Molecular Modification of Kex2 P1’ Site Enhances Expression and Druggability of Fungal Defensin

Abstract

Pichia pastoris is the widely used expression system for producing recombinant secretory proteins. It is known that Kex2 protease plays a vital role in the process of protein secretion, in which the P1’ site affects its cleavage efficiency. To enhance the expression level of fungal defensin-derived peptide NZ2114, this work attempts to optimize the P1’ site of Kex2 by replacing it with 20 amino acids in turn. The results showed that when the amino acid of the P1’ site was changed to Phe (F), the yield of target peptide significantly increased from 2.39 g/L to 4.81 g/L. Additionally, the novel peptide F-NZ2114 (short for FNZ) showed strong antimicrobial activity against Gram-positive (G+) bacteria, especially for Staphylococcus aureus and Streptococcus agalactiae (MIC: 4–8 μg/mL). The FNZ was very stable and retained high activity in various conditions; in addition, a low cytotoxicity and no hemolysis were observed even at a high concentration of 128 μg/mL, and a longer postantibiotic effect was reached. The above results indicate that this engineering strategy provided a feasible optimization scheme for enhancing the expression level and druggability of this antimicrobial peptide from fungal defensin and other similar targets by this updated recombinant yeast.

Site Mutation Improves the Expression and Antimicrobial Properties of Fungal Defense

“…Plectasin binds to lipid II to inhibit cell wall biosynthesis, thereby altering the bacterial morphology of S. aureus [ 33 ]. It is worth noting that the activity of AMPs is not limited to a single targeting mechanism; Many membrane-damaging cationic AMPs have secondary intracellular mechanisms of action [ 1 , 26 , 40 ]. P2 is derived from Pyronema omphalodes and has a similarity of 92% with plectasin, and it killed bacteria via interacting with plasma membrane by molecular oligomerization and destroying genomic DNA [ 40 ].…”

“…The experimental operation of the expression of the Plectasin variant in P. pastoris in well plates, shaking flasks, and 5 L level fermentation was performed as referenced in previous studies [ 26 , 40 , 64 ]. Candidates were identified through three different levels of screening.…”

“…Animal-derived antimicrobial peptides are synthesized in small amounts in response to stress and are useful for fine-tuning immunity [ 25 , 26 ], but their druggability is relative weak, and thus their development direction should be focused on immune regulation as expected. The microbial-derived peptides that they are more close to peptide antibiotics especially for the narrow-spectrum antibiotics as vancomycin, nosiheptide, polymyxin-, daptomycin and others, have good clear antibacterial mechanism and drug-forming properties, i.e., druggability, which is more suitable for development as a new source of candidate antibacterial drugs, and were produced by synthetic biology including gene and metabolic engineering and gene cluster transfer by the present feasible way [ 26 , 27 , 28 , 29 , 30 ]. Plectasin is the first fungal defensin that was isolated from the secreted protein of Pseudoplectania nigrella by Mygind and co-workers in 2005 [ 31 ].…”

Plectasin: from evolution to truncation, expression, and better druggability

“…The sequences of the 15 novel peptides from plectasin truncation were analyzed for their physicochemical properties using biological software. These new derived peptides were constructed by composing of 34-38 amino acids lacking five different fragments, with an isoelectric point range of 6.00-8.62, a positive charge number of −1 ~ +3, and increased hydrophobicity compared to plectasin (Table 1), from our experiences of the recombination expression of many defensin AMPs (Zhang et al, 2011(Zhang et al, , 2014Li et al, 2017;Yang et al, 2019;Liu et al, 2020;Hao et al, 2023;Jin et al, 2023). We cannot exactly estimate and know their possible expression results but only try them one by one, even though they have shown good feasibility of recombination expression based on their net charge number, pI, instability index, and other parameters, and also we cannot know whether they have normal biological activities expect testing.…”

Polylactic Glycolic Acid-Mediated Delivery of Plectasin Derivative NZ2114 in Staphylococcus epidermidis Biofilms

“…NZ2114 ( Table 1 ), a derivative of Plectasin, showed an excellent antibacterial activity against Gram-positive bacteria, especially Staphylococcus aureus ( S. aureus ) and Staphylococcus epidermidis ( S. epidermidis ), with the minimum inhibitory concentration (MIC) as low as 4 μg/mL. However, it is not resistant to trypsin and has a low druggability in mature biofilm [ 15 , 16 ]. Therefore, it is urgent to design a reasonable drug delivery system (DDS) to improve the stability of AMP and the accessibility of delivery in biofilm in order to improve the bioavailability of AMP [ 17 , 18 ].…”