Engineering a bifunctional Phr60-Rap60-Spo0A quorum-sensing molecular switch for dynamic fine-tuning of menaquinone-7 synthesis in Bacillus subtilis

Shixiu Cuia,b, Xueqin Lva,b, Yaokang Wua,b, Jianghua Lia,b, Guocheng Dua,b, Rodrigo Ledesma-Amaroc, Long Liua,b†

aKey Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

bKey Laboratory of industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China

c Department of Bioengineering, Imperial College London, London SW7 2AZ, UK.

†corresponding author: Long Liu, Tel: +86-510-85918312, Fax: +86-510-85918309, E-mail: longliu@jiangnan.edu.cn

Abstract: 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 were 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, PabrB (down-regulation by Spo0A-P) and PspoiiA (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.

Keywords: Menaquinone-7; Spo0A; Rap60-Phr60; quorum sensing; Bacillus subtilis

Introduction

Metabolic engineering aims to redistribute the metabolic flux of desired pathways by rewiring metabolic networks to achieve maximum production of valuable pharmaceuticals, functional nutraceuticals, and fine chemicals1. Because of the complexity of microbial metabolic networks, commonly-used static metabolic engineering strategies (i.e. gene overexpression and knockout) are prone to overload or disrupt the native metabolic network and cause metabolic imbalances, resulting in accumulation of toxic intermediates, reduced substrate conversion and compromised product yield. Compared to static regulation, dynamic pathway regulation has been successfully applied to balance the tradeoff between cell growth and synthesis of desired products. This is particularly useful when the engineered system needs to adapt to the changing intracellular or extracellular environments2, which has been proven effective for fine-tuning of metabolic fluxes3,4.

Currently, dynamic regulation systems could be categorized into three groups: metabolite responsive promoters, biosensors, and quorum sensing (QS) systems2,5. Metabolite responsive promoters are typically identified by screening for native promoters that are responsive to a specific metabolite. For example, using the farnesyl pyrophosphate-responsive promoter, the amorphadiene was increased by more than 2-fold compared to that obtained from inducible or constitutive promoters6. Protein-based biosensors, which are typically derived from transcription factors, are capable of binding to DNA and up-regulate/down-regulate gene expression upon interacting with metabolites7-9. For instance, FadR, an acyl-CoA sensor, was used to repress the expression of adhb and pdc and prevent the accumulation of toxic intermediates, ultimately improving fatty acid ethyl ester production by 3-fold10. By using metabolite-responsive promoters or biosensors, the titer of target products can be greatly improved8; however, these promoters belong to pathway-specific regulation systems11, severely limiting their application in other metabolic pathways. The QS system overcomes the limitations of metabolite-responsive promoters and transcriptional factor-based biosensors12. A QS system could be rewired to control cell density-dependent processes such as biofilm formation, cellular motility, and sporulation13,14, which has the benefit of not depending on a metabolic pathway or requiring inducers or other interventions15. For instance, the Esa QS from Pantoea stewartia has been engineered for transcriptional repression of phosphofructokinase in Escherichia coli, resulting in a 5.5-fold increase in the myo-inositol titer15. However, most reported QS systems tend to inhibit gene expression, while there are few reports of the simultaneous up-regulation and down-regulation of gene expression by using a bifunctional QS system15-16. Such a system would be helpful for fine-tuning complex metabolic networks.

Bacillus subtilis 168 is a gram-positive model microorganism that is widely used to produce nutraceuticals. The transcription factor Spo0A is responsible for the initiation of spore formation in B. subtilis and can regulate the expression of 121 genes16. Spo0A could be activated by histidine auto-kinases (KinA–E) and two phosphorelay proteins Spo0F and Spo0B17-18. In addition, Rap60 can inhibit Spo0A-P activity and KinA phosphorylation14,19. The activity of Rap60 is inhibited by the signal molecule Phr60, which depends on the cell density. Therefore, we hypothesize that a QS system can be developed by controlling the phosphorylation of Spo0A with the regulation protein Rap60 and the signal molecule Phr60 (Fig.1). When Phr60 accumulates above a certain density during cell growth, Rap60 activity is greatly inhibited, after which phosphorylated Spo0A (Spo0A-P) binds the target promoter and performs its function. Notably, Spo0A can only be phosphorylated under carbon starvation conditions, while most bioprocesses are conducted in nutrient-rich medium; therefore, the Spo0A-based QS system should be preferentially engineered to function in nutrient-rich medium.

