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Applied Microbiology and Biotechnology

Applied Microbiology and Biotechnology

IF: 3.9
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Heterologous expressing melittin in a probiotic yeast to evaluate its function for promoting NSC-34 regeneration

Published:28 October 2024 DOI:
Hsiao-Yun Huang, Hung-Yi Hsu, Cheng-Yu Kuo, Mao-Lun Wu, Chien-Chen Lai, Gary Ro-Lin Chang, Yu-Ju Lin

Abstract

Melittin is a bioactive peptide and the predominant component in bee venom (BV), studied for its many medical properties, such as antibacterial, anti-inflammatory, anti-arthritis, nerve damage reduction, and muscle cell regeneration. Melittin is primarily obtained through natural extraction and chemical synthesis; however, both methods have limitations and cannot be used for mass production. This study established a heterologous melittin expression system in the probiotic yeast Kluyveromyces marxianus. This yeast was selected for its advantages in stress tolerance and high secreted protein yields, simplifying purification. A?>?95% high-purity melittin (MET) and its precursor promelittin (ProMET) were successfully produced and purified at 1.68 μg/mL and 3.33 μg/mL concentrations and verified through HPLC and mass spectrum. The functional test of the NSC-34 cell regeneration revealed that MET achieved the best activity compared to ProMET and the natural-extracted BV groups. Growth-related gene expressions were evaluated, including microtubule-associated protein 2 (MAP2), microtubule-associated protein Tau (MAPT), growth-associated protein 43 (GAP-43), choline acetyltransferase (ChAT), vesicular acetylcholine transporter (VAChT), and acetylcholine esterase (AChE). The results indicated that treating MET increased MAP2, GAP-43, and VAChT expressions, in which cholinergic signaling is related to neurological functions. A heterologously expressed melittin in a probiotic yeast and its potential for promoting NSC-34 regeneration described here facilitate commercial and therapeutic use.

? MET and its precursor ProMET were successfully hetero-expressed in K. marxianus

? ?>?95% high-purity MET and ProMET were purified at 1.68?μg/mL and 3.33?μg/mL

? MET has no cytotoxicity toward NSC-34 and significantly promotes NSC-34 growth

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