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

Abstract

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 .

Epsilon-poly-L-lysine: Recent Advances in Biomanufacturing and Applications

“…Currently, increasing numbers of ε-PL-producing strains have been screened out as the development of novel screening methods. Efforts in ε-PL biomanufacturing research have mainly focused on Streptomyces , especially on S. albulus , which gives the highest reported ε-PL production (70 g/L) in liquid fermentation after strain evolution and fermentation process optimization ( Wang et al, 2019 ). Some other actinomycetes, such as Kitasatospora , Streptomyces ahygroscopicus , Streptomyces griseofuscus and Streptomyces diastatochromogenes have also been investigated in some studies, with various degrees of success ( Ouyang et al, 2006 ; Chen J. et al, 2019 ; Shu et al, 2019; Wang et al, 2021 ).…”

“…It has been reported that the introduction of resistance to some macrolide antibiotics can strongly activate the production of various secondary metabolites, including actinorhodin and salinomycin ( Ochi, 2007 ; Zhang et al, 2019 ). Through this technique, mutations conferring resistance to streptomycin and five other antibiotics were successively induced in S. albulus to generate the S. albulus R6 mutant strain, which exhibited the highest reported ɛ-PL production of 70.3 g/L, 2.79-fold greater than the original strain, using an optimized acidic pH shock strategy ( Wang et al, 2019 ). Mutations in protein S12 (Q856H) and RNA polymerase (R99P) of the R6 strain could be responsible for the increased transcription of the pls gene and elevated activities of key enzymes in ɛ-PL biosynthetic pathway, consequently increasing the level of Pls activity, as well as L-lysine and ATP concentrations available for ɛ-PL production ( Wang et al, 2019 ).…”

“…Through this technique, mutations conferring resistance to streptomycin and five other antibiotics were successively induced in S. albulus to generate the S. albulus R6 mutant strain, which exhibited the highest reported ɛ-PL production of 70.3 g/L, 2.79-fold greater than the original strain, using an optimized acidic pH shock strategy ( Wang et al, 2019 ). Mutations in protein S12 (Q856H) and RNA polymerase (R99P) of the R6 strain could be responsible for the increased transcription of the pls gene and elevated activities of key enzymes in ɛ-PL biosynthetic pathway, consequently increasing the level of Pls activity, as well as L-lysine and ATP concentrations available for ɛ-PL production ( Wang et al, 2019 ). To explore the mechanism by which streptomycin resistance leads to higher productions, a high ɛ-PL-producing mutant was generated by continuous selection for streptomycin resistance (and hence increasing mutations) ( Liu et al, 2019b ).…”

Recent Advances in Epsilon-Poly-L-Lysine and L-Lysine-Based Dendrimer Synthesis, Modification, and Biomedical Applications

“…However, limitations, such as small-scale and complicated polymerization processes, hinder the extensive use of chemically synthesized EPL. EPL, as a natural existing product, its production mainly relies on biosynthesis with few kinds of microorganisms, such as Streptomycetaceae, Kitasatospora sp ., and Ergot fungi (Shukla et al, 2012 ; Wang et al, 2019 ). Furthermore, the process of commercially producing EPL especially relies on Streptomyces albulus fermentation ( Figure 2 ) as the metabolic process of this microbe combating acid stress (Wang C. Y. et al, 2020 ).…”

“…The former additives could be astaxanthin, which works as an antioxidant to reduce intracellular oxidant response and downregulated relevant gene transcription (Li S. et al, 2020 ), while the latter pointing to replacing the carbon sources to cyclodextrins and glycerol for producing low-molecular-weight EPL, which exhibits more powerful antimicrobial activity than its larger counterparts (Chen et al, 2018 ); (ii) optimizing the producing strategies by combining other computation methodologies. For example, researchers used artificial neural network modeling and surface response methodology to assist to the optimization of the medium composition then enlarging the yield (Bhattacharya et al, 2017 ; Guo et al, 2018 ); or (iii) cloning or mutating the key genes of those microorganism, activating a larger production of EPL (Liu et al, 2019 ; Wang et al, 2019 ; Xu et al, 2019 ). Furthermore, with a more precise production or synthesis mechanism, such as determining the opposite and negative elements that may influence the production, higher yield with a more time-saving and effective production process could be achieved, which could be the preferred pathway for further studies.…”