In this study, we developed a bifunctional Phr60-Rap60-Spo0A-based QS system for dynamic fine-tuning of complex pathways in B. subtilis 168. First, we reconstituted the Spo0A regulatory system and Rap60-Phr60 QS system to effectively down-regulate and up-regulate gene expression in nutrient-rich medium. Next, we systematically optimized the biosynthesis of the nutraceutical menaquinone -7 (MK-7) using the Rap60-Phr60-Spo0A QS system. MK-7 has been used in prevention of osteoporosis, arterial calcification, cardiovascular disease and parkinsons disease20,21. Applying this dynamic control led to a 40-fold improvement of MK-7 production in shaker flask. Our Phr60-Rap60-Spo0A QS system highlights the importance of using quorum-sensing based genetic circuits for fine-tuning of complex metabolic pathways in industrially-relevant species. This strategy could also be generalized to improve metabolite production and construct efficient microbial cell factories for various bioproduction applications.

Results and discussion

Reconstitution of Spo0A-based gene expression regulation system in B. subtilis. Spo0A, a member of the response regulator family of DNA-binding protein, is activated at the start of sporulation and can directly regulate the expression of approximately 121 genes17. KinA and KinB are mainly responsible for phosphorylating Spo0A to activate sporulation pathways under carbon starvation conditions24. The KinA amino terminus contains sensor domains with three PAS motifs (PAS-A, -B, and -C), and PAS-A senses carbon starvation and changes the conformation of KinA to cause self-phosphorylation of KinA25 (Fig. 2a). To control Spo0A phosphorylation mainly by KinA, we first deleted the KinB gene and then deleted the PAS-A domain from KinA (marked as KinAΔPAS-A) to enable phosphorylation of Spo0A in nutrient-rich medium. We then replaced the native KinA promoter with the constitutive promoter Pveg to control the expression of KinAΔPAS-A and yield the strain BS1. When the mutant strains BS1 was cultured in DSM medium (favorable for sporulation) and LB medium (nutrient-rich medium), which results in the sporulation process regardless of whether the media was nutrient-rich (Fig. 2b), indicating that Spo0A was phosphorylated, although the number of spores was significantly decreased in LB medium. The spore early genes spoIIA and spo0IIE in the BS1 strain were knocked out to prevent or decrease spore formation in nutrient-rich medium and the strain BS2 was obtained (Fig. 2b). The results indicated that regardless of the nutrient conditions, signaling in strain BS2 resulted in Spo0A phosphorylation, but spore synthesis was significantly inhibited.

To verify whether different concentrations of Spo0A-P would regulate gene expression to different extents, spo0A was expressed under control of the IPTG inducible promoter Pgrac100, yielding the strain SP1. A transcriptional unit with spoIIA promoter droves the expression of GFP (Spo0A-P can bind to the spoIIA promoter and activate the transcription of spoIIA) was inserted into the pHT01 vector (Fig. S1) and transformed into SP1 for transcriptional activity detection. As shown in Fig. 3a, as the IPTG concentration was increased, GFP fluorescence intensity forms a quantitative dose-response curve with IPTG. Additionally, after 6–7 h of IPTG induction, strong GFP fluorescence signals were detected, indicating that Spo0A-P can effectively regulate the transcription of PspoIIA. Considering that the native PspoIIA promoter contains four spo0A binding sites named as the “0A box” sequence, we mutated or deleted the “0A box” to obtain a series of promoters with different affinities for Spo0A-P. Fig. 3b shows that the activity of Pspoiia(cs-3) was 3.5-fold higher than that of native PspoIIA when the “0A box” sequence was changed to conserved binding sites, surprisingly, an 8.0-fold transcriptional activity was observed when the “0A box” sequence at 1 and 3 positions was changed to conserved binding sites. When the binding site at 3 location was knocked out to create new promoter Pspoiia(OA-3), the activity of the Pspoiia(OA-3) promoter decreased to 64% of the native PspoIIA (Fig. 3c). Additionally, the binding site sequence of Spo0A-P has a significant effect on the affinity of Spo0A-P to promoter. By altering the Spo0A-P binding site sequence in Pspo0iia promoter, we obtained a series of promoters with different intensity of regulation by Spo0A-P.

In addition to activating gene expression, Spo0A-P can repress gene expression26. For quantitative detection, we created a transcriptional unit where the abrB promoter drove the expression of GFP (Spo0A can bind to the abrB promoter and inhibit the transcription of abrB). As expected, the expression level of GFP driven by the PabrB promoter decreased in the presence of IPTG (Fig. 3d). There are two binding sites in the native PabrB promoter, and we obtained a series of promoters with different repression intensities by changing the sequence and number of the "0A box" in PabrB (Fig. 3e). First, we changed the “0A box” sequence of site 1 and obtained the promoter PabrB(cs-1), whose activity was only 15% of the native PabrB, and then changed the “0A box” sequence of site 2 and obtained the promoter PabrB(cs-2), whose activity was only 46% of the native PabrB (Fig. 3f). Next, a binding site was knocked out to produce PabrB(OA-2), which significantly reduced the inhibition intensity to a greater extent than native PabrB. These results demonstrate that increasing the affinity of Spo0A-P to the promoter PabrB leads to stronger inhibition.

