The project focused on optimizing key regulatory parts—five promoters (AOX1, CAT1, FDH1, AOX713) and four signal peptides (α-factor, SP4, SP14, 0030)—to enhance the expression and secretion of the antimicrobial peptide Plectasin NZ2114 in Pichia pastoris. By constructing recombinant plasmids combining different promoter and signal peptide parts, the team screened for the most effective part combinations that enabled high-level, functional expression of NZ2114, demonstrating the critical role of selecting and engineering these genetic parts in developing an efficient production platform.
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Name |
Part Number |
Type |
Description |
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BBa_25DAT2H3 |
Basic part |
Plectasin, the first fungal Plectasin, the first fungal defensin discovered in 2005, is effective against Gram-positive bacteria without harming human cells or causing antibiotic resistance. NZ2114 is a modified version with three amino acid changes (D9N, M13L, Q14R). |
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BBa_K5384004 |
Exist Basic part |
The AOX1 promoter is a strong inducible promoter from the alcohol oxidase 1 gene, commonly used for high-level expression of heterologous genes in methanol-utilizing yeast. |
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BBa_25Y7VR7B |
Exist Basic part |
The FDH1 promoter is a strong, constitutive promoter derived from the formate dehydrogenase 1 gene, commonly used to drive stable gene expression in yeast. |
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BBa_250YWZY2 |
Basic part |
The CAT1 promoter is a constitutive promoter and commonly used to drive gene expression in yeast under various conditions. |
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BBa_2509YDPZ |
Basic part |
The AOX713 promoter is a modified version of the AOX1 promoter from Pichia pastoris, induced by methanol to drive high-level expression of target genes. |
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BBa_25W90YSW |
Basic part |
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BBa_25EJSAXC |
Basic part |
Endogenous signal peptide of Pichia pastoris cells |
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BBa_259J4DW3 |
Exist Basic part |
Endogenous signal peptide of Pichia pastoris cells |
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BBa_K4077005 |
Exist Basic part |
The alpha factor signal peptide is used in *Pichia pastoris* cells to direct the secretion of recombinant proteins. |
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BBa_252FQI57 |
Plasmid backbone |
pPIC9K is a shuttle vector used in Pichia pastoris for high-level expression of recombinant proteins. |
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BBa_25P0CGVJ |
Composite part |
AOX1 promoter drives the expression of Plectasin NZ2114 |
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BBa_25NJWF2D |
Composite part |
FDH1 promoter drives the expression of Plectasin NZ2114 |
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BBa_25Y7DCBM |
Composite part |
CAT1 promoter drives the expression of Plectasin NZ2114 |
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BBa_25FBRV07 |
Composite part |
AOX713 promoter drives the expression of Plectasin NZ2114 |
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BBa_25EMVDZF |
Composite part |
SP4 signal peptide drives the secretion of Plectasin NZ2114 |
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BBa_25YORBVC |
Composite part |
SP14 signal peptide drives the secretion of Plectasin NZ2114 |
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BBa_25D2294J |
Composite part |
0030 signal peptide drives the secretion of Plectasin NZ2114 |
[1] Yang Y, Teng D, Zhang J, et al. Characterization of recombinant plectasin: solubility, antimicrobial activity and factors that affect its activity[J]. Process biochemistry, 2011, 46(5): 1050-1055.
Mygind P H, Fischer R L, Schnorr K M, et al. Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus[J]. Nature, 2005, 437(7061): 975-980.
[2] Jing, X. L., Luo, X. G., Tian, W. J., Lv, L. H., Jiang, Y., Wang, N., & Zhang, T. C. (2010). High-level expression of the antimicrobial peptide plectasin in Escherichia coli. Current microbiology, 61(3), 197–202. https://doi.org/10.1007/s00284-010-9596-3
[3] Zhang J, Yang Y, Teng D, et al. Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus[J]. Protein expression and purification, 2011, 78(2): 189-196.
[4] Wang N, Wang Y J, Li G Q, et al. Expression, characterization, and antimicrobial ability of T4 lysozyme from methylotrophic yeast Hansenula polymorpha A16[J]. Science China Life Sciences, 2011, 54: 520-526.
[5] Zhang, Y., Teng, D., Mao, R. et al. High expression of a plectasin-derived peptide NZ2114 in Pichia pastoris and its pharmacodynamics, postantibiotic and synergy against Staphylococcus aureus. Appl Microbiol Biotechnol 98, 681–694
[6] Nong L, Zhang Y, Duan Y, Hu S, Lin Y, Liang S. Engineering the regulatory site of the catalase promoter for improved heterologous protein production in Pichia pastoris. Biotechnol Lett. 2020 Dec;42(12):2703-2709. doi: 10.1007/s10529-020-02979-x. Epub 2020 Aug 17. PMID: 32808199.
[7] Patent application publication. No.: US/2024/0141363 A1