Efficiently activated ε‐poly‐L‐lysine production by multiple antibiotic‐resistance mutations and acidic pH shock optimization in Streptomyces albulus

ε‐Poly‐L‐lysine (ε‐PL) is a food additive produced by Streptomyces and is widely used in many countries. Working with Streptomyces albulus FEEL‐1, we established a method to activate ε‐PL synthesis by successive introduction of multiple antibiotic‐resistance mutations. Sextuple mutant R6 was finally developed by screening for resistance to six antibiotics and produced 4.41 g/L of ε‐PL in a shake flask, which is 2.75‐fold higher than the level produced by the parent strain. In a previous study, we constructed a double‐resistance mutant, SG‐31, with high ε‐PL production of 3.83 g/L and 59.50 g/L in a shake flask and 5‐L bioreactor, respectively. However, we found that R6 did not show obvious advantages in fed‐batch fermentation when compared with SG‐31. For further activation of ε‐PL synthesis ability, we optimized the fermentation process by using an effective acidic pH shock strategy, by which R6 synthetized 70.3 g/L of ε‐PL, 2.79‐fold and 1.18‐fold greater than that synthetized by FEEL‐1 and SG‐31, respectively. To the best of our knowledge, this is the highest reported ε‐PL production to date. This ε‐PL overproduction may be due to the result of R99P and Q856H mutations in ribosomal protein S12 and RNA polymerase, respectively, which may be responsible for the increased transcription of the ε‐poly‐lysine synthetase gene (pls) and key enzyme activities in the Lys synthesis metabolic pathway. Consequently, ε‐PL synthetase activity, intracellular ATP, and Lys concentrations were improved and directly contributed to ε‐PL overproduction. This study combined ribosome engineering, high‐throughput screening, and targeted strategy optimization to accelerate ε‐PL production and probe the fermentation characteristics of hyperyield mutants. The information presented here may be useful for other natural products produced by Streptomyces.