Design and construction of Phr60-Rap60-Spo0A QS system. After constructing a series of promoters with different transcriptional activity under the control of Spo0A-P, we further controlled the expression of Spo0A-P using the regulatory protein Rap60 and cell density-responsive molecule Phr60. Rap60 is known to inhibit Spo0A-P activity19 and KinA phosphorylation14. To verify the inhibitory effect of Rap60 on Spo0A-P, we first knocked out the other two Spo0A-P activity inhibitors, RapA and RapB, and then fused the PspoIIA promoter with GFP to monitor Spo0A-P activity. Next, by constructing a Pgrac100 promoter-rap60 DNA fragment and integrating it into the genome, we obtained strain SP2; when the concentration of IPTG reached 40 µM, the fluorescence intensity of GFP decreased significantly to only 17% of the initial intensity, indicating that Rap60 severely repressed Spo0A-P activity (Fig. 4a). To investigate the relationship between Phr60 and Rap60, rap60 and phr60 were respectively expressed by the constitutive promoter Phag, yielding strain SP3, and the Phr60 peptide bound to and inhibited Rap60, thereby restoring the ability of Spo0A-P to regulate gene expression. We also observed that the expression of Phr60-Rap60 caused the expression level of PspoiiA-GFP to be slightly higher than that of Rap60 alone, which may be because Phr60 did not completely restore the inhibition of Rap60 on Spo0A-P activity. To maximize the regulatory capacity of Spo0A-P, we evaluated the ratio of the copy number of Phr60 and Rap60 at 2:1 and found that Spo0A-P activity was restored to 85% of the native level when the cells entered the stationary phase, which supported our hypothesis (Fig. 4b). To examine the effect of cell density on Spo0A-P activity, Phr60 and spo0A were expressed by their native promoters. Promoters spoiiA and abrB were fused with GFP to monitor Spo0A-P activity (Fig. 4c). When the cell density reached an OD600 of 1.433, the GFP fluorescence signal of PspoiiA-GFP increased with increasing cell density in the shaking flask (Fig. 4d), while cell density reached an OD600 of 1.289, the GFP fluorescence signal of PabrB-GFP decreased as cell density increased (Fig. 4e). A QS system controls cell density-dependent processes and does not depend on a metabolic pathway or require inducers or other interventions15. In this study, we have constructed promoter Pspoiia(cs-1,3) and PabrB(cs-1) whose activities were dependent on the cell density. When the cell dry weight reaches 0.36 g/L, the transcription of Pspoiia (cs-1,3) was activated, while the cell dry weight reaches 0.25 g/L, transcription of PabrB(cs-1) was inhibited (Fig.4f,g). These results indicate that the Phr60-Rap60-Spo0A QS system can be used as a bifunctional switch to increase or inhibit the transcription of target genes in response to cell density.

Boosting the synthesis of MK-7 precursors in B. subtilis. MK-7 is a highly valuable vitamin K2 with wide applications in the prevention of osteoporosis, arterial calcification, cardiovascular disease, and Parkinson’s disease20,21. The biosynthetic pathway of MK-7 in B. subtilis 168 is summarized in Fig. 5. The quinone skeleton of MK-7 is synthesized from chorismite via the men gene cluster (including menF, menD, ytxM, menC, menE, and menB)27, and the variable side chain (C35) is derived from isoprenoid pathway with ispA and HepS/T28. To improve the biosynthesis of MK-7, menF and menE were overexpressed by the strong constitutive promoter P43, yielding strain BS3; menB was overexpressed by the constitutive promoter Phbs in the genome, yielding strain BS4 (Fig. 6a). Additionally, we used the P43 promoter to overexpress the heterogenous gene entC (Km = 11.93 μM, kcat = 2.12s-1)29, which encodes isochorismate synthase in E. coli K12 to replace the isochorismatase gene dhbB in BS4, strengthening the synthesis of isochorismate and yielding the strain BS5. Strain BS5 yields MK-7 titer at 9.3 mg/L in shaking flask after 6 days of cultivation, which was nearly the same as that of wild-type B. subtilis 168 (9 mg/L) (Fig. 6b), and the growth of BS5 cells was similar to that of BS3 and BS4 (Fig. 6c), indicating that the men gene cluster is not a rate-limiting step in MK-7 synthesis. Additionally, the glucose and glycerin consumption rate of BS5 was significantly lower than that of B. subtilis 168 (Fig. S2a, b).

