Abstract
BackgroundThe bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. Export and proteolytic cleavage of the leader peptide results in release of active nisin. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products.ResultsWe established heterologous nisin production using the biotechnological workhorse organism Corynebacterium glutamicum in a two-step process. We demonstrate successful biosynthesis and export of fully modified prenisin and its activation to mature nisin by a purified, soluble variant of the nisin protease NisP (sNisP) produced in Escherichia coli. Active nisin was detected by a L. lactis sensor strain with strictly nisin-dependent expression of the fluorescent protein mCherry. Following activation by sNisP, supernatants of the recombinant C. glutamicum producer strain cultivated in standard batch fermentations contained at least 1.25 mg/l active nisin.ConclusionsWe demonstrate successful implementation of a two-step process for recombinant production of active nisin with C. glutamicum. This extends the spectrum of bioactive compounds that may be produced using C. glutamicum to a bacteriocin harboring complex posttranslational modifications. Our results provide a basis for further studies to optimize product yields, transfer production to sustainable substrates and purification of pharmaceutical grade nisin.
Highlights
The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in fivelanthionine rings characteristic for class Ia bacteriocins
C. glutamicum is not suitable for production of active nisin previous studies reported C. glutamicum to be resistant to up to 40 μg/ml of nisin [46], our own results suggest that growth of C. glutamicum is completely inhibited by ~ 1 μg/ml of nisin [47, 48]
To corroborate these results and to determine the nisin concentration that is required for formation of pores in the membrane of C. glutamicum, we devised a biosensor expressing the pH-dependent fluorescent protein pHluorin2 [49] in a similar fashion as described recently for L. monocytogenes [50]
Summary
The bacteriocin nisin is naturally produced by Lactococcus lactis as an inactive prepeptide that is modified posttranslationally resulting in five (methyl-)lanthionine rings characteristic for class Ia bacteriocins. By targeting the universal peptidoglycan precursor lipid II, nisin has a broad target spectrum including important human pathogens such as Listeria monocytogenes and methicillin-resistant Staphylococcus aureus strains. Industrial nisin production is currently performed using natural producer strains resulting in rather low product purity and limiting its application to preservation of dairy food products. Bacteriocins are a group of ribosomally synthesized peptides produced by different bacteria that show high. Weixler et al Microbial Cell Factories (2022) 21:11 antimicrobial activity against various bacteria including antibiotic resistant human pathogens [5, 6]. One of the best characterized bacteriocins is the lanthipeptide nisin, which is naturally produced by different L. lactis strains [8]. The mature peptides consist of 34 amino acids with multiple posttranslational modifications including dehydrated amino acids and formation of so-called (methyl-)lanthionine rings [8]
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