Zhang Bin, Yu Miao, Zhou Ying, Ye Bang-Ce
Laboratory of Biosystems and Microanalysis, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237, China.
School of Chemistry and Chemical Engineering, Shihezi University, Xinjiang, 832000, China.
AMB Express. 2018 Feb 24;8(1):26. doi: 10.1186/s13568-018-0557-8.
L-Ornithine, a non-essential amino acid, has enormous industrial applications in food, pharmaceutical, and chemical industries. Currently, L-ornithine production is focused on microorganism fermentation using Escherichia coli or Corynebacterium glutamicum. In C. glutamicum, development of high L-ornithine producing C. glutamicum was achieved by deletion of argF, but was accompanied by growth deficiency and arginine auxotrophy. L-Arginine has been routinely added to solve this problem; however, this increases production cost and causes feedback inhibition of N-acetyl-L-glutamate kinase activity. To avoid the drawbacks of growth disturbance due to disruption of ArgF, strategies were adopted to attenuate its expression. Firstly, ribosome binding site substitution and start codon replacement were introduced to construct recombinant C. glutamiucm strains, which resulted in an undesirable L-ornithine production titer. Then, we inserted a terminator (rrnB) between argD and argF, which significantly improved L-ornithine production and relieved growth disturbance. Transcription analysis confirmed that a terminator can be used to downregulate expression of argF and simultaneously improve the transcriptional level of genes in front of argF. Using disparate terminators to attenuate expression of argF, an optimal strain (CO-9) with a T4 terminator produced 6.1 g/L of L-ornithine, which is 42.8% higher than that produced by strain CO-1, and is 11.2-fold higher than that of the parent CO strain. Insertion of terminators with gradient termination intensity can be a stable and powerful method to exert precise control of the expression level of argF in the development of L-ornithine producing strains, with potential applications in metabolic engineering and synthetic biology.
L-鸟氨酸是一种非必需氨基酸，在食品、制药和化工行业有着广泛的工业应用。目前，L-鸟氨酸的生产主要集中在利用大肠杆菌或谷氨酸棒杆菌进行微生物发酵。在谷氨酸棒杆菌中，通过缺失argF实现了高产L-鸟氨酸谷氨酸棒杆菌的开发，但伴随着生长缺陷和精氨酸营养缺陷。常规添加L-精氨酸来解决这个问题；然而，这增加了生产成本，并导致N-乙酰-L-谷氨酸激酶活性的反馈抑制。为了避免由于ArgF缺失导致的生长干扰缺点，采取了减弱其表达的策略。首先，引入核糖体结合位点替换和起始密码子替换来构建重组谷氨酸棒杆菌菌株，这导致了不理想的L-鸟氨酸生产效价。然后，我们在argD和argF之间插入了一个终止子（rrnB），这显著提高了L-鸟氨酸的产量并缓解了生长干扰。转录分析证实，终止子可用于下调argF的表达，同时提高argF之前基因的转录水平。使用不同的终止子来减弱argF的表达，带有T4终止子的最优菌株（CO-9）产生了6.1 g/L的L-鸟氨酸，比菌株CO-1产生的L-鸟氨酸高42.8%，比亲本CO菌株高11.2倍。插入具有梯度终止强度的终止子可以是一种稳定且强大的方法，在L-鸟氨酸生产菌株的开发中对argF的表达水平进行精确控制，在代谢工程和合成生物学中具有潜在应用。