Based on the above results, we attempted to increase the supply of the precursors chorismate acid and heptaprenyl diphosphate (HDP) to improve MK-7 synthesis. To increase the supply of chorismate, we first enhanced the synthesis of erythrose-4-phosphate and phosphoenolpyruvate (PEP) by overexpressing transketolase encoded by tkt through in situ replacement of the native promoter with Phbs, and then overexpressed phosphoenolpyruvate synthase encoded by ppsA with P43 and deleted ptsG to obtain strains BS6 and BS7, respectively. Next, we overexpressed aroA and the feedback-inhibition-resistant aroG encoded by aroGfbr 30, yielding strain BS8. Moreover, shkimate kinase aroK was expressed under the P43 promoter, yielding strain BS9 to eliminate the feedback-inhibition of phenylalanine, tyrosine, and tryptophan. The titer of MK-7 was increased to 19.1 ± 0.7 mg/L in strain BS9, a 2-fold increase compared to in strain B. subtilis 168 (Fig. 6d). The growth of BS9 was delayed at an early stage of culture, but the cell concentration was consistent with that of B. subtilis 168 after 30 h of cultivation (Fig. 6e). We then integrated ispA and hepS/T in the genome to promote HDP synthesis, yielding strains BS10 and BS11, respectively. To generate additional metabolites such as PYR and G3P for the MEP pathway, we also integrated the heterologous gene kdpG (encode 2-dehydro-3-deoxy-phosphogluconate aldolase in Zymomonas mobilis) in the genome, yielding the recombinant strain BS12. dxr and dxs were overexpressed under the P43 promoter in situ to eliminate the bottlenecks of the MEP pathway, yielding BS13, and we further overexpressed fni using the P43 promoter in situ in BS13, yielding BS14. The MK-7 titer of BS14 reached 33.1 ± 0.6 mg/L (Fig. 6f), which was approximately 4-fold than that in wild-type B. subtilis 168. The cell growth of BS11, BS13, and BS14 was slow before 30 h but is comparable to wild-type B. subtilis 168 after 30 h (Fig. 6g). Additionally, by increasing the copy number of menA at the lacA, thrC, and dacA locus in BS14, we obtained the recombinant strains BS15, BS16, and BS17, respectively (Fig. 6h). The titer of MK-7 was increased to 75 mg/L in strain BS17 (Fig. 6i) without cell growth inhibition (Fig. 6j) presumably due to the consumption of toxic metabolite 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate (HMBPP). In addition, the glucose and glycerol consumption rates of strains BS15 and BS17 were significantly higher than those of BS14, but lower than those of the wild-type (Fig. S2). These results demonstrate that menA is a critical player determining the efficiency of MK-7 synthesis pathway.

Dynamic fine-tuning of MK-7 synthesis in B. subtilis by bifunctional Phr60-Rap60-Spo0A QS system. Although there are lots of progress in recent decades to improve MK-7 production31-35, the titer of MK-7 remained low because of the inherent constraints of the metabolic pathway. For example, PEP is an important precursor for the synthesis of chorismate acid36, but most PEP forms pyruvate in the TCA cycle37, and thus balancing the flux distribution at the PEP branch will be a critical intervention. In contrast, UppS is a prenyltransferase that catalyzes the condensation of farnesyl diphosphate with eight molecules of isopentenyl diphosphate to generate undecaprenyl pyrophosphate (UPP, C55), which plays an essential role in cell wall formation and serves as a carrier in the synthesis of peptidoglycan and another cell wall component; if this step is blocked, cell growth will be significantly inhibited38. Additionally, the MK-7 precursors HMBPP and DMAPP in the MEP pathway are toxic to cells39, and thus controlling the concentrations of HMBPP and DMAPP are also critical for efficient synthesis of MK-7 without significantly compromising growth fitness. Therefore, it posts a significant challenge to achieve the maximal synthesis of MK-7 using traditional metabolic engineering strategies such as direct gene overexpression and knockout.

