A pathway independent multi-modular ordered control system based on thermosensors and CRISPRi improves bioproduction in Bacillus subtilis

Abstract

Dynamic regulation is an effective strategy for control of gene expression in microbial cell factories. In some pathway contexts, several metabolic modules must be controlled in a time dependent or ordered manner to maximize production, while the creation of genetic circuits with ordered regulation capacity still remains a great challenge. In this work, we develop a pathway independent and programmable system that enables multi-modular ordered control of metabolism in Bacillus subtilis. First, a series of thermosensors were created and engineered to expand their thresholds. Then we designed single-input-multi-output circuits for ordered control based on the use of thermosensors with different transition points. Meanwhile, a repression circuit was constructed by combining CRISPRi-based NOT gates. As a proof-of-concept, these genetic circuits were applied for multi-modular ordered control of 2′-fucosyllactose (2′-FL) biosynthesis, resulting in a production of 1839.7 mg/l in shake flask, which is 5.16-times that of the parental strain. In a 5-l bioreactor, the 2′-FL titer reached 28.2 g/l with down-regulation of autolysis. Taken together, this work provides programmable and versatile thermosensitive genetic toolkits for dynamic regulation in B. subtilis and a multi-modular ordered control framework that can be used to improve metabolic modules in other chassis cells and for other compounds.

Module-Guided Metabolic Rewiring for Fucosyllactose Biosynthesis in Engineered Escherichia coli with Lactose De Novo Pathway

“…Recently, Yu et al developed a temperature-sensitive multimodule time-sequential bidirectional regulatory system in Bacillus subtilis and applied it to optimize the metabolic network of 2′-FL. Finally, the stability and robustness of the system were verified in a 5 L fermenter, and the yield of 2′-FL reached 28.2 g/L . Based on previous reports, the current work reconstructed the biosynthetic pathway from glucose to lactose in E.…”

“…Finally, the stability and robustness of the system were verified in a 5 L fermenter, and the yield of 2′-FL reached 28.2 g/L. 42 Based on previous reports, the current work reconstructed the biosynthetic pathway from glucose to lactose in E. coli BZWNDPAL. Green production of 2′-FL and 3-FL was achieved in antibiotic-free strains by iterative optimization of modules and metabolic modulation.…”

Fine-Tuning of Carbon Flux and Artificial Promoters in Bacillus subtilis Enables High-Level Biosynthesis of d-Allulose

“…The −35 and −10 regions are σ factor binding sites, which mediate the recruitment and assembly of the RNA polymerase complex . The modification of the spacer region between the −35 and −10 regions of the promoter was reported to have a great influence on gene expression .…”

“…The −35 and −10 regions are σ factor binding sites, which mediate the recruitment and assembly of the RNA polymerase complex. 40 The modification of the spacer region between the −35 and −10 regions of the promoter was reported to have a great influence on gene expression. 41 We further enhanced the promoter strength by creating random mutations in the spacer region between the −35 and −10 regions of the dual promoter P amyE -P HpaII (Table S2) using a degeneracy primer (Figure 5A).…”

Characterization of the endogenous promoters in Yarrowia lipolytica for the biomanufacturing applications

“…Noticeably, a stable and reliable reporter system is essential for accurate analysis of promoter strength. [21] Currently, commonly used reporter systems for promoter analysis mainly include: [19] fluorescent proteins (generate fluorescence) and enzymes (generate chromogenic products), such as X-gluc (5-Bromo-4chloro-3-indolyl-β-glucuronide). However, Y. lipolytica could generate severe fluorescence background, which interferes with the reliabilities of the fluorescent protein.…”