pH-DO组合调控策略提高小白链霉菌ε-聚赖氨酸的生物合成

Enhancing the epsilon-poly-L-lysine biosynthesis of Streptomyces albulus by a pH-DO combined regulation strategy

ε-聚赖氨酸(ε-poly-L-lysine, ε-PL)是一种主要由小白链霉菌生产的广谱性天然食品防腐剂,具有广泛的工业应用价值。该研究对ε-PL高产菌Streptomyces albulus WG-608在5 L罐发酵不同阶段的pH与溶解氧(dissolved oxygen, DO)进行了系统优化,并构建了一种新的pH-DO组合调控策略。该策略将发酵分为2个阶段:第一阶段DO被控制在40%,pH维持在4.0;第二阶段DO被控制在20%,pH维持在4.3。经240 h的补料-分批发酵,WG-608的ε-PL产量与平均比生产速率为(68.77±2.53) g/L和(2.33±0.08) d-1,比对照策略分别提高了28.23%和12.02%。为进一步探究该策略提高WG-608 ε-PL产量的细胞生理代谢差异的原因,对不同时期细胞的关键酶活力、呼吸链活力和辅因子水平等进行了分析。结果表明,葡萄糖消耗、中心碳代谢途径、L-赖氨酸生物合成途径、呼吸链活性和胞内辅因子的增强是pH-DO组合调控策略增强ε-PL产量的原因。总之,pH-DO组合调控策略是一种通过增强碳代谢和能量代谢提高ε-PL产量的有效方法。该方法的建立为其他链霉菌发酵生产ε-PL提供了新的思路。

ε-Poly-L-lysine(ε-PL) is a kind of food preservatives mainly produced by Streptomyces albulus, which possesses excellent antimicrobial activity and has broad industrial applications. In this study, the pH and dissolved oxygen(DO) levels in the fed-batch fermentation by an ε-PL high-producing strain S. albulus WG-608 were systemically optimized, and a novel pH-DO regulation strategy was subsequently established. Specifically, the fermentation process was divided into two stages: stage I, DO concentration and pH level were respectively kept at 40% and 4.0, for the accumulation of biomass and ε-PL;stage II, DO and pH levels were maintained at 20% and 4.3, to improve the specific formation rate and ε-PL production. After 240 h of fed-batch fermentation, the ε-PL production and average specific formation rate of WG-608 reached(68.77±2.53) g/L and(2.33±0.08) d-1, which were 28.23% and 12.02% higher than those of the control strategy. The total glucose consumption rate increased, especially the substrate consumption rate increased from 315.5 g/L to 528.8 g/L in the stage II, which increased by 67.6%. And the glucose conversion rate increased by 6.7%. Changes in key enzyme activities, respiratory chain activity, and intracellular nucleotide levels were detected and analyzed to further investigate the metabolic differences between the pH-DO regulation strategy and control strategy. The result showed that the enhanced glucose consumption rate, key enzyme activity of central carbon metabolism(include glucose-6-phosphate dehydrogenase, phosphoenolpyruvate carboxylase, citrate synthase, aspartokinase and ε-poly-L-lysine synthase), respiratory chain activity, L-lysine biosynthesis pathway are responsible for the improved ε-PL production by pH-DO regulation strategy. Overall, the novel pH-DO regulation strategy is a convenient and effective approach for promoting ε-PL biosynthesis by strengthening carbon and energy metabolism. As mentioned above, the new strategy increases the demand for energy, and the demand in the cell may be in short supply. To further increase the yield of ε-PL in S. albulus, balancing the intracelluar cofactors by metabolic engineering may become the next step research. This developed method has shed new light on the ε-PL production by other Streptomyces strains.