To maximize carbon flux toward MK-7 synthesis pathway, we use the bifunctional Phr60-Rap60-Spo0A QS system to fine-tune key genes in the MK-7 biosynthesis pathway. We first expressed Rap60 with Phag and expressed Phr60 under its native promoter in BS17, yielding strain BS18. By in situ replacement of pyk and uppS promoters with the PabrB(cs-1) promoter in strain BS18, we obtained strain BS19, where we expect that the metabolic flux from PEP to the TCA pathway would be limited and consumption of IPP would be reduced. Compared to BS17, the synthesis of MK-7 was increased by approximately 110% to 169 ± 3.8 mg/L (Fig. 7a). In the late stage, cell growth decreased rapidly because of the limited pyruvate synthesis (Fig. 7b). Considering that the MEP pathway is the only route to synthesize IPP in B. subtilis and that the intermediate of HMBPP and dimethylallyl pyrophosphate (DMAPP) are toxic to cells, we expressed ispH and hepS/T driven by the Pspoiia(cs-1,3) promoter, yielding strain BS, in which the yield of MK-7 reached 360 mg/L (Fig. 7a). The cell growth of BS20 decreased and the maximum OD600 was 60% of B. subtilis 168 (Fig. 7b). MK-7 was predicted to play a crucial role in shuttling electrons between membranes bound to protein complexes in the electron transport chain. Whether a high concentration of MK-7 can switch the physiology of cells and lead to cell lysis remains unclear. Because the titer of MK-7 changed significantly in 3–4 days, we detected the relative expression level of genes on the third day. Fig. 7c shows that the transcription level of genes was ranging from 2 to 24-fold; particularly, the changes in aroA and menE were more than 20-fold. The transcriptional level of hepS/T and ispH gradually increased over the culture time and the transcription level of hepS/T increased by 5.0-fold compared to in BS18. As expected, the transcription of pyk and uppS was inhibited by Spo0A-P (Fig. 7d). These results indicated that the Phr60-Rap60-Spo0A QS system can effectively fine-tune gene expression and improve MK-7 production in B. subtilis 168.

Based on the flask culture results, the recombinant strain BS20 was used to produce MK-7 in a 15-L bioreactor via fed-batch culture. Fig.8a shows the production of MK-7 in fed-batch culture with control of glucose concentration, and the maximum production of MK-7 reached 200 mg/L on the 3rd day. On the first day of fermentation, no MK-7 was found in the fermentation supernatant, while it appeared in the fermentation supernatant on the second day. The MK-7 titer in fermentation supernatant is almost as much as that in the cell precipitate. In fed-batch cultivation with control of glucose concentration between 3-6 g/L (Fig. 8b), a total of 1500 mL of feeding solution was added to the bioreactor during the culture period. Cell density reached OD600=30 at 17 h, then decreased at 50 h and stabilized at OD600=20. These results demonstrated that fed-batch cultivation with control of glucose concentration was favorable for MK-7 production by strain BS20 and provided a basis for large-scale production of MK-7.

Other reported QS systems including LuxR system from Vibrio fischeri and Esa system from Pantoea stewartia have been well-documented. Using the LuxR system, Liu et al. achieved delayed induction of key enzymes in the production of 1,4-butanediol from xylose40, and Soma et al. has constructed a cell concentration-dependent toggle switch that could divert carbon flux from TCA cycle to maximize isopropanol production41. Gupta et al. used the Esa system to inhibit the transcription of phosphofructokinase in E. coli, which improved glucaric acid production by dynamically shifting carbon flux away from glycolysis13. Compared to other reported QS regulation systems, our Phr60-Rap60-Spo0A system can be used as a bifunctional switch for simultaneous upregulation and down-regulation of gene expression. We used the Phr60-Rap60-Spo0A system to inhibit the expression of pyk and uppS, which prevented the adverse effect on cell growth. This bifunctional Phr60-Rap60-Spo0A system can be used to dynamically fine-tune metabolic networks in B. subtilis to produce other chemicals.

Conclusions

In this study, we developed a bifunctional Phr60-Rap60-Spo0A QS switch to simultaneously increase and reduce the expression of multiple genes in B. subtilis. With this QS-based genetic switch, we also systematically optimized MK-7 synthesis pathways in B. subtilis and dynamically tuned the expression of key enzymes with the Phr60-Rap60-Spo0A system. The MK-7 titer in shaking flask was improved to 360 mg/L, which is the highest level ever reported, demonstrating the effectiveness of the Phr60-Rap60-Spo0A system and brings the biotechnological production of MK-7 one step closer to industrially relevant titers.

Methods

Microorganism and medium. All strains with the genotypes of the constructed and engineered B. subtilis and plasmids used in this study are listed in Table 1. All strains were cultivated in Luria–Bertani (LB) liquid culture or on LB agar plates at 37°C for genetic experiments. The fermentation medium consisted of 5% (w/v) glucose, 5% (w/v) glycerol, 5% (w/v) soy peptone, and 0.06% (w/v) KH2PO4. Media were sterilized for 30 min at 121°C. Appropriate antibiotics were added to the medium: kanamycin (50 g/mL), zeocin (20 g/mL), ampicillin (100 g/mL), spectinomycin (50 g/mL), and chloramphenicol (5 g/mL). All chemicals were purchased from Sangon Biotech Co., Ltd (Shanghai, China). Pure MK-7 was purchased from ChromaDex (Irvine, CA, USA). Soy peptone, glycerol, and glucose were purchased from Sinopharm Chemical Reagent Co., Ltd (shanghai, China). Methanol, dichloromethane, 2-propanol, and n-hexane were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Shake flask culture of engineered B. subtilis. The engineered B. subtilis strains were grown in 3 mL LB at 37°C with shaking at 220 rpm for 10 h in a 15-mL tube. A seed culture (1 mL) was inoculated into a 250-mL baffled flask containing 20 mL of fermentation medium and grown at 40°C with shaking at 220 rpm for 7 days. A volume of 0.5 mL of fermentation liquid was sampled every 24 h for MK-7 concentration analysis.

