微液滴适应性进化强化大肠杆菌耐受高浓度L-山梨糖

Enhanced tolerance of Escherichia coli to L-sorbose by microdroplet aided adaptive laboratory evolution

前期研究构建了1株组成型表达山梨糖脱氢酶(sorbose dehydrogenase,SDH)的大肠杆菌工程菌,该菌株能利用L-山梨糖生产维生素C前体2-酮基-L-古龙酸(2-keto-L-gulonic acid,2-KLG),但对底物L-山梨糖的耐受性较差。为解决这一问题,对该菌株进行适应性进化,并强化了进化菌株发酵生产2-KLG的能力。首先,应用基于微流控技术的全自动高通量微生物液滴培养系统,将出发菌株在不同浓度梯度的L-山梨糖培养基中生长、传代,获得能够耐受高浓度L-山梨糖的进化菌株。在摇瓶上进一步验证,最终获得了1株能耐受高浓度L-山梨糖的进化菌株2-F6。然后在2-F6中共表达了能促进2-酮基-L-古龙酸积累的山梨酮脱氢酶(sorbosone dehydrogenase,SNDH),并在摇瓶水平上对接种量、发酵温度、SNDH诱导时间、IPTG诱导浓度以及L-山梨糖添加量进化了优化,在最优条件下,2-KLG的产量达6. 05 g/L。最终,将摇瓶发酵条件放大至5 L发酵罐后,2-KLG的产量为5. 70 g/L。研究结果为一菌一步发酵法生产维生素C前体2-KLG提供了参考。

An engineered Escherichia coli constitutively expressing sorbose dehydrogenase(SDH)was previously constructed for producing the vitamin C precursor 2-keto-L-gulonic acid(2-KLG)with L-sorbose as substrate.However,this strain has a poor tolerance to L-sorbose.In order to solve this problem,adaptive laboratory evolution was carried out.Firstly,to enhance the tolerance of L-sorbose,the starting strain was grown and subcultured in the medium with different gradient concentration of L-sorbose by applying Microbial Microdroplet Culture System designed based on microfluidic technology.An evolutionary strain 2-F6 that could tolerate high concentration of L-sorbose was obtained after further verifying on the shaking flasks.Then,the sorbosone dehydrogenase(SNDH)was expressed in 2-F6 to promote the strength of 2-KLG production.The inoculation amount,fermentation temperature,induction time for SNDH,IPTG concentration and L-sorbose addition were optimized in the shaking flasks,and the titer of 2-KLG reached 6.05 g/L under the optimized optimal conditions.Finally,after scaling up in a 5 L fermenter,2-KLG of 5.70 g/L was obtained.The results obtained could provide references for the production of vitamin C precursor 2-KLG with one-step fermentation process.