Moderate expression of the transcriptional regulator ALsR enhances acetoin production by 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.

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Conformational transitions induced by γ-amino butyrate binding in GabR, a bacterial transcriptional regulator

Scientific Reports ◽

10.1038/s41598-019-55581-1 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 2

Author(s):

Mario Frezzini ◽

Leonardo Guidoni ◽

Stefano Pascarella

Keyword(s):

Molecular Dynamics ◽

Bacillus Subtilis ◽

Closed Form ◽

Molecular Mechanism ◽

Aspartate Aminotransferase ◽

Transcriptional Regulator ◽

Transcription Activation ◽

Conformational Transitions ◽

Winged Helix ◽

Gaba Binding

AbstractGabR from Bacillus subtilis is a transcriptional regulator of the MocR subfamily of GntR regulators. The MocR architecture is characterized by the presence of an N-terminal winged-Helix-Turn-Helix domain and a C-terminal domain folded as the pyridoxal 5′-phosphate (PLP) dependent aspartate aminotransferase (AAT). The two domains are linked by a peptide bridge. GabR activates transcription of genes involved in γ-amino butyrate (GABA) degradation upon binding of PLP and GABA. This work is aimed at contributing to the understanding of the molecular mechanism underlying the GabR transcription activation upon GABA binding. To this purpose, the structure of the entire GabR dimer with GABA external aldimine (holo-GABA) has been reconstructed using available crystallographic data. The structure of the apo (without any ligand) and holo (with PLP) GabR forms have been derived from the holo-GABA. An extensive 1 μs comparative molecular dynamics (MD) has been applied to the three forms. Results showed that the presence of GABA external aldimine stiffens the GabR, stabilizes the AAT domain in the closed form and couples the AAT and HTH domains dynamics. Apo and holo GabR appear more flexible especially at the level of the HTH and linker portions and small AAT subdomain.

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Crystal structure of YfiR, an unusual TetR/CamR-type putative transcriptional regulator from Bacillus subtilis

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.21073 ◽

2006 ◽

Vol 65 (1) ◽

pp. 255-257 ◽

Cited By ~ 8

Author(s):

Shyamala S. Rajan ◽

Xiaojing Yang ◽

Ludmilla Shuvalova ◽

Frank Collart ◽

Wayne F. Anderson

Keyword(s):

Crystal Structure ◽

Bacillus Subtilis ◽

Transcriptional Regulator

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High acetoin production by a newly isolated marine Bacillus subtilis strain with low requirement of oxygen supply

Process Biochemistry ◽

10.1016/j.procbio.2015.07.010 ◽

2015 ◽

Vol 50 (11) ◽

pp. 1730-1734 ◽

Cited By ~ 21

Author(s):

Jian-Ying Dai ◽

Ling Cheng ◽

Qing-Feng He ◽

Zhi-Long Xiu

Keyword(s):

Bacillus Subtilis ◽

Oxygen Supply ◽

Subtilis Strain ◽

Acetoin Production

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The LysR-type transcriptional regulator (LTTR) AlsR indirectly regulates expression of the Bacillus subtilis bdhA gene encoding 2,3-butanediol dehydrogenase

Applied Microbiology and Biotechnology ◽

10.1007/s00253-013-4871-4 ◽

2013 ◽

Vol 97 (16) ◽

pp. 7307-7316 ◽

Cited By ~ 5

Author(s):

Rafael R. de Oliveira ◽

Wayne L. Nicholson

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Regulator ◽

Gene Encoding ◽

Butanediol Dehydrogenase ◽

Bdha Gene

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A Mn-sensing riboswitch activates expression of a Mn2+/Ca2+ ATPase transporter in Streptococcus

Nucleic Acids Research ◽

10.1093/nar/gkz494 ◽

2019 ◽

Vol 47 (13) ◽

pp. 6885-6899 ◽

Cited By ~ 8

Author(s):

Julia E Martin ◽

My T Le ◽

Nabin Bhattarai ◽

Daiana A Capdevila ◽

Jiangchuan Shen ◽

...

