The major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.

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.

BackgroundGlucosamine-6-phosphate N-acetyltransferase (GNA1) is the key enzyme that causes overproduction of N-acetylglucosamine in Bacillus subtilis. Previously, we increased GlcNAc production by promoting the expression of GNA1 from Caenorhabditis elegans (CeGNA1) in an engineered B. subtilis strain BSGN12. In this strain overflow metabolism to by-products acetoin and acetate had been blocked by mutations, however pyruvate accumulated as an overflow metabolite. Although overexpression of CeGNA1 drove carbon flux from pyruvate to the GlcNAc synthesis pathway and decreased pyruvate accumulation, the residual pyruvate reduced the intracellular pH, resulting in inhibited CeGNA1 activity and limited GlcNAc production.ResultsIn this study, we attempted to further overcome pyruvate overflow by enzyme engineering and host engineering for enhanced GlcNAc production. To this end, the key enzyme CeGNA1 was evolved through error-prone PCR under pyruvate stress to enhance its catalytic activity. Then, the urease from Bacillus paralicheniformis was expressed intracellularly to neutralize the intracellular pH, making it more robust in growth and more efficient in GlcNAc production. It was found that the activity of mutant CeGNA1 increased by 11.5% at pH 6.5–7.5, with the catalytic efficiency increasing by 27.5% to 1.25 s−1 µM−1. Modulated expression of urease increased the intracellular pH from 6.0 to 6.8. The final engineered strain BSGN13 overcame pyruvate overflow, produced 25.6 g/L GlcNAc with a yield of 0.43 g GlcNAc/g glucose in a shake flask fermentation and produced 82.5 g/L GlcNAc with a yield of 0.39 g GlcNAc/g glucose by fed-batch fermentation, which was 1.7- and 1.2-times, respectively, of the yield achieved previously.ConclusionsThis study highlights a strategy that combines pathway enzyme engineering and host engineering to resolve overflow metabolism in B. subtilis for the overproduction of GlcNAc. By means of modulated expression of urease reduced pyruvate burden, conferred bacterial survival fitness, and enhanced GlcNAc production, all of which improved our understanding of co-regulation of cell growth and metabolism to construct more efficient B. subtilis cell factories.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-1049-x) contains supplementary material, which is available to authorized users.

The clustered regularly interspaced short palindromic repeats and CRISPR‐associated proteins (CRISPR‐Cas) systems have been widely used in genome editing and transcriptional regulation. In this study, by engineering the Francisella novicida U112 CRISPR/Cpf1 system, a powerful tool called CRISPR/Cpf1 assisted multiple‐genes editing and regulation system for B. subtilis was constructed for engineering Bacillus subtilis, and a synthetic oligos mediated assembly of CRISPR RNA (crRNA) array method was created to build crRNA array. This system can achieve the double genes in‐frame knocking out, multiple point mutations (up to six), or single gene insertion at a time with 100% efficiency. In addition, transcriptional regulation systems were also developed using the DNase deactivated Cas protein (dCpf1) and a transcription factor RemA, which can implement repression and activation on multiple‐genes concurrently. Finally, as a proof‐of‐concept demonstration, the synthesis pathways of N‐acetylglucosamine and acetoin in B. subtilis were engineered by using this system. Overall, we provide effective tools for genome editing and metabolic engineering of B. subtilis cell factories to produce various biochemicals.

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.

These authors contributed equally to this work. SUMMARYArabidopsis hypersensitive-induced reaction (AtHIR) proteins function in plant innate immunity. However, the underlying mechanisms by which AtHIRs participate in plant immunity remain elusive. Here, using VA-TIRFM and FLIM-FRET, we revealed that AtHIR1 is present in membrane microdomains and co-localizes with the membrane microdomain marker REM1.3. Single-particle tracking analysis revealed that membrane microdomains and the cytoskeleton, especially microtubules, restrict the lateral mobility of AtHIR1 at the plasma membrane and facilitate its oligomerization. Furthermore, protein proximity index measurements, fluorescence cross-correlation spectroscopy, and biochemical experiments demonstrated that the formation of the AtHIR1 complex upon pathogen perception requires intact microdomains and cytoskeleton. Taken together, these findings suggest that microdomains and the cytoskeleton constrain AtHIR1 dynamics, promote AtHIR1 oligomerization, and increase the efficiency of the interactions of AtHIR1 with components of the AtHIR1 complex in response to pathogens, thus providing valuable insight into the mechanisms of defense-related responses in plants.

Lacto-N-neotetraose (LNnT), one of the oligosaccharides in human milk, has many beneficial effects on infant health. In a recent work, we have constructed a recombinant Bacillus subtilis strain for the production of LNnT. Here, we further improved LNnT production with a xylose-induced clustered regularly interspaced short palindromic repeats interference system. In particular, the expressions of pf kA and pyk genes in the Embden−Meyerhof−Parnas pathway module, zwf gene in the pentose phosphate pathway module, and mnaA gene in the teichoic acid synthesis module were downregulated. The LNnT titer was increased from 1.32 to 1.55 g/L. Furthermore, to improve the conversion efficiency of lacto-N-triose II to LNnT, we knocked out tuaD gene in branch pathway and improved the expression of lgtB gene, resulting in the further increase of LNnT titer to 2.01 g/L. Finally, the addition time and amount of inducer xylose were optimized, and LNnT titer reached 2.30 g/L in shake flask and 5.41 g/L in 3 L bioreactor.

Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.