Highly efficient expression and secretion of human lysozyme using multiple strategies in Pichia pastoris

Abstract

BackgroundHuman lysozyme (hLYZ), an emerging antibacterial agent, has extensive application in the food and pharmaceutical industries. However, the source of hLYZ is particularly limited.ResultsTo achieve highly efficient expression and secretion of hLYZ in Pichia pastoris, multiple strategies including G418 sulfate screening, signal sequence optimization, vacuolar sorting receptor VPS10 disruption, and chaperones/transcription factors co‐expression were applied. The maximal enzyme activity of extracellular hLYZ in a shaking flask was 81,600 ± 5230 U mL−1, which was about five times of original strain. To further reduce the cost, the optimal medium RDMY was developed and the highest hLYZ activity reached 352,000 ± 16,696.5 U mL−1 in a 5 L fermenter.ConclusionThis research provides a very useful and cost‐effective approach for the hLYZ production in P. pastoris and can also be applied to the production of other recombinant proteins.

High‐activity recombinant human carboxypeptidase B expression in Pichia pastoris through rational protein engineering and enhancing secretion from the Golgi apparatus to the plasma membrane

“…Surprisingly, both Δ Vps10 and Δ Vps10 ‐domain I strains did not show improved performance in secreting pre‐pro‐α‐GFP but reduced extracellular GFP to 50%−60% of GS115 levels (Figure 2E), contradicting our expectations based on a previous report where deletion of Vps10 promoted the secretion of human lysozyme. [ 30 ] Differences in vacuolar morphology between Δ Vps10 and Δ Vps10 ‐domain I strains suggest a potential role of Vps10 ‐domain II in maintaining P. pastoris vacuolar morphology, although this phenomenon has not been confirmed in other yeast such as Saccharomyces cerevisiae .…”

“…[ 28 ] Recent research on P. pastoris has further enhanced its advantages in protein expression, especially with the knockout of the vacuolar protein sorting receptor Vps10 and the cell wall permeability regulation gene PAS_0305, which respectively reduced mis‐sorting of secreted proteins and improved methanol utilization efficiency, thereby enhancing the expression of recombinant proteins. [ 29,30 ] This has positioned P. pastoris as a more powerful host for recombinant protein expression. [ 30 ] At the same time, P. pastoris distinguishes itself as an advantageous host for recombinant hCPB1 expression due to precise regulation with methanol promoters, [ 31 ] efficient extracellular secretion of proteins, well‐established molecular tools, high genetic stability, and capabilities for high‐density fermentation.…”

“…[ 29,30 ] This has positioned P. pastoris as a more powerful host for recombinant protein expression. [ 30 ] At the same time, P. pastoris distinguishes itself as an advantageous host for recombinant hCPB1 expression due to precise regulation with methanol promoters, [ 31 ] efficient extracellular secretion of proteins, well‐established molecular tools, high genetic stability, and capabilities for high‐density fermentation. [ 32–34 ]…”

Dual cytoplasmic‐peroxisomal compartmentalization engineering and multiple metabolic engineering strategies for high yield non‐psychoactive cannabinoid in Saccharomyces cerevisiae

“…Microbial metabolic engineering is a crucial technology for addressing environmental concerns and overcoming resource limitations, providing sustainable solutions. [14][15][16] Saccharomyces cerevisiae has experienced significant advancements and has become a wellestablished cell factory for the synthesis of natural products in recent years, such as cembratriene-ol, [17] 7-dehydrocholesterol, [18] lycopene, [19] valencene, [20] vincristine, [21] artemisinic acid, [22] and cannabinoids. [23] S. cerevisiae is an ideal host for producing natural products due to its numerous advantages.…”

Recombinant human fibroblast growth factor 7 obtained from stable Chinese hamster ovary cells enhances wound healing

“…[18,19] Post-translational modifications, especially glycosylation, play a crucial role in the function and efficacy of recombinant proteins, making them an important focus of research. [20,21] Therefore, developing and producing recombinant proteins with appropriate glycosylation in animal cells is essential for human applications.…”