B. subtilis transformation and DNA assembly. DNA constructs were generated using previously described20 assembly methods and transformed into B. subtilis by electroporation. The modified DNA fragments and plasmids were sequenced by GENEWIZ (South Plainfield, NJ, USA). Primers used for genetic modification are listed in Supplementary Table 1.

In situ replacement of promoter, genomic integration of heterogeneous genes and gene knockout. A marker-free genetic modification strategy was used as previously reported20. The expression of homologous genes was strengthened by replacing the native promoter with the strong constitutive promoter P43 or Phbs. The upstream, target sequences, and P43 or Phbs promoter sequences were amplified from B. subtilis. These three fragments and the lox71–zeo–lox66 cassettes were joined by triple-fusion PCR. The purified PCR products were used to transform competent B. subtilis cells. The resistance marker from the host strain was removed by using the Cre/lox system. The PDG148 plasmid was introduced into zeor clones to promote the recombination of lox71 and lox66, which removed the resistance marker cassette. Next, the intracellular plasmid PDG148 was removed by incubation at 50°C for 12 h.

Heterogeneous genes were expressed under control of the strong constitutive promoter P43 or Phbs. Five fragments, including the upstream and downstream sequences (1000 base pairs, bp) flanking the integration locus, lox71–zeo–lox66 cassettes, P43 or Phbs strong constitutive promoter, and target gene sequences, were joined by triple-fusion PCR, and the purified PCR products were used to transform competent B. subtilis cells. Heterologous genes were synthesized with codon optimization by GenScript Biotech (Piscataway, NJ, USA).

The method used for gene knockout was similar to that used for gene overexpression. Briefly, the upstream and downstream sequences (both approximately 1000 bp) flanking the deletion targets were amplified from B. subtilis. These two fragments and the lox71–zeo–lox66 cassettes, which were amplified from the plasmid p7Z6, were joined by triple-fusion PCR. Purified PCR products were used to transform competent B. subtilis cells. The promoter sequence used in this study was the same as that used by Yang et al21.

MK-7 extraction. Centrifugal fermentation media collects supernatant as fermentation supernatant, then resuspend the cell pellet as cell precipitate using an equal volume of sterile water. MK-7 was extracted from the fermentation supernatant and cell precipitate using a mixture of 2-propanol and n-hexane (1:2, v/v) in a 4:1 ratio (organic:liquid, v/v). The mixture was vigorously shaken with a vortex mixer for 10 min and then centrifuged at 5,000×g for 5 min to separate the two phases. The organic phase was then separated to recover the extracted MK-7.

Sporulation assays. Overnight cultures were inoculated into Difco nutrient broth sporulation medium (DSM; Detroit, MI, USA) and fresh LB cultures were grown at 37 °C with vigorous aeration for approximately 20 h after entry into the stationary phase and heated to 80 °C for 20 min. Serial dilutions were prepared with each culture using sterile water, and 100 L of the culture was spread onto LB plates. Viable colony-forming units were evaluated after overnight incubation at 37°C.

Analytical methods. Cell density was determined from the optical density at 600 nm with a spectrophotometer after suitable dilution with deionized water. The concentration of glycerol was assayed with free glycerol determination Kit (Jiancheng, Nanjing, China). The glucose concentration in the medium was measured with a glucose–glutamate analyzer (SBA-40C; Biology Institute of Shandong Academy of Sciences, Shandong, China). High-performance liquid chromatography (Agilent 1260, Santa Clara, CA, USA) equipped with a photon diode array UV detector and C18 ODS column (5 µm, 250 × 4.6 mm, Thermo Fisher Scientific, Waltham, MA, USA) was used at 40°C to determine the MK-7 concentration. Methanol: dichloromethane (9:1, v/v) was used as the mobile phase at a flow rate of 1 mL/min. A wavelength of 254 nm was used for calibration and analysis. The MK-7 calibration curve was linear between 1 mg/L and 100 mg/L (R2 = 0.998).

