<jats:title>Summary</jats:title><jats:p>ε‐Poly‐<jats:styled-content style="fixed-case">L</jats:styled-content>‐lysine (ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content>), showing a wide range of antimicrobial activity, is now industrially produced as a food additive by a fermentation process. A new strain capable of producing ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> was isolated from a soil sample collected from <jats:styled-content style="fixed-case">G</jats:styled-content>utian, <jats:styled-content style="fixed-case">F</jats:styled-content>ujian <jats:styled-content style="fixed-case">P</jats:styled-content>rovince, <jats:styled-content style="fixed-case">C</jats:styled-content>hina. Based on its morphological and biochemical features and phylogenetic similarity with 16<jats:styled-content style="fixed-case">S rRNA</jats:styled-content> gene, the strain was identified as <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptomyces albulus</jats:italic> and named <jats:styled-content style="fixed-case">NK</jats:styled-content>660. The yield of ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> in 30 l fed‐batch fermentation with <jats:styled-content style="fixed-case">pH</jats:styled-content> control was 4.2 g l<jats:sup>−1</jats:sup> when using glycerol as the carbon source. The structure of ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> was determined by nuclear magnetic resonance (<jats:styled-content style="fixed-case">NMR</jats:styled-content>) and matrix‐assisted laser desorption/ionization–time of flight mass spectrometry (<jats:styled-content style="fixed-case">MALDI‐TOF MS</jats:styled-content>). Previous studies have shown that the antimicrobial activity of ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> is dependent on its molecular size. In this study, the polymerization degree of the ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> produced by strain <jats:styled-content style="fixed-case">NK</jats:styled-content>660 ranged from 19 to 33 <jats:styled-content style="fixed-case">L</jats:styled-content>‐lysine monomers, with the main component consisting of 24–30 <jats:styled-content style="fixed-case">L</jats:styled-content>‐lysine monomers, which implied that the ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> might have higher antimicrobial activity. Furthermore, the ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> synthetase gene (<jats:styled-content style="fixed-case"><jats:italic>pls</jats:italic></jats:styled-content>) was cloned from strain <jats:styled-content style="fixed-case">NK</jats:styled-content>660 by genome walking. The <jats:styled-content style="fixed-case"><jats:italic>pls</jats:italic></jats:styled-content> gene with its native promoter was heterologously expressed in <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>treptomyces lividans</jats:italic> <jats:styled-content style="fixed-case">ZX</jats:styled-content>7, and the recombinant strain was capable of synthesizing ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content>. Here, we demonstrated for the first time heterologous expression of the <jats:styled-content style="fixed-case"><jats:italic>pls</jats:italic></jats:styled-content> gene in <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>. lividans</jats:italic>. The heterologous expression of <jats:styled-content style="fixed-case"><jats:italic>pls</jats:italic></jats:styled-content> gene in <jats:styled-content style="fixed-case"><jats:italic>S</jats:italic></jats:styled-content><jats:italic>. lividans</jats:italic> will open new avenues for elucidating the molecular mechanisms of ε‐<jats:styled-content style="fixed-case">PL</jats:styled-content> synthesis.