小白链霉菌(Streptomyces albulus)是天然抗菌肽ε-聚赖氨酸(ε-poly-L-lysine, ε-PL)的主要生产菌株。为了提高小白链霉菌生产ε-PL效率,该文构建并优化了全细胞转化L-赖氨酸合成ε-PL体系:葡萄糖质量浓度80 g/L,菌龄12 h,反应温度30 ℃,L-赖氨酸质量浓度15 g/L,柠檬酸浓度15 g/L,初始反应pH 4.0,硫酸铵质量浓度6 g/L,湿菌体量为1 900 g/L。基于该转化体系,实现小白链霉菌在96 h合成ε-PL产量和底物转化率达到13.80 g/L和38.9%,分别是常规摇瓶发酵的4.1、3.2倍。最后,在小白链霉菌中异源表达来自大肠杆菌的L-赖氨酸特异性通透蛋白基因lysp,获得的重组菌S.albulus OE-lysp实现L-赖氨酸利用能力和底物转化率较出发菌株分别提升26%和33%,ε-PL产量增加至17.21 g/L,约为常规摇瓶发酵ε-PL产量的6.4倍,这是文献报道的最高摇瓶规模ε-PL产量。该研究结果一方面说明了通过全细胞转化L-赖氨酸生产ε-PL的可行性,另一方面为S.albulus转化大宗氨基酸L-赖氨酸生产高值ε-PL奠定了坚实的技术基础,具有重要的理论意义和经济价值。
Streptomyces albulus is the main producer of ε-poly-L-lysine (ε-PL). ε-PL is a natural antimicrobial peptide used primarily as a biological preservative in food industry, but its widespread application is limited by its high costs. To improve the production efficiency of S. albulus, this study used a whole-cell transformation method to produce ε-PL. Firstly, the synthesis efficiency of ε-PL in different fermentation systems was compared, and the whole-cell transformation system was found to produce the highest ε-PL yield, reaching 10.74 g/L. Then, the transformation medium and conditions were systematically optimized, and the optimal transformation conditions was obtained: glucose 80 g/L, cell culture time 12 h, reaction temperature 30 ℃, L-lysine 15 g/L, citric acid 15 g/L, initial pH 4.0, (NH4)2SO4 6 g/L, and wet biomass 1 900 g/L. In the optimal system, the ε-PL yield and substrate conversion rate reached 13.80 g/L and 38.9%, respectively, which were 4.1 and 3.2 folds higher than those in shake flask fermentation system. Finally, the L-lysine specific permease gene (lysp) from Escherichia coli BL21 was heterologous expressed to enhance the strain's ability to uptake exogenous L-lysine. Compared with the starting strain, the recombinant strain S. albulus OE-lysp showed a 26% increase in L-lysine utilization rate and a 33% increase in substrate conversion rate. The ε-PL yield also increased by 17.21 g/L, about 6.4 times higher than that in shake flask fermentation system, which is to our knowledge the highest reported ε-PL production in shake flask fermentation system. The results demonstrate the feasibility of producing ε-PL using a whole-cell transformation method and provide a solid technical foundation for the production of high-value ε-PL using L-lysine. Therefore, this study has important theoretical significance and high economic value.
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