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

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.

Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine

“…Recently, Liu et al built a coupled ADC system consisting of a GlcN6P-responsive biosensor and CRISPRi. Using this system, a genetic feedback circuit was constructed to fine-tune the metabolic flow toward GlcNAc synthesis and competing modules, which increased the titer of GlcNAc in a 15-L fed-batch bioreactor to 131.6 g/L [ 67 ].…”

Dynamic control over feedback regulatory mechanisms improves NADPH fluxes and xylitol biosynthesis in engineeredE. coli

“…18 Dynamic control over metabolism has become a popular approach in metabolic engineering, and has been used for the production of various products from 3-hydroxypropionic acid to myo-inositol and many others. [19][20][21][22] We have recently reported an extension of dynamic metabolic control to two-stage bioprocesses, where products are made in a metabolically productive phosphate depleted stationary phase. 23,24 The implementation of this approach relies on combined use of controlled proteolysis and gene silencing, using degron tags and CRISPR interference respectively.…”

Coupling metabolic addiction with negative autoregulation to improve strain stability and pathway yield

“…Indeed, dynamic feedback control has been successfully applied to various organisms to autonomously partition carbon flux and optimize cell metabolism for chemical production in recent years. The development of the metabolator ( Fung et al, 2005 ), malonyl-CoA inverter gate ( Liu et al, 2015 ), malonyl-CoA oscillatory switches ( Xu et al, 2014 ; Xu et al, 2014 ; Johnson et al, 2017 ; Xu, 2019 ), burden-driven feedback control ( Ceroni et al, 2015 ; Ceroni et al, 2018 ), quorum-sensing based genetic circuits ( Gupta et al, 2017 ; Doong et al, 2018 ; Dinh and Prather, 2019 ), antisense RNA-based control ( Yang et al, 2018 ), and dCas9-CRIRPRi-based circuits ( Berckman and Chen, 2019 ; Wu et al, 2020 ) have been implemented to relieve metabolic stress and boost cell's productivity in recent years. To combat cellular heterogeneity, one promising area that is largely underexplored is to consider the mutualistic social interactions and enhance microbial cooperation.…”