Quantitative reverse-transcriptase PCR (qRT-PCR) analysis. Cells were harvested during different growth phases from the shaking flask cultures. Total RNA was purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). A ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs, Ipswich, MA, USA) was used to synthesize cDNA from total RNA according to the manufacturer`s instructions. qRT-PCR amplifications were carried out using the Bio-Rad IQ5 Real-Time PCR system (Hercules, CA, USA). The PCR samples were prepared in a total volume of 20 µL using 2×Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with 1.5 µL of cDNA and a final primer concentration of 200 nM. The 16 S rRNA gene was used as an internal standard. PCR conditions were 95°C for 3 min, followed by 50 three-step cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s.

Fed-Batch culture in 15-L bioreactor. The production of MK-7 by fed-batch culture of BS20 was performed at an initial glucose concentration of 10 g/L. The fermentation medium used for fed-batch culture consisted of 50 g/L soy peptone, 5 g/L glycerol and 0.6 g/L KH2PO4. The feeding solution contained 500 g/L glucose and 500 g/L glycerol. Seed culture was carried out in 2 L shake flasks containing 500 mL of seed medium with shaking at 220 rpm and 37 °C for 12 h on rotary shakers. The seed culture was inoculated into 15-L fermenter containing 10 L fermentation medium. The pH was natural, and the temperature was maintained at 40 °C. The aeration rate and agitation speed were 10 vvm and 500 rpm, respectively.

In fed-batch culture, whenever the residual glucose concentration fell to blew 5 g/L, the feeding solution was pumped into the fermenter to restore the glucose concentration to 3-6 g/L. The feeding rates were adjusted every 2 h based on the concentration of residual glucose in the fermentation medium.

Statistical analysis. All experiments were independently carried out at least three times and the results were expressed as mean±standard deviation (SD).

Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (31871784, 31870069, 21676119 and 31671845). Postgraduate Research & Practice Innovation Program of Jiangsu Provence (KYCX18_1786)

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21

Figures

Figure 1 | Schematic diagram of quorum sensing regulation system based on Spo0A. The activity of Spo0A is governed by KinA and KinB, two intermediate protein Spo0F and Spo0B. KinA and KinB phosphorylate the intermediate regulator Spo0F, the phosphoryl group is transferred from Spo0F-P to Spo0B, then to the response regulator Spo0A. Moreover, Rap60 influence Spo0A activity by modulating the phosphoryl system. As the cell grows, the Phr60 gradually increases and enters into the cell by the Opp transporter. Phr60 inhibits the activity of Rap60, and so Spo0A is activated to regulate gene expression.

Figure 2 | Analysis of spore formation rate. (a) Domain architectures of KinA. PAS domains of the amino terminal signal input domain. H405 is catalytic histidine and ATP-binding site of the kinase domain are indicated. (b) Comparison of spore germination rates of different strains. CFU was measured by the production of heat-resistant (80 °C for 20 min) in DSM and LB medium All experiments were performed in triplicate and error bars show standard deviation (SD)

Figure 3 | Spo0A regulates gene expression. (a) The different concentrations of IPTG effect on PspoiiA-GFP expression. Pgrac100-Spo0A was constructed to obtain different concentrations of Spo0A-P. The GFP fluorescence signals increased significantly with increasing IPTG concentration (b) The schematic of PspoiiA, delete the “0Abox” in different locations of PspoiiA. Replace the sequence of “0Ab ox” in the promoter with a conserved sequence. (c) Different promoters are regulated by Spo0A to varying degrees. The indicated strains SP1 (spo0A::Pgrac100-spo0A) were grown in LB medium. T0 represents the time at which IPTG (20 uM) was added to induce expression of Spo0A. (d) The different concentrations of IPTG effect on PabrB-GFP expression. The GFP fluorescence signals decreased significantly with increasing IPTG concentration (e) The schematic of PabrB delete one “0Abox” of PabrB. Replace the sequence of “0Ab ox” in the promoter with a conserved sequence. (f) Different promoters are regulated by Spo0A to varying degrees. Induction conditions are the same as (b). The blue rectangle represents “0Abox” in promoter. The “+1” represent the transcription initiation site. The purple rectangle represents the conserved sequence of “0A box”. All experiments were performed in triplicate and error bars show standard deviation (SD).

