温度调节基因开关调控大肠杆菌发酵合成L-丙氨酸

L-alanine production in recombinant Escherichia coli with thermo-regulated genetic switch

【目的】L-丙氨酸的存在导致Escherichia coli的生长速率显著降低,最终会降低发酵过程中L-丙氨酸的体积合成速率。用温度调节基因开关(λpR-pL)高效、动态调控重组E.coli菌株菌体生长与L-丙氨酸合成过程,使两者相协调。【方法】以野生型E.coli B0016为出发菌株,敲除乙酸、甲酸、乙醇、琥珀酸、乳酸代谢产物合成途径以及丙氨酸消旋酶编码基因(ack A-pta、pfl B、adh E、frd A、ldh A、dad X),获得菌株B0016-060B。将嗜热脂肪地芽孢杆菌(Geobacillus stearothermophilus)来源的L-丙氨酸脱氢酶基因(ala D)克隆于pL启动子下游,并在B0016-060B菌株中表达,获得菌株B0016-060B/p PL-ala D,进行摇瓶和发酵罐发酵考察菌体生长和L-丙氨酸发酵性能。【结果】竞争代谢途径的敲除显著降低了副产物合成量,仅形成极少量的乙酸、琥珀酸和乙醇。28°C下菌株B0016-060B/p PL-ala D几乎不合成L-丙氨酸,可保证菌体快速生长;而在42°C下可高效合成L-丙氨酸。经发酵罐发酵,可合成67.2 g/L L-丙氨酸,体积生产强度达到2.06 g/(L·h)。【结论】通过发酵培养温度的简单切换,分阶段实现了细胞的快速增量和L-丙氨酸的高强度合成。

[Objective] Specific growth rate of Escherichia coli could be significantly decreased by L-alanine, which would result in reduction in L-alanine volumetric productivity. Therefore, the λpR-pLpromoter was used as a thermo-controllable genetic switch to coordinate the processes of cell growth and L-alanine production in E. coli. [Methods] Synthetic routes for acetate, formate, ethanol, succinate and lactate as well as for L-alanine recemization were inactivated by deleting the corresponding genes（ack A-pta, pfl B, adh E, frd A, ldh A, dad X） in the wild-type E. coli B0016 to generate B0016-060 B. Subsequently, alanine dehydrogenase derived from Geobacillus stearothermophilus was cloned downstream of the pL promoter and expressed in B0016-060 B to produce B0016-060B/p PL-ala D. Shake-flask and bioreactor experiments were conducted to investigate the properties of cell growth and L-alanine fermentation. [Results] Deletions of the competing pathways significantly decreased by-products accumulation with formation of low levels of acetate, succinate and ethanol. Strain B0016-060B/p PL-ala D hardly produced L-alanine during cell growth phase at 28 °C, which facilitated high growth rate. Meanwhile, efficient L-alanine production was obtained when cultured at 42 °C under oxygen-limited conditions. In bioreactor experiment, strain B0016-060B/p PL-ala D produced 67.2 g/L L-alanine, with a productivity of 2.06 g/（L·h）. [Conclusion] Efficient cell growth and L-alanine production were realized simply by switching the cultivation temperature.