基于2-C-甲基-D-赤藓糖醇-4磷酸途径的产紫槐二烯大肠杆菌构建及其补糖策略优化

Engineering MEP pathway in Escherichia coli for amorphadiene production and optimizing the bioprocess through glucose feeding control

摘要:

2-C-甲基-D-赤藻糖醇-4-磷酸 (2-methyl-D-erythritol-4-phosphate, MEP) 途径是大肠杆菌Escherichia coli唯一的萜类前体合成途径，研究表明它比甲羟戊酸(Mevalonate, MVA)途径具有更高的理论产率。但目前有关MEP途径的调控所知非常有限，故单独强化MEP途径对萜类异源合成产量的提高效果并不理想。研究中通过引入外源MEP途径基因强化E. coli萜类合成的遗传改造策略和发酵过程补糖控制优化，尝试更有效地释放MEP途径的潜力，建立青蒿素前体——紫槐二烯的高密度发酵过程。研究结果表明共表达阿维链霉菌Streptomyces avermitilis dxs2基因和枯草芽胞杆菌Bacillus subtilis idi基因可使紫槐二烯的摇瓶发酵产量比野生菌株提高12.2倍。随后针对该菌株建立了高密度发酵过程，发现稳定期的中前期 (24?72 h) 是产物合成的关键期，通过稳定期补糖速率的调整，明显改善了产物合成速度，使紫槐二烯的产量从2.5 g/L提高到了4.85 g/L，但不影响产物积累的周期。考虑到72 h后菌体老化可能会影响产物合成，进一步采取了调整对数期的补糖速率控制菌体生长的策略，使紫槐二烯的产量达到6.1 g/L。研究结果为基于MEP途径的萜类异源合成工程菌构建及其发酵工艺的建立奠定了基础。

Abstract:

The pathway of 2-methyl-D-erythritol-4-phosphate (MEP) is the exclusive isoprenoid precursor biosynthetic pathway in Escherichia coli, with a higher theoretical yield than mevalonate (MVA) pathway. However, due to lack of information about the regulation of MEP pathway, only engineering MEP pathway in E. coli achieved limited improvement of heterologous isoprenoid production. We used exogenous MEP pathway genes to improve MEP pathway in E. coli and optimized the glucose feeding to release the potential of MEP pathway. The results demonstrate that co-expression of dxs2 from Streptomyces avermitilis and idi from Bacillus subtilis can increase amorphadiene production with 12.2-fold compared with the wild-type strain in shake flask fermentation. Then we established a high-cell density fermentation process for the engineered strain, and found that the phase from 24 to 72 h is important for product biosynthesis. The optimization of glucose feeding rate during 24 to 72 h significantly improved product accumulation, which was improved from 2.5 to 4.85 g/L, within the same process time. Considering the attenuation of strain metabolism after 72 h, this study further modulated the glucose feeding rate during exponential phase to control strain growth and the amorphadiene yield eventually reached to 6.1 g/L. These results provided useful information to develop engineered E. coli for isoprenoid production through MEP pathway engineering.