Distribution of Epsilon-Polylysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin

Epsilon-polylysine (ε-PL) is an antimicrobial commercially produced by Streptomyces fermentation and widely used in Asian countries for food preservation. Here we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the ε-PL synthetase from Streptomyces in terms of enzymatic domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus ε-PL synthetase promoter, we confirmed that its product is indeed ε-PL. A comprehensive sequence analysis suggests that ε-PL synthetases are widely spread among coryneform bacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain Pls gene. This discovery raises the possibility that ε-PL as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems. IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of foodborne illness. ε-PL is an excellent food preservative as it is potent, wide spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those in the EU) partially because it was not a natural composition of food but rather originated from the soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The unexpected finding of ε-PL synthetases in cheese and skin bacteria suggests that ε-PL may naturally exist in cheese and on our skin.

IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and 23 millions of cases of foodborne illness. ε-PL is an excellent food preservative as it is potent, wide 24 spectrum and is heat stable and biodegradable. It has not been accepted by all countries (e.g those 25 in the EU) partially because it was not a natural composition of food but rather originated from the 26 soil bacteria Streptomyces, a famous producer of various antibiotic drugs and toxins. The 27 unexpected finding of ε-PL synthetases in cheese and skin bacteria suggests that ε-PL may 28 naturally exist in cheese and on our skin. Epsilon-polylysine (ε-PL) is a small cationic isopeptide made from the essential amino acid L-35 lysine (Fig. 1A). It exhibits antimicrobial activity against a wide spectrum of bacteria, yeast, and 36 fungi and is heat stable and active in different food matrices (1). ε-PL has been a popular food 37 preservative in Japan since the late 1980s, followed by Korea and China, and has been given 38 Generally Regarded As Safe (GRAS) status in the USA. As a secondary metabolite, ε-PL was first 39 discovered from the soil bacterium Streptomyces albulus, which is still used in its commercial 40 production (2). Later, more producers of ε-PL were identified from the family of   Cheese prepared by fermentation of milk is an ancient food with a history of at least eight thousand 47 years (6).
The microorganisms on and in cheese and their secondary metabolites play key roles for 48 the flavor, quality, preservation and safety of the cheese.

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We tested ε-PL production from C. variabile using a two-stage culture method which was efficient 80 in finding Streptomyces producers (18). However, no ε-PL production was detected in the culture. 81 We reasoned that the cheese bacteria may have different regulation of ε-PL biosynthesis to the soil 82 bacteria Streptomyces. Therefore, we cloned the C. variabile gene onto a plasmid with a pBAD 83 promoter. The recombinant plasmid was transferred into model organism Corynebacterium 84 glutamicum. However, again ε-PL production could not be observed. 85 In Streptomyces, the promoter sequence is critical for ε-PL production. It has been demonstrated 86 that expression of pls in the native host S. albulus with an altered promoter did not lead to ε-PL 87 production, but the use of the original promoter resulted in ε-PL production even in a heterologous    Concluding remarks. In this study, we confirmed that cheese bacterium C. variabile has a ε-PL 117 synthetase gene. We did not observe ε-PL production by C. variabile in our artificial culturing 118 conditions. This is probably because the production of this secondary metabolite is regulated by a 119 mechanism we are yet to understand, which is not uncommon for antimicrobial secondary 120 metabolites. Furthermore, Pls were widely found in cheese-and skin-isolated coryneform bacteria.    Table   134 S2.

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Culturing conditions. Streptomyces and Corynebacterium strains were maintained on ISP2 agar 136 (BD Difco™). They were assayed for ε-PL production by a two-stage culture method (18). S. 137 coelicolor strains were inoculated in M3G medium (26) at pH6.8 for 24 h at 30°C, then the pH 138 was adjusted to 4.0 by HCl and cultured for another 3 days with shaking at 120 rpm.

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Corynebacterium strains were cultured similarly with GMPY medium (Malt extract 10 g/L;

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Peptone 10 g/L; Yeast extract 0.1 g/L autoclaved added with 10 g/L glucose as carbon source) and