptone, 2% dextrose) plates containing 0.25, 0.50, 1.00, 2.00, and 4.00 mg/mL of geneticin. Some positive transformants with high resistance to geneticin were confirmed by polymerase chain reaction(PCR).The copy number of the pc-iLys1 gene in P. pastoris genome was determined by realtime fluorescent quantitative PCR and calculated according to the double standard curves. The glyceraldehyde 3-phosphate dehydrogenase (GAPDH)gene in P.pastoris was chosen as the reference gene.

1.4 Cultivation and condition optimization of recombinant P.pastoris in a shaking flask

The selected P. pastoris transformants were inoculated with 3 mL of YPD medium in conical tubes and grown at 30 ℃for 24 h.Then, 1 mL of the culture was transferred to 100 mL of BMGY [100 mmol/L potassium phosphate (pH 6.0), 1% yeast extract,2%peptone,1.34%yeast nitrogen base,0.004‱biotin, 1% glycerol] medium in a 500 mL shake flask.After growth for 24 h, the culture was centrifuged at 3 000 r/min for 10 min at the room temperature to harvest the cells. Next, the cells were resuspended in 100 mL of BMMY [100 mmol/L potassium phosphate(pH 6.0), 1% yeast extract, 2% peptone, 1.34% yeast nitrogen base, 0.004‱ biotin, 0.5% methanol] medium and cultivated at 30 ℃for 72 h to induce the pciLys1 expression. To maintain the methanol induction,100% methanol was added every 24 h at a final concentration of 1.0% throughout the induction period. At the end of the induction, the culture supernatant was sampled for subsequent analyses.The P. pastoris SMD1168 transformed with an empty pPIC9K plasmid was used as the control.

Four key parameters, including induction time,induction temperature, pH, and methanol concentration,were optimized at the shaking flask level using onefactor-at-a-time (OFAT) approach. First, different induction temperatures (20, 24, 28, and 32 ℃) and induction time(48,72,96,and 120 h)were applied at the constant pH 7.0 and 1.0% methanol concentration.Then, different pH (3.0, 4.0, 5.0, 6.0, 7.0, and 8.0)and methanol concentrations (0.25%, 0.50%, 1.00%,1.50%,2.00%,and 2.50%)were examined successively.

1.5 High cell density fed-batch fermentation and purification of recombinant pc-iLys1

A three-phase fed-batch fermentation was carried out. The selected transformant was inoculated into 100 mL of BMGY medium and cultivated at 30 ℃. After D (600 nm)≈6.0, the culture broth was transferred to 3 L of basal salt medium in a 5 L bioreactor [CSBio (Shanghai) Ltd.] for high cell density fermentation.The parameters optimized at the shaking flask level above were applied. The glycerol batch phase was implemented after inoculation and continued for 24 h. After glycerol exhaustion, 50%glycerol was supplemented at a rate of 20 mL/(L·h)for 5 h.Pure methanol was added at a rate of 4 ml/(L·h)for 2-3 h after glycerol was depleted. When the dissolved oxygen (DO) level was stabilized, the methanol feeding rate was increased to maintain the methanol concentration at 1.5%.The culture broth was sampled every 12 h for further analyses of dry cell mass(DCM)and lytic activity.

The pc-iLys1 fermentation supernatant and Ni-NTA agarose (Invitrogen, USA) were mixed and loaded into a gravity flow column. The mixture was kept at 4 ℃for 4 h and then washed with a linear gradient of imidazole (10-40 mmol/L). Finally, the His6-tagged pc-iLys1 was eluted by 500 mmol/L imidazole and desalted by using the Amicon Ultra 3K device(Millipore,USA).

1.6 Antimicrobial activity assay

Gram-positive bacteria (Staphylococcus aureus,Bacillus subtilis, and Micrococcus luteus) and Gramnegative bacteria (Pseudomonas aeruginosa, Vibrio vulnificus, and E. coli) were utilized for antimicrobial activity assay. Each bacterium was cultured overnight,collected, and diluted using 3% trypticase soy broth(TSB) to a density of 2.5×105CFU/mL. Then, 180 mL of the bacterial suspension was mixed with 20 mL of the purified pc-iLys1(10 μmol/L),seeded into a 96-well polypropylene plate, and cultured at 37 ℃for 24 h. The bacterial density was estimated at the absorbance of 630 nm.

2 Results

2.1 Screening of yeast transformants for pciLys1 expression

Six highly geneticin-resistant clones (two of them and the rest were resistant to 4.0 and 2.0 mg/mL of geneticin, respectively) were selected for a flaskscale expression test. The integration of the plasmid pPIC9K-pc-iLys1 was confirmed by PCR with 5´and 3´ AOX1 sequencing primers, and the genomic DNA of P. pastoris recombinants was used as the template. Lytic activity was detected in the culture supernatants of all transformants.The transformant with the highest lytic activity in its culture supernatant was selected for subsequent experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis of the culture supernatant of the excellent transformant revealed the efficiently secretory expression of pc-iLys1, which was indicated by a protein band of about 17 kDa (Fig. 2). The expressed pc-iLys1 protein was also recognized through an anti-His6-tag mouse monoclonal antibody by Western blotting analysis (Fig. 2). No apparent lytic activity and corresponding protein bands were detected in the supernatant of the transformant with the empty pPIC9K vector.To simplify the purification process,a His6-tag, which allowed for one-step purification by immobilized metal-affinity chromatography, was introduced to the C-terminus of the recombinant pciLys1.Finally,the recombinant enzyme was eluted by 0.5 mol/L imidazole under natural conditions(Fig.2).

