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

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.

[1]  L. Schurgers,et al.  High-Dose Menaquinone-7 Supplementation Reduces Cardiovascular Calcification in a Murine Model of Extraosseous Calcification , 2015, Nutrients.

[2]  F. Dehghani,et al.  Enhanced Production of Menaquinone 7 via Solid Substrate Fermentation from Bacillus subtilis , 2011 .

[3]  Peng Xu,et al.  Engineering metabolite-responsive transcriptional factors to sense small molecules in eukaryotes: current state and perspectives , 2019, Microbial Cell Factories.

[4]  H. Shimizu,et al.  Systems metabolic engineering: the creation of microbial cell factories by rational metabolic design and evolution. , 2013, Advances in biochemical engineering/biotechnology.

[5]  W. Quax,et al.  Metabolic engineering of Bacillus subtilis for terpenoid production , 2015, Applied Microbiology and Biotechnology.

[6]  Gholson J. Lyon,et al.  Peptide signaling in Staphylococcus aureus and other Gram-positive bacteria , 2004, Peptides.

[7]  Shane T. Jensen,et al.  The Spo0A regulon of Bacillus subtilis , 2003, Molecular microbiology.

[8]  E. Oldfield,et al.  Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase , 2016, ChemMedChem.

[9]  Fuzhong Zhang,et al.  Biosensors and their applications in microbial metabolic engineering. , 2011, Trends in microbiology.

[10]  Kevin L. Griffith,et al.  Novel mechanisms of controlling the activities of the transcription factors Spo0A and ComA by the plasmid‐encoded quorum sensing regulators Rap60‐Phr60 in Bacillus subtilis , 2015, Molecular microbiology.

[11]  D. Hilbert,et al.  Sporulation of Bacillus subtilis. , 2004, Current opinion in microbiology.

[12]  J. Keasling,et al.  Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids , 2012, Nature Biotechnology.

[13]  Xueli Zhang,et al.  Balanced activation of IspG and IspH to eliminate MEP intermediate accumulation and improve isoprenoids production in Escherichia coli. , 2017, Metabolic engineering.

[14]  B. Panda,et al.  Development of menaquinone-7 enriched nutraceutical: inside into medium engineering and process modeling , 2015, Journal of Food Science and Technology.

[15]  Kristala L. J. Prather,et al.  Dynamic pathway regulation: recent advances and methods of construction. , 2017, Current opinion in chemical biology.

[16]  Jian Chen,et al.  Coupling feedback genetic circuits with growth phenotype for dynamic population control and intelligent bioproduction. , 2019, Metabolic engineering.

[17]  Peng Xu,et al.  Production of chemicals using dynamic control of metabolic fluxes. , 2018, Current opinion in biotechnology.

[18]  Hao Song,et al.  Modular Pathway Engineering of Bacillus subtilis To Promote De Novo Biosynthesis of Menaquinone-7. , 2018, ACS synthetic biology.

[19]  Huaiwei Liu,et al.  Autonomous production of 1,4-butanediol via a de novo biosynthesis pathway in engineered Escherichia coli. , 2015, Metabolic engineering.

[20]  A. Grossman,et al.  Different roles for KinA, KinB, and KinC in the initiation of sporulation in Bacillus subtilis , 1995, Journal of bacteriology.

[21]  A. Demirci,et al.  Strain and plastic composite support (PCS) selection for vitamin K (Menaquinone-7) production in biofilm reactors , 2017, Bioprocess and Biosystems Engineering.

[22]  Taizo Hanai,et al.  Self-induced metabolic state switching by a tunable cell density sensor for microbial isopropanol production. , 2015, Metabolic engineering.

[23]  J. Keasling,et al.  Engineering dynamic pathway regulation using stress-response promoters , 2013, Nature Biotechnology.

[24]  Peng Xu,et al.  Design and kinetic analysis of a hybrid promoter-regulator system for malonyl-CoA sensing in Escherichia coli. , 2014, ACS chemical biology.

[25]  J. Naoum,et al.  Vitamin k dependent proteins and the role of vitamin k2 in the modulation of vascular calcification: a review. , 2014, Oman medical journal.

[26]  K. Tsujimoto,et al.  Mutational analysis of the feedback sites of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase of Escherichia coli , 1997, Applied and environmental microbiology.

[27]  G. Du,et al.  Characterization and application of endogenous phase-dependent promoters in Bacillus subtilis , 2017, Applied Microbiology and Biotechnology.

[28]  R. Mahadevan,et al.  Estimating optimal profiles of genetic alterations using constraint-based models. , 2005, Biotechnology and bioengineering.

[29]  J. Hoch,et al.  PAS-A domain of phosphorelay sensor kinase A: A catalytic ATP-binding domain involved in the initiation of development in Bacillus subtilis , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  Shuyi Yang,et al.  Magnetic immobilization of Bacillus subtilis natto cells for menaquinone-7 fermentation , 2015, Applied Microbiology and Biotechnology.

[31]  W. R. Cluett,et al.  Dynamic metabolic engineering for increasing bioprocess productivity. , 2008, Metabolic engineering.

[32]  Qipeng Yuan,et al.  Microbial biosynthesis of the anticoagulant precursor 4-hydroxycoumarin , 2013, Nature Communications.

[33]  C. Yang,et al.  Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains , 2014, Microbial Cell Factories.

[34]  S. Bron,et al.  A plasmid-borne Rap-Phr system of Bacillus subtilis can mediate cell-density controlled production of extracellular proteases. , 2003, Microbiology.

[35]  Hideki Kobayashi,et al.  Analysis and design of a genetic circuit for dynamic metabolic engineering. , 2013, ACS synthetic biology.

[36]  Francisco Bolívar,et al.  Inactivation of Pyruvate Kinase or the Phosphoenolpyruvate: Sugar Phosphotransferase System Increases Shikimic and Dehydroshikimic Acid Yields from Glucose in Bacillus subtilis , 2013, Journal of Molecular Microbiology and Biotechnology.

[37]  Kristala L. J. Prather,et al.  Dynamic regulation of metabolic flux in engineered bacteria using a pathway-independent quorum-sensing circuit , 2017, Nature Biotechnology.

[38]  A. Grossman,et al.  Identification of AbrB‐regulated genes involved in biofilm formation by Bacillus subtilis , 2004, Molecular microbiology.

[39]  G. Stephanopoulos,et al.  Improving fatty acids production by engineering dynamic pathway regulation and metabolic control , 2014, Proceedings of the National Academy of Sciences.