Inhibition of Epsilon-Poly- l -Lysine Biosynthesis in Streptomycetaceae Bacteria by Short-Chain Polyols

Abstract

Antimicrobial epsilon-poly-L-lysine (ePL) is secreted by Streptomycetaceae bacteria, and the mechanism of ePL biosynthesis remains to be elucidated. We previously reported that an unknown ePL derivative accumulates in the culture medium of ePL-producing bacteria when glycerol is added to the culture medium (Nishikawa and Ogawa, Appl. Environ. Microbiol. 68: [3575][3576][3577][3578][3579][3580][3581] 2002). In this study, by using matrix-assisted laser desorption ionization-time of flight mass spectrometry and nuclear magnetic resonance, we identified the unknown derivative as the ester formed between the hydroxyl group of a glycerol molecule and the terminal carboxyl group of an ePL molecule. When a short-chain aliphatic polyol, such as ethylene glycol, propanediol, or butanediol, was added instead of glycerol, a corresponding ePL-polyol monoester accumulated in the culture medium of ePL-producing bacteria. ePL esterification was accompanied by ePL synthesis in intact cells and a cell-free system, but no esterification of exogenous ePL was observed. ePL-polyol esters were formed during lysine polymerization. The number of lysine residues of ePL-polyol esters decreased with increasing polyol concentration. Taken together, these results indicate that ePL synthesis is inhibited by polyols via esterification and that ePL elongation occurs via the incorporation of lysine monomers into the carboxyl terminus of ePL.ε-Poly-L-lysine (ePL) is a strong basic poly(amino acid) secreted by various Streptomycetaceae bacteria and a few filamentous fungi (16,(20)(21)(22)(23). This linear polymer is constructed from L-lysine monomers by the formation of amide bonds between ε-amino and ␣-carboxyl groups (5, 21). Industrially, ePL is produced by fermentation. Because ePL is harmless to humans (7), its antimicrobial activity has been utilized in food preservation. Furthermore, the ability of ePL to suppress fat absorption in the small intestine (10, 24) is of interest for use in health foods. However, natural long-chain ePL tastes bitter. Thus, control of the chain length is important for improving the taste of ePL for consumption (23). Efforts to alter ePL chain length in the industrially established strain Streptomyces albulus 346 (20) have not been successful. This is probably because of the present poor understanding of ePL biosynthesis.There are two possible mechanisms underlying the activation of amino acids for peptide biosynthesis: adenylation (AMP forming) by nonribosomal peptide synthetases (NRPSs) and phosphorylation (ADP forming) by amide ligases. NRPSs usually produce fixed-length peptides except for streptothricin (nourseothricin), which has a variable-length poly(␤-lysine) chain (9, 25). In contrast, ADP-forming amide ligase produces variable-length peptides, such as poly-␥-DL-glutamate (2), in addition to dipeptides, such as D-alanyl-D-alanine and ␥-Lglutamyl-L-cysteine (14, 15). In contrast to poly-␥-glutamate produced by Bacillus species (1), ePL has a relatively low molecular mass and a narrow molecular mass distr...

ε-Poly- l -Lysine Peptide Chain Length Regulated by the Linkers Connecting the Transmembrane Domains of ε-Poly- l -Lysine Synthetase

“…Thus, mutating the linker regions connecting the TM domains should be a good strategy for effective production of short-chain ε-PL. In addition to this strategy, it is known that the use of ε-PL production media containing polyols such as glycerol, ethylene glycol, and trimethylene glycol resulted in the shortening of the ε-PL chain length (6,26); the S. albulus NBRC14147 strain produced a short-chain ε-PL (13-to 28-mer) in the ε-PL production medium containing 2.5% pentamethylene glycol (1,5-pentanediol). However, the polymer was produced as an ester formed between the terminal carboxyl group of ε-PL and a hydroxyl group of pentamethylene glycol.…”

A Novel Peptide Hydrogel for an Antimicrobial Bandage Contact Lens

“…A feature which makes this hydrogel unique is the incorporation of two naturally occurring components; pεK is an edible, non-toxic material currently used as an emulsifi er and preservative in foodstuffs and classifi ed as "generally regarded as safe" (GRAS) by the US Food and Drug Administration since 2004. [16][17][18] Short chain bis-carboxy fatty acids are found in both plants and animals. In the latter they are products of the oxidative degradation of longer chain fatty acids.…”

Recent advances in the biotechnological production of microbial poly(ɛ-l-lysine) and understanding of its biosynthetic mechanism

“…Additional short-chain polyols or modified cyclodextrins in the culture broth were also observed to regulate the chain length of ɛ-PL. Nishikawa and Ogawa found that ɛ-PL elongation was inhibited by the polyols via esterification; the ɛ-PL esters were formed during L-lysine polymerization, and the number of L-lysine residues of ɛ-PL-polyol esters decreased with increasing polyol concentration (Nishikawa and Ogawa 2006). Thus, it is feasible to control the number of Llysine residues in ɛ-PL by regulating the polyol concentration in the culture broth.…”

“…In 2002, a high-efficiency method was developed to screen ɛ-PL-producing strains by detecting the color zone formed by the secreted ɛ-PL (Nishikawa and Ogawa 2002). Since then, more and more studies have been performed to exploit the microbial ɛ-PL, including screening of new ɛ-PL-producing strains, development of new fermentation processes, control of the molecular weight of ɛ-PL, and discovery of ɛ-PL degrading enzymes and synthetase (Hamano et al 2006;Hirohara et al 2006Hirohara et al , 2007Nishikawa 2009;Nishikawa and Ogawa 2006;Shih and Shen 2006a, b;Shih et al 2006;Yamanaka et al 2008). In recent years, with the development of genetic engineering, bioinformatics, and advanced precision instruments and testing equipment, the biosynthetic mechanism of microbial ɛ-PL has been investigated more and more.…”