2.2 Optimization of expression condition

The best enzymatic activity was attained when the temperature was maintained at 28 ℃and the induction time was 96 h (Fig. 3A).The preferable pH for pc-iLys1 expression was 7.0 (Fig. 3B).The effect of methanol concentration on pc-iLys1 expression showed that the volume fraction with 1.5% was the best inducing concentration (Fig. 3C). When the best fermentation parameters were combined, the extracellular expression level of pc-iLys1 reached to 96.5 U/mL and the DCM concentration reached to 4.8 g/L in the shaking flask. Through the optimization of expression condition, the pc-iLys1 activity of culture supernatant under the best condition increased by 30.7%.

Fig.2 SDS-PAGE and Western blotting analysis of the secretory expression of pc-iLys1 in P.pastoris

2.3 Fed-batch fermentation of pc-iLys1 in a 5 L bioreactor

To achieve the high-yield production of pciLys1,the glycerol batch phase was first implemented after inoculation and continued for 24 h, resulting in an increase in DO because of carbon source limitation. Subsequently, the glycerol fed-batch phase started and lasted for 5 h. The DCM concentration increased to 33.6 g/L. After that, the methanol fed-batch phase was initiated to induce the expression of pc-iLys1. No apparent lytic activity was detected in the culture supernatant before the methanol fed-batch phase. During the induction phase of fed-batch fermentation, a higher expression level of pc-iLys1 was achieved compared with the shake flask cultivation. By the end of the fermentation process, the lytic activity and the DCM concentration reached to 2 052.6 U/mL and 98.1 g/L,respectively(Fig.4).

2.4 Antimicrobial activity of the purified recombinant pc-iLys1

The antimicrobial activities of the purified recombinant pc-iLys1 and the hen egg-white lysozyme(HEWL) against Gram-positive and Gram-negative bacteria were assayed. The results (Fig. 5) showed that both pc-iLys1 and HEWL exhibited antimicrobial activities against S. aureus, B. subtilis, M. luteus, P.aeruginosa, V. vulnificus and E. coli. Furthermore,compared with the HEWL, B. subtilis, P. aeruginosa,and V. vulnificus were more susceptible to the pciLys1. Bovine serum albumin (BSA) as a control did not exhibit obvious antimicrobial activity against any of the examined bacterial species.

Fig.4 Time-course profile of three-stage fermentation for the high-yield production of pc-iLys1 by recombinant P. pastoris in a 5 L bioreactor

Fig.5 Antimicrobial activities of recombinant pc-iLys1 in vitro

3 Discussion

Procambarus clarkiiaquaculture in China has developed rapidly in recent years[10]. However, the rapid expansion and intensification of P. clarkii farming have led to the emergence of several infectious diseases[10-11]. The discovery and characterization of antimicrobial molecules is essential for designing novel therapeutic agents on controlling these diseases.The purification of i-type lysozyme from the body of P. clarkii is difficult since the native lysozymes have a very low abundance in the tissues. Researchers had tried to use the E. coli expression system to produce recombinant pc-iLys1, but the protein was mostly expressed as inclusion bodies in E. coli. In this study,the P. pastoris system was successfully utilized for high-yield extracellular production of the recombinant pc-iLys1. Recently, a large-scale production of an itype lysozyme from the sea urchin (Strongylocentrotus purpuratus) in P. pastoris has also been achieved by the high cell density fermentation[12]. Its highest lytic activity of culture supernatant in a 5 L bioreactor reached to 960 U/mL[12], which was less than that of this study.

In the P. pastoris system, the expression level of recombinant protein was generally growth-associated.The pc-iLys1 expression level in the shaking flask culture was relatively low due to the limitations of volume, oxygen transfer, and substrate addition, as well as inefficient monitoring of these factors[8]. To achieve high-level production, the bioreactor was preferentially utilized in the fermentation process. In this work, the fed-batch fermentation in a 5 L bioreactor was employed to boost the biomass and production level of the recombinant pc-iLys1.Ultimately, the biomass of P. pastoris and the lytic activity of the pc-iLys1 in the fermentation culture supernatant increased by 20.4- and 21.2-fold,respectively, compared with the corresponding levels in the shaking flask cultivation.One problem commonly existing in the high cell density fermentation of P.pastoris is the degradation of recombinant protein due to proteases[13]. In this study, the proteasedeficient host strain P. pastoris SMD1168 was used,which decreased the proteolysis degree possibly.

In conclusion,the i-type lysozyme from P.clarkii,pc-iLys1,is successfully expressed as a secreted protein in P. pastoris. The high-yield production of bioactive pc-iLys1 is achieved by combining codon modification,strain screening, expression condition optimization,and high cell density fed-batch fermentation. This P.pastoris recombinant strain is promising for the industrial production of pc-iLys1, which can provide an adequate quantity of protein for future studies, and the pc-iLys1 can serve as a candidate for a novel antimicrobial agent for treating the infectious diseases in P.clarkii.