Full
Outline
To establish a co-expression system combining episomal and integrative vectors, enhance the expression efficiency of antimicrobial peptides in Pichia pastoris, and address the issue of insufficient production caused by copy number limitations and expression instability.
The 2 kanamycin-resistant episomal vectors, pPICKαA-panARS-JS21T and pGAPKαA-panARS-JS21T, were constructed using the Kluyveromyces lactis panARS autonomously replicating sequence. These were co-electroporated with the integrative vector pPICZαA-JS21T into Pichia pastoris X-33. Positive transformants were selected on Zeocin plus G418 double-antibiotic plates. Shake-flask cultures were run for more than 120 h with methanol supplementation every 24 h to maintain induction. Secreted total protein was quantified by the Bradford assay, target bands were visualized by trimethylglycine-sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine-SDS-PAGE), and antimicrobial activity against Staphylococcus aureus was measured by agar diffusion.
After 120 h of fermentation, the integrative plus episomal pGAPKαA-panARS-JS21T co-expression group yielded 107.22 µg/mL of extracellular total protein, 52% higher than 70.56 µg/mL of the integrative-only control. The corresponding inhibition-zone assay showed a 129% increase in antimicrobial activity (P<0.05). The integrative plus pPICKαA-panARS-JS21T group also achieved a 15.8% improvement over the integrative-only group.
A combined episomal/integrative co-expression strategy synergistically elevates JS21T antimicrobial peptide levels in Pichia pastoris, outperforming single-vector systems and offering a new route for high-level production of small peptide molecules.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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