Design of a programmable biosensor-CRISPRi genetic circuits for dynamic and autonomous dual-control of metabolic flux in Bacillus subtilis

• Published in: Nucleic acids research 2020-01, Vol.48 (2), p.996-1009
• Main Authors: Wu, Yaokang, Chen, Taichi, Liu, Yanfeng, Tian, Rongzhen, Lv, Xueqin, Li, Jianghua, Du, Guocheng, Chen, Jian, Ledesma-Amaro, Rodrigo, Liu, Long
• Format: Article
• Language: English
• Subjects: Acetoin - metabolism; Acetylglucosamine - metabolism; Bacillus subtilis - isolation & purification; Bacillus subtilis - metabolism; Biosensing Techniques; Clustered Regularly Interspaced Short Palindromic Repeats - genetics; Gene Regulatory Networks - genetics; Glucosamine - analogs & derivatives; Glucosamine - genetics; Glucosamine - metabolism; Glucose - chemistry; Glucose - genetics; Glucose-6-Phosphate - analogs & derivatives; Glucose-6-Phosphate - genetics; Glucose-6-Phosphate - metabolism; Metabolic Engineering - methods; Metabolic Networks and Pathways - genetics; Synthetic Biology and Bioengineering
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkz1123

Abstract Dynamic regulation is an effective strategy for fine-tuning metabolic pathways in order to maximize target product synthesis. However, achieving dynamic and autonomous up- and down-regulation of the metabolic modules of interest simultaneously, still remains a great challenge. In this work, we created an autonomous dual-control (ADC) system, by combining CRISPRi-based NOT gates with novel biosensors of a key metabolite in the pathway of interest. By sensing the levels of the intermediate glucosamine-6-phosphate (GlcN6P) and self-adjusting the expression levels of the target genes accordingly with the GlcN6P biosensor and ADC system enabled feedback circuits, the metabolic flux towards the production of the high value nutraceutical N-acetylglucosamine (GlcNAc) could be balanced and optimized in Bacillus subtilis. As a result, the GlcNAc titer in a 15-l fed-batch bioreactor increased from 59.9 g/l to 97.1 g/l with acetoin production and 81.7 g/l to 131.6 g/l without acetoin production, indicating the robustness and stability of the synthetic circuits in a large bioreactor system. Remarkably, this self-regulatory methodology does not require any external level of control such as the use of inducer molecules or switching fermentation/environmental conditions. Moreover, the proposed programmable genetic circuits may be expanded to engineer other microbial cells and metabolic pathways.