Abstract
Human lysozyme (hLYZ), known for its bacteriolytic activity, is widely applied in the food and pharmaceutical industries as an antimicrobial agent. However, its extensive application was limited by its low large-scale production efficiency. In this study, a combinational method of integrating codon optimization, multiple gene copies, and ER molecular chaperone co-expression was developed to improve the heterologous production of hLYZ in Pichia pastoris GS115. Our results showed that increasing the copy number of the optimized hLYZ gene in P. pastoris could enhance its secretory production level up to 1.57-fold. The recombinant opt-hLYZ-6C strain that contains six copies of opt-hLYZ gene exhibited the highest mRNA transcription levels, giving the highest production of 0.22 ± 0.02 mg/mL of hLYZ in the medium supernatant with a bacteriolytic activity of 14,680 ± 300 U/mL against Micrococcus lysodeikticus in the shaking flask experiment. Moreover, co-overexpression of ER retention molecular chaperones, such as Pdi1 or Ero1, in the recombinant opt-hLYZ-6C strain both presented positive effects on the secretory production of hLYZ. Our further characterization indicated that tandem co-expression of Ero1 and Pdi1 together presented an added-up effect. The secretory production of hLYZ in the medium supernatant reached 0.34 ± 0.02 mg/mL of the recombinant opt-hLYZ-6C-EP strain in the shaking flask experiment, with a bacteriolytic activity of 21,200 ± 400 U/mL. Compared to the recombinant opt-hLYZ-1C strain, these final improvements were calculated as 2.43-fold and 2.30-fold on secretory protein levels and antibacterial activity, respectively. Finally, the recombinant opt-hLYZ-6C-EP strain was applied for high-density cultivation in 5 L of fermenter, in which the secretory yield of hLYZ reached 2.34 ± 0.02 mg/mL in the medium supernatant, with a bacteriolytic activity of 1.76 ± 0.02 × 105 U/mL against M. lysodeikticus. All these numbers presented the highest heterologous production levels of hLYZ in microbial systems.
Highlights
Lysozyme (EC 3.2.1.17) belongs to ovo-antimicrobials, which has a well-characterized bacteriolytic characteristic mainly against Gram + bacteria, such as Lactobacillus brevis, Micrococcus lysodeikticus, and Pediococcus damnosus (Ercan and Demirci, 2016)
In improving the heterologous protein production levels in P. pastoris, the most simple and efficient strategies are optimizing the target gene codons based on the genetic preference of P. pastoris and increasing its gene copies integrated into the P. pastoris genome
Using a combinational strategy of gene codon optimization, multiple gene copies, and molecular chaperone co-expression, here, we constructed a robust recombinant P. pastoris GS115 strain, opt-hLYZ6C-EP, in which the heterologous human lysozyme was extracellularly secreted with high antibacterial activity
Summary
Lysozyme (EC 3.2.1.17) belongs to ovo-antimicrobials, which has a well-characterized bacteriolytic characteristic mainly against Gram + bacteria, such as Lactobacillus brevis, Micrococcus lysodeikticus, and Pediococcus damnosus (Ercan and Demirci, 2016). The c-type lysozymes from chicken and human lysozyme were mostly studied, due to their good antibacterial properties and serving as models for enzyme structure and function studies (Peters et al, 1989; Prager and Jollès, 1996; Callewaert and Michiels, 2010). Chicken lysozyme is widely used, while its bacteriolytic activity is almost four times lower than that of human lysozyme (hLYZ) (Ercan and Demirci, 2016). There are reports that hLYZ has better thermostability (Li et al, 1995), and it is safer and less antigenic than chicken lysozyme, especially for use in human food and therapeutics (Morita et al, 1995; Ercan and Demirci, 2016). It is necessary to develop heterologous production strategies of hLYZ in microbial systems to meet market demands
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