Engineering a PhrC-RapC-SinR quorum sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis

Abstract

Background: Menaquinone-7 (MK-7) is a valuable vitamin K₂ produced by Bacillus subtilis. Although many strategies have been adopted to increase the yield of MK-7 in B. subtilis, the effectiveness of these common approaches is not high because long metabolic synthesis pathways and numerous bypass pathways competing for precursors with MK-7 synthesis. Regarding the modification of bypass pathways, studies of common static metabolic engineering method such as knocking out genes involved in side pathway have been reported previously. Since byproductsphenylalanine(Phe), tyrosine (Tyr), tryptophan (Trp), folic acid, dihydroxybenzoate, hydroxybutanone in the MK-7 synthesis pathway are indispensable for cell growth, the complete knockout of the bypass pathway restricts cell growth, resulting in limited increase in MK-7 synthesis. Dynamic regulation via quorum sensing (QS) provides a cost-effective strategy to harmonize cell growth and product synthesis, eliminating the need for pricey inducers. SinR, a transcriptional repressor, is crucial in suppressing biofilm formation, a process closely intertwined with MK-7 biosynthesis. Given this link, we targeted SinR to construct a dynamic regulatory system, aiming to modulate MK-7 production by leveraging SinR's regulatory influence.

Results: A modular PhrC-RapC-SinR QS system is developed to dynamic regulate side pathway of MK-7. In this study, first, we analyzed the SinR-based gene expression regulation system in B. subtilis 168 (BS168). We constructed a promoter library of different abilities, selected suitable promoters from the library, and performed mutation screening on the selected promoters. Furthermore, we constructed a PhrC-RapC-SinR QS system to dynamically control the synthesis of Phe, Tyr, Trp, folic acid, dihydroxybenzoate, hydroxybutanone in MK-7 synthesis in BS168. Cell growth and efficient synthesis of the MK-7 production can be dynamically balanced by this QS system. Using this system to balance cell growth and product fermentation, the MK-7 yield was ultimately increased by 6.27-fold, from 13.95 mg/L to 87.52 mg/L.

Conclusion: In summary, the PhrC-RapC-SinR QS system has been successfully integrated with biocatalytic functions to achieve dynamic metabolic pathway control in BS168, which has potential applicability to a large number of microorganisms to fine-tune gene expression and enhance the production of metabolites.

Keywords: Bacillus subtilis; MK-7; PhrC-RapC; SinR.

