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

Nucleic Acids Res. 2020 Jan 24;48(2):996-1009. 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 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*

Substances

  • glucosamine 6-phosphate
  • Glucose-6-Phosphate
  • Acetoin
  • Glucose
  • Glucosamine
  • Acetylglucosamine