Genome shuffling筛选ε-聚赖氨酸高产菌及其对代谢流量分配的影响

Effect of genome shuffling on ε-poly-L-lysine synthesis and metabolic flux

摘要:

【目的】从代谢流量分配的角度，探讨Genome shuffling导致链霉菌ε-聚赖氨酸合成量提升的原因。【方法】从葡萄糖耐受型的亲本菌株Streptomyces sp. AS32和ε-聚赖氨酸耐受型的亲本菌株Streptomyces albulus F15出发，进行三轮Genome shuffling，筛选得到ε-聚赖氨酸产量提高的链霉菌株Streptomyces sp. AF3-44，采用通量分析方法构建链霉菌ε-聚赖氨酸合成代谢网络，并对上述3株菌的代谢通量进行比较。【结果】AF3-44的ε-聚赖氨酸摇瓶产量为3.1 g/L，较AS32和F15分别提高了34%和29%。3株菌株中AS32三羧酸循环(TCA)的代谢通量最高；F15磷酸戊糖途径(PPP)代谢通量最高；AF3-44流向赖氨酸合成前体天冬氨酸以及ε-聚赖氨酸的通量最高，TCA和PPP通量位于两亲本菌株的中间水平，其中TCA中流向异柠檬酸的通量分别为AS32和F15的77%和116%，PPP中流向5-磷酸核酮糖的通量分别为AS32和F15的149%和92%。【结论】Genome shuffling导致了代谢流的重新分布，流向前体赖氨酸和ε-聚赖氨酸通量的增加，以及PPP和TCA通量配比的改变是链霉菌ε-聚赖氨酸合成量增加的重要因素。

Abstract:

[Objective] To explain the reason of the improvement of ε-poly-L-lysine (ε-PL) in Streptomyces by genome shuffling (GS) through the analysis of ε-PL metabolic flux distribution between the parent strains and the high yield strain. [Methods] Glucose tolerant Streptomyces sp. AS32 and ε-PL tolerant Streptomyces albulus F15 were selected as parent strains for GS. Three rounds of GS were carried out and a higher ε-PL producing shuffled strain Streptomyces sp. AF3-44 was obtained. The ε-PL synthetic network was constructed by metabolic flux analysis method and the metabolic fluxes among the above strains were compared. [Results] Shuffled strain, AF3-44, had a flask yield of 3.1 g/L which was increased by 34% and 29% compared with those of parent strains AS32 and F15 respectively. The fluxes to the tricarboxylic acid cycle (TCA) were highest in AS32, whereas and the fluxes to the pentose phosphate pathway (PPP) are highest in F15. AF3-44 had highest fluxes from oxaloacetic acid to aspartic acid and ε-PL synthesis, while it had moderate TCA and PPP fluxes, where the flux to isocitrate in TCA was 77% and 116% of those in AS32 and F15 respectively, and the flux to ribulose-5-phosphate was 149% and 92% of those in AS32 and F15 respectively. [Conclusion] Genome shuffling led to increase of flux to the precursor lysine and ε-PL, and the redistribution of metabolic fluxes in PPP and TCA which contributed to the improvement of ε-PL in the shuffled strain.