Fig. 1

Biosynthetic pathway of menaquinone-7 in Bacillus subtilis. Enzymes: GlpF: glycerol uptake facilitator, GlpK: glycerol kinase, GlpD: glycerol-3-phosphate dehydrogenase, Tpi: triosephosphate isomerase, Dxs: 1-deoxyxylulose-5-phosphate synthase, Dxr: 1-deoxyxylulose-5-phosphate reductoisomerase, YgfY: 4-hydroxy-3-methylbut-2-enyl diphosphate synthase, YqfP: 4-hydroxy-3-methylbut-2-enyl diphosphate reductase, YpgA: isopentenyl-diphosphateδ-isomerase, YqiD: farnesyl diphosphate synthase, AroA: 3-deoxy-7-phosphoheptulonate synthase, AroB: 3-dehydroquinate synthase, AroC: 3-dehydroquinate dehydratase, AroD: shikimate dehydrogenase, AroK: shikimate kinase, AroE: 3-phosphoshikimate-1-carboxyvinyltransferase, AroF: chorismate synthase, MenF: isochorismate synthase, MenD: 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase, MenH: 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate synthase, MenC: o-succinylbenzoate synthase, MenE: o-succinylbenzoic acid-CoA ligase, MenB: 1,4-dihydroxy-2-naphthoyl-CoA synthase, MenI: 1,4-dihydroxy-2-naphthoyl-CoA hydrolase, MenA: 1,4-dihydroxy-2-naphthoate heptaprenyltransferase, MenG: demethylmenaquinone methyltransferase, HepS/HepT: heptaprenyl diphosphate synthase component I/II. Ldh: lactate dehydrogenase, AlsS: acetolactate synthase, AlsD: acetolactate decarboxylase, AroH: chorismate mutase, TrpE: anthranilate synthase, PabB/PabA: para-aminobenzoate synthase component I/II, DhbB: bifunctional isochorismate lyase/aryl carrier protein. Abbreviations of metabolites: Gly: glycerol, Gly-3P: glycerol-3-phosphate, DAHP 3-deoxy-arabino-heptulonate 7-phosphate, G3P: glyceraldehyde-3-phosphate, PEP: phosphoenolpyruvate, PYR: pyruvate, E4P: erythrose 4-phosphate, DHQ: 3-dehydroquinate, DHS 3-dehydroshikimate, SA shikimate, S3P: shikimate 3-phosphate, CHA: chorismate, DXP: 1-deoxyxylulose-5-phosphate, MEP: methyl-eryth ritol-4-diphosphate, HMBPP: 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, DMAPP: dimethylallyl diphosphate, IPP: isopentenyl diphosphate, GPP: geranyl diphosphate, FPP: farnesyl diphosphate, ICHA: isochorismate, SEPHCHC: 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate, OSB: 2-succinylbenzoate, DHNA-CoA: 1,4-dihydroxy-2-naphthoyl-CoA, DHNA: 1,4-dihydroxy-2-naphthoate, DMK: 2-demethylmenaquinone, MK-7: menaquinone-7, DHDHB: (2 S,3 S)-2,3-dihydro-2,3-dihydroxybenzoate, Phe: phenylalanine, Tyr: tyrosine, Trp: tryptophan

Fig. 2

Comparison of yield of menaquinone-7 (MK-7) and OD600 values for recombinant strain BS01-BS06 and original strain BS168. (A) final MK-7 yield after 132 h of BS01-BS06 and BS168 fermentation; (B) OD600 change of BS01-BS06 and BS168 during fermentationFig. 2A has too many reserved digits after the decimal point, which is different from the full-text number format

Fig. 3

Verification of the SinR Target Promoter. (A) SinR structure, the red dashed box shows the N-terminal domain; (B) simulated structure of SinR bound DNA; (C) the process of SinR regulation after induction by IPTG: in the absence of IPTG induction, BX00 expressed a small amount of SinR, unable to regulate eGFP expression in BX01-BX09 (two plots of the upper layer); upon the addition of IPTG, a large amount of SinR was produced, which bent the DNA in the target promoter region and inhibited the expression of eGFP(two plots of the lower layer); (D) before and after the addition of the IPTG, relative fluorescence intensity change of BX01-BX09 strain; (E) relative fluorescence intensity change of strains BX01, BX02 and BX04 with IPTG concentration

Fig. 4

Effect of the number of SinR binding sites on transcription efficiency. A) schematic diagram of promoter mutation; B) effect of different number of binding sequences on promoter transcription efficiency

Fig. 5

Effect of the core region on the promoter transcription efficiency. A) schematic diagram of promoter mutation; B) fold change in promoter transcription efficiency after mutation in the − 35 and − 10 regions of P_e3; C) fold change in promoter transcription efficiency after mutation in the − 16 region of P_e9;D) comparison of promoter transcription efficiency of P_epsA and P_e9

Fig. 6

Construction of the RapC-PhrC-SinR. A) genotype schematic representation of the strain BX00-BX05; B) the regulatory effect of rapC on sinR; C) the regulatory effect of PhrC-RapC on sinR; D) the response of the PhrC-RapC-SinR system to bacterial density

Fig. 7

Regulation of the MK-7 metabolic process by PhrC-RapC-SinR system. A) change of MK-7 yield and OD₆₀₀ values over time during BS168 fermentation; B) MK-7 yield of strain BW1-BW5; C) genotype schematic representation of the strain BW5-BW8; D) MK-7 yield of strain BW5-BW8; E) comparison of OD₆₀₀ values during fermentation of BS168 and BW7