Physiological analysis of the improved ε-polylysine production induced by reactive oxygen species.

INTRODUCTION: Epsilon-poly-L-lysine (ε-PL) is produced by Streptomyces species in acidic and aerobic conditions, which inevitably induces rapid generation of reactive oxygen species (ROS). The devastating effects of ROS on biomolecules and cell vitality have been well-studied, while the positive effects of ROS are rarely reported. RESULTS: In this study, we found that a proper dose of intracellular ROS (about 3.3 µmol H2O2 /g DCW) could induce a physiological modification to promote the ε-PL production (from 1.2 to 1.5 g/L). It resulted in larger sizes of colony and mycelial pellets as well as vibrant, aggregated, and more robust mycelia, which were of high capability of ROS detoxication. Physiological studies showed that appropriate doses of ROS activated the metabolism of the pentose phosphate pathway at both transcriptional and enzymatic levels, which was beneficial for biomass accumulation. The biosynthesis of lysine was also promoted in terms of transcriptional regulatory overexpression, increased transcription and enzymatic activity of key genes, larger pools of metabolites in the TCA cycle, replenishment pathway, and diaminoheptanedioic acid pathway. In addition, energy provision was ensured by activated metabolism of the TCA cycle, a larger pool of NADH, and higher activity of the electron transport system. Increased transcription of HrdD and pls further accelerated the ε-PL biosynthesis. SIGNIFICANCE: These results indicated that ROS at proper intracellular dose could act as an inducing signal to activate the ε-PL biosynthesis, which laid a foundation for further process regulation to maintain optimal ROS dose in industrial ε-PL production and was of theoretical and practical significance. KEY POINTS: • A proper dose of intracellular ROS positively influences the ε-PL production. • Proper dose of ROS enhanced the mycelial activity and antioxidative capability. • ROS increased lysine synthesis metabolism, energy provision and pls expression.

Transcriptome and Metabolome Analysis Revealing the Improved ε-Poly-l-Lysine Production Induced by a Microbial Call from Botrytis cinerea.

ε-Poly-l-lysine (ε-PL) is a wide-spectrum antimicrobial agent, while its biosynthesis-inducing signals are rarely reported. This study found that Botrytis cinerea extracts could act as a microbial call to induce a physiological modification of Streptomyces albulus for ε-PL efficient biosynthesis and thereby resulted in ε-PL production (34.2 g/liter) 1.34-fold higher than control. The elicitors could be primary isolated by ethanol and butanol extraction, which resulted in more vibrant, aggregate and stronger mycelia. The elicitor-derived physiological changes focused on three aspects: ε-PL synthase, energy metabolism, and lysine biosynthesis. After elicitor addition, upregulated sigma factor hrdD and improved transcription and expression of pls directly contributed to the high ε-PL productivity; upregulated genes in tricarboxylic acid (TCA) cycle and energy metabolism promoted activities of citrate synthase and the electron transport system; in addition, pool enlargements of ATP, ADP, and NADH guaranteed the ATP provision for ε-PL assembly. Lysine biosynthesis was also increased based on enhancements of gene transcription, key enzyme activities, and intracellular metabolite pools related to carbon source utilization, the Embden-Meyerhof pathway (EMP), the diaminopimelic acid pathway (DAP), and the replenishment pathway. Interestingly, the elicitors stimulated the gene transcription for the quorum-sensing system and resulted in upregulation of genes for other antibiotic production. These results indicated that the Botrytis cinerea could produce inducing signals to change the Streptomyces mycelial physiology and accelerate the ε-PL biosynthesis. IMPORTANCE This work identified the role of microbial elicitors on ε-PL production and disclosed the underlying mechanism through analysis of gene transcription, key enzyme activities, and intracellular metabolite pools, including transcriptome and metabolome analysis. It was the first report for the inducing effects of the "microbial call" to Streptomyces albulus and ε-PL biosynthesis, and these elicitors could be potentially obtained from decayed fruits infected by Botrytis cinerea; hence, this may be a way of turning a biohazard into bioproduct wealth. This study provided a reference for application of microbial signals in secondary metabolite production, which is of theoretical and practical significance in industrial antibiotic production.