Keyword(s):

Bacillus Subtilis ◽

Transcriptional Regulator ◽

Respiratory Pathogen ◽

Rna Aptamer ◽

Gene Encoding ◽

Protein Levels ◽

Activation Of Transcription ◽

In Vitro Activation ◽

Aptamer Domain

Abstract Maintaining manganese (Mn) homeostasis is important for the virulence of numerous bacteria. In the human respiratory pathogen Streptococcus pneumoniae, the Mn-specific importer PsaBCA, exporter MntE, and transcriptional regulator PsaR establish Mn homeostasis. In other bacteria, Mn homeostasis is controlled by yybP-ykoY family riboswitches. Here, we characterize a yybP-ykoY family riboswitch upstream of the mgtA gene encoding a PII-type ATPase in S. pneumoniae, suggested previously to function in Ca2+ efflux. We show that the mgtA riboswitch aptamer domain adopts a canonical yybP-ykoY structure containing a three-way junction that is compacted in the presence of Ca2+ or Mn2+ at a physiological Mg2+ concentration. Although Ca2+ binds to the RNA aptamer with higher affinity than Mn2+, in vitro activation of transcription read-through of mgtA by Mn2+ is much greater than by Ca2+. Consistent with this result, mgtA mRNA and protein levels increase ≈5-fold during cellular Mn stress, but only in genetic backgrounds of S. pneumoniae and Bacillus subtilis that exhibit Mn2+ sensitivity, revealing that this riboswitch functions as a failsafe ‘on’ signal to prevent Mn2+ toxicity in the presence of high cellular Mn2+. In addition, our results suggest that the S. pneumoniae yybP-ykoY riboswitch functions to regulate Ca2+ efflux under these conditions.

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

10.21203/rs.3.rs-409769/v1 ◽

2021 ◽

Author(s):

Jing Wu ◽

Wei Li ◽

Shiguang Zhao ◽

Sen-he Qian ◽

Zhou Wang ◽

...

Keyword(s):

Bacillus Subtilis ◽

Electron Transport ◽

Biofilm Formation ◽

Stable State ◽

Transcriptional Regulator ◽

Site Directed Mutagenesis ◽

Expression Level ◽

Transcriptional Analysis ◽

Directed Mutagenesis ◽

Nadh Dehydrogenases

Abstract Background: MK-7 is a highly valuable vitamin K2 produced by Bacillus subtilis. Biofilm forming caused by quorum sensing (QS) system was beneficial for MK-7 synthesis. However, the specific regulator in QS system which play the most significant role in biofilm formation and MK production have not been revealed at genetic level, and this limits the possibility of further increasing MK-7 production.Results: In this study, the influence of different mutants in QS system on biofilm formation and MK-7 production was investigated. The transcriptional regulator SinR was chosen finally because the complete knocking out of sinR (KO-SinR) maximized the biofilm biomass and increased the yield of MK-7. However, KO-SinR led to a large number of spore and wrinkle forming, which slow down even stop the MK-7 biosynthesis. Five SinR mutants (E97K, Y101L, W104K, R105S, SinRquad) were constructed by site-directed mutagenesis of four aromatic residues (Glu97, Tyr101, Trp104 and Arg105). Among these, SinRquad formed more wrinkly but smooth biofilm, and obtained the maximum MK-7 value (102.56±2.84 mg/L), which was ten times of the parent strain. Comparative transcriptional analysis showed that SinRquad promoted the synthesis of extracellular polymeric substances and inhibited the swimming motility of late-flagellar. In addition, SinRquad upregulated the expression level of ctaC-G operator and qcrA-C operator which encode the cytochromes. Upregulation of components in the oxidative respiratory chain of B. subtilis was due to the upregulation of NADH dehydrogenases. Most NADH dehydrogenases especially sdhA-C and glpD, increased 1.01-, 3.93-, 1.87-, 1.11- fold, respectively. The increase in expression level of NADH dehydrogenases indicated the ratio of NADH/NAD+ decreased and more electrons produced for the electron transport system (ETM). Wrinkly and smooth biofilm formed a network of interconnected channels with low resistance to liquid flow was beneficial to obtain a more stable state of electron transport chain, which facilitate the metabolic flux of four modules of MK-7 synthesis pathways.Conclusions: In this study, we report for the first time that SinRquad has significant effects on the MK-7 synthesis by forming wrinkly and smooth biofilm, upregulating the expression level of most NADH dehydrogenases, providing more stable state of the electron transport.

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