Engineering a Bifunctional Phr60-Rap60-Spo0A Quorum-Sensing Molecular Switch for Dynamic Fine-Tuning of Menaquinone-7 Synthesis in Bacillus subtilis

Abstract

Quorum sensing (QS)-based dynamic regulation has been widely used as basic tool for fine-tuning gene expression in response to cell density changes without adding expensive inducers. However, most reported QS systems primarily relied on down-regulation rather than up-regulation of gene expression, significantly limiting its potential as a molecular switch to control metabolic flux. To solve this challenge, we developed a bifunctional and modular Phr60-Rap60-Spo0A QS system, based on two native promoters, P abrB (down-regulation by Spo0A-P) and P spoiiA (up-regulation by Spo0A-P). We constructed a library of promoters with different capacities to implement down-regulation and up-regulation by changing the location, number, and sequences of the binding sites for Spo0A-P. The QS system can dynamically balance the relationship between efficient synthesis of the target product and cell growth. Finally, we validated the usefulness of this strategy by dynamic control of menaquinone-7 (MK-7) synthesis in Bacillus subtilis 168, a model Gram-positive bacterium, with the bifunctional Phr60-Rap60-Spo0A quorum sensing system. Our dynamic pathway regulation led to a 40-fold improvement of MK-7 production from 9 to 360 mg/L in shake flasks and 200 mg/L in 15-L bioreactor. Taken together, our bilayer QS system has been successfully integrated with biocatalytic functions to achieve dynamic pathway regulation in B. subtilis 168, which may be extended for use in other microbes to fine-tune gene expression and improve metabolites production.

Comparative transcriptional analysis site-directed mutagenesis of transcriptional regulator SinR boosts productivity of menaquinone synthesis in Bacillus subtilis

“…The result indicated that AroA, D, K played the most important role in CHA synthesis. It was consistent with previous study which showed that simultaneous overexpression of aroA and aroK in B. subtilis resulted in 2-fold increase in MK-7 compared with that in strain BS168 [34].…”

Titrating bacterial growth and chemical biosynthesis for efficient N-acetylglucosamine and N-acetylneuraminic acid bioproduction

“…Efforts have been made to solve these issues through various metabolic engineering strategies, such as de-coupling cell growth with biosynthesis based on genetic circuits, co-coupling cell growth with biosynthesis by coupling production to overall energy and biomass formation, and parallel metabolic pathway engineering via separating bioproduction from cell growth by implementing parallel dual carbon sources pathways 11 – 13 . De-coupling cell growth with biosynthesis commonly uses toggles, autonomous and quorum-sensing system-based genetic circuits, or optogenetic circuits to shift growth and production phases 11 , 14 – 17 . However, a potential interference of intracellular metabolites or the culture environment with genetic circuits and the requirement of large-scale fermentation equipment redesign for optogenetic circuit implementation impact either orthogonality or large-scale applicability 18 , 19 .…”

“…dual carbon sources pathways [11][12][13] . De-coupling cell growth with biosynthesis commonly uses toggles, autonomous and quorumsensing system-based genetic circuits, or optogenetic circuits to shift growth and production phases 11,[14][15][16][17] . However, a potential interference of intracellular metabolites or the culture environment with genetic circuits and the requirement of large-scale fermentation equipment redesign for optogenetic circuit implementation impact either orthogonality or large-scale applicability 18,19 .…”

Systems metabolic engineering of Bacillus subtilis for efficient biosynthesis of 5‐methyltetrahydrofolate

“…Medium E: 50 g/L glucose, 50 g/L sucrose, 50 g/L soy peptone, 0.6 g/L KH 2 PO 4 , 1 g/L glutamate, and 0.3 g/L p ABA (Cui et al, 2019).…”