DataSheet1_Identification of key genes through the constructed CRISPR-dcas9 to facilitate the efficient production of O-acetylhomoserine in Corynebacterium glutamicum.pdf

O-Acetylhomoserine (OAH) is an important platform chemical for the synthesis of L-methamidophos and l-methionine. It has been produced efficiently in Corynebacterium glutamicum. However, a wider range of key factors had not been identified, limiting further increases in OAH production. This study successfully identified some limiting factors and regulated them to improve OAH titer. Firstly, an efficient clustered regularly interspaced short palindromic repeats/dead CRISPR associated protein 9 (CRISPR-dCas9) system was constructed and used to identify the key genes in central metabolism and branch pathways associated with OAH biosynthesis. Then, the gltA gene involved in TCA cycle was identified as the most critical gene. A sequential promoter PNCgl2698, which showed different transcriptional intensity in different strain growth periods, was used to control the expression of gltA gene, resulting in OAH production of 7.0 g/L at 48 h. Finally, the OAH titer of the engineered strain reached 25.9 g/L at 72 h in a 5-L bioreactor. These results show that the identification and regulation of key genes are critical for OAH biosynthesis, which would provide a better research basis for the industrial production of OAH in C. glutamicum.

O-乙酰高丝氨酸（O-Acetylhomoserine, OAH）是合成L-甲胺磷与L-甲硫氨酸的重要平台化合物，目前已可通过谷氨酸棒杆菌（Corynebacterium glutamicum）实现高效合成。然而，此前尚未明确更多关键影响因素，这限制了OAH产量的进一步提升。本研究成功筛选出若干限速因子，并通过对其进行调控以提高OAH效价。首先，本研究构建了一套高效的成簇规律间隔短回文重复序列/失活CRISPR相关蛋白9（CRISPR-dCas9）系统，用于挖掘与OAH生物合成相关的中心代谢及分支通路关键基因。随后，研究确定三羧酸（TCA）循环中的gltA基因为最关键的调控靶点。研究采用可在菌株不同生长阶段呈现不同转录强度的时序启动子PNCgl2698，对gltA基因的表达进行精准调控，最终使菌株在48小时时的OAH产量达到7.0 g/L。最后，在5升生物反应器中，工程菌株的OAH效价在72小时时可达25.9 g/L。上述研究结果表明，关键基因的筛选与调控对OAH生物合成至关重要，可为谷氨酸棒杆菌工业化生产OAH提供更为坚实的研究基础。