利用CRISPRi挖掘大肠杆菌生物合成D-泛酸的关键基因

Mining key genes for biosynthesis of D-pantothenic acid in Escherichia coli by CRISPRi

摘要:

【目的】D-泛酸(D-pantothenic acid, DPA)是一种重要的功能化合物，被广泛应用于医疗保健、化妆品、动物食品和饲料等领域，具有良好的市场前景及应用。本研究以实验室保藏的大肠杆菌菌株DPAP10为底盘菌株，利用CRISPR干扰(clustered regularly interspaced palindromic repeats interference, CRISPRi)技术，筛选影响工程菌株DPA生物合成的内源性基因靶点。【方法】构建了pTarget和pdCas9的双质粒CRISPRi系统，可以实现对基因单个或组合表达抑制，摇瓶发酵检测基因抑制对DPA合成的影响；通过实时荧光定量聚合酶链式反应(real-time fluorescence quantitative polymerase chain reaction, RT-qPCR)检测了基因抑制后的转录水平；通过高效液相色谱(high performance liquid chromatography, HPLC)检测了中间代谢物分析代谢通路变化。【结果】成功从126个靶基因中筛选得到5个显著影响DPA合成的关键基因pgk、gltA、ptsH、ptsI、crp和7个基因组合pgk-gltA、pgk-ptsH、gltA-ptsH、pgk-ptsI、gltA-ptsI、pgk-crp、gltA-crp，其中菌株DPAP10/ pdCas9+pT-gltA-ptsH相对于原始菌株DPAP10，在摇瓶中DPA产量提高了49.5%达到5.3 g/L，进一步在基因组上对gltA和ptsH的表达进行下调，得到工程菌株DPAP10-gltA^TTG-ptsH^TTG，最终在5 L罐中DPA产量相较于对照菌株DPAP10在相同培养条件下提高了19.5%达到75.4 g/L。【结论】本研究证实CRISPRi筛选可以得到对DPA合成有益的基因靶点；中间代谢物检测分析发现，改变丙酮酸通量分布和降低三羧酸(tricarboxylic acid, TCA)循环速率，会导致碳流更多流向DPA合成代谢路径中，从而提高DPA的产量，这一发现为构建更高产菌株提供了新思路。

Abstract:

[Objective] D-pantothenic acid (DPA) is a functional compound widely used in medical care, cosmetics, animal food and feed and other fields, with good market prospects. This study aims to use CRISPRi to mine the endogenous gene targets affecting the biosynthesis of DPA by the lab-stored Escherichia coli strain DPAP10. [Methods] We constructed a dual-plasmid CRISPRi system with pTarget and pdCas9 to achieve the inhibition of gene expression individually or in combination. Shaking flask fermentation was carried out to validate the effect of gene blocking on DPA production. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was employed to determine the transcription level after gene blocking. The changes of metabolic pathways were analyzed by high performance liquid chromatography (HPLC). [Results] We screened out 5 key genes (pgk, gltA, ptsH, ptsI, and crp) and 7 gene combinations (pgk-gltA, pgk-ptsH, gltA-ptsH, pgk-ptsI, gltA-ptsI, pgk-crp, and gltA-crp) from 126 target genes. The engineered strain DPAP10/pdCas9+pT-gltA-ptsH showed the DPA yield of 5.3 g/L in a shaking flask, which increased by 49.5% compared with that by the start strain DPAP10. Furthermore, we down-regulated the expression of gltA and ptsH to construct an engineered strain DPAP10-gltA^TTG-ptsH^TTG. This strain showed the DPA yield of 75.4g/L in 5 L fermentor which compared with the control strain DPAP10 under the same culture conditions increased by 19.5%. [Conclusion] We confirmed that CRISPRi could mine the genes involved in DPA synthesis. Changing the pyruvate flux and reducing the tricarboxylic acid (TCA) cycling rate would promote the carbon flow into the DPA synthesis, thus increasing DPA production. This finding provides a new idea for constructing higher yielding strains.