Figure 4 | Phr60-Rap60 affects the activity of protein Spo0A. (a) Effects of different concentrations of Rap60 on the activity of Spo0A. Expression of Rap60 with Pgrac-100, and spo0A was expressed in genome with native promoter. The IPTG added in LB medium at first. (b) The relationship between Phr60 and Rap60. Expression of Rap60 and Phr60 with Phag promoter in genome, two Phr60 tandem expressions with two operons. (c) The schematic of Spo0A-P regulated PspoiiA and PabrB promoter. Phosphorylation level of Spo0A is regulated by Rap-Phr quorum sensing gene pair. Promoter spoiiA and abrB fused GFP to monitor Spo0A-P activity, Spo0A-P activate the transcription of PspoiiA-GFP, Spo0A-P inhibit transcription of PabrB-GFP. (d) Relationship between Phr60-Rap60 QS and cell concentration. When cells grown to an OD between 1.433 and 4.562, Spo0A-P significantly activated the transcription of PspoiiA-GFP. (e) When cells grown to an OD between 1.289 and 6.187, the transcription of PabrB-GFP was decreased. (f) The relationship between the transcription level of promoter Pspoiia (cs-1,3) and the dry weight of cells. Activation of Pspoiia (cs-1,3) transcription when the cell dry weight reaches 0.36 g/L. (g) The relationship between the transcription level of promoter PabrB(cs-1) and the dry weight of cells.

Figure 5 | The pathway of MK-7 in B. subtilis 168. MK-7 synthesis is a complex process involving the synergy of multiple metabolic pathways, including glycolysis, pentose phosphate, shikimic acid, MEP and MK-7 pathways. Enzymes: MenF, isochorismate synthase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate synthase; YtxM, 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; YuxO, 1,4-dihydroxy-2-naphthoyl-CoA hydrolase; MenA, 1,4-dihydroxy-2-naphthoate heptaprenyltransferase; MenH, demethylmenaquinone methyltransferase; EntC, isochorismate synthase from E. coli K12; PpsA, phosphoenolpyruvate synthase; AroGfbr, feedback-inhibition-resistant aroG; IspA, farnesyl diphosphate synthase; HepS/HepT, heptaprenyl diphosphate synthase component I/II; Dxs, 1-deoxyxylulose-5-phosphate synthase; Dxr, 1-deoxyxylulose-5-phosphate reductoisomerase; IspD, 2-C-methylerythritol 4-phosphate cytidylyltransferase; IspE, 4-diphosphocytidyl-2-C-methylerythritol kinase; IspF, 2-C-methylerythritol 2,4-cyclodiphosphate synthase; IspG, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase; IspH, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; DhbB, isochorismatase; Abbreviation of metabolites: Glu, glucose; G-6P, glucose-6- phosphate; F6P, fructose-6- phosphate; F-1,2P, fructose -1,6- bisphosphate; Gly, glycerol; G3P, glyceraldehyde-3-phosphate; PEP, phosphoenolpyruvate; Pyr, pyruvate; E4P, erythrose 4-phosphate; DAHP, 3-deoxy-arabino-heptulonate 7-phosphate; DHQ, 3-dehydroquinate; DHS, 3-dehydroshikimate; SA, shikimate; S3P, shikimate 3-phosphate; EPSP, 5-O-(1-carboxyvinyl)-3-phosphoshikimate; CHA, Chorismite; DXP, 1-deoxyxylulose-5-phosphate; MEP, methyl-erythritol-4-diphosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GPP, Geranyl diphosphate; FPP, farnesyl diphosphate; UPP, undecaprenyl phosphate; HDP, heptaprenyl diphosphate; ICHA, isochorismate; SEPHCHC, 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate; SHCHC, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate; OSB, 2-succinylbenzoate; OSB-CoA, 2-succinyl benzoyl-CoA; DHNA-CoA, 1,4-dihydroxy-2-naphthoyl-CoA; DHNA, 1,4-dihydroxy-2-naphthoate; DMK, 2-demethylmenaquinone; MK-7, menaquinone-7;

Figure 6 | Strengthening precursor supply to push carbon flux to the MK-7 synthesis pathway. Panels a indicate the schematic of overexpression gene with promoter P43 or Phbs and gene knockout. Panels b and c indicate the production of MK-7 and cell growth of MK-7 after modular optimization men cluster by overexpression gene with P43-menF P43-menB Phbs-menE P43-entC and ΔdhbB. Panels d and e indicate the production of MK-7 and cell growth of MK-7 after gene overexpression with P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK. Panels f and g indicate overexpression of MEP pathway (P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr::lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni) impact on MK-7 production and cell growth. Panels h indicate the schematic of increasing the copy numbers of the menA gene at lacA, thrC and dacA locus. Panels i and j indicate the different copies of menA in strain BS12 effect on MK-7 production and cell growth. Integration sites are lacA, thrC and dacA. Slashes indicate cell precipitate, rectangular indicate of fermentation supernatant All experiments were performed in triplicate and error bars show standard deviation (SD).

Figure 7 | Spo0A dynamically regulates key genes in the MK-7 synthesis pathway. (a) The titers of MK-7 synthesized by strains BS19 and BS20. Spo0A inhibits the expression of pyk and uppS at appropriate time points to yield strain BS19. Spo0A promotes the expression of ispH and hepS/T basis on strain BS20. Slashes indicate cell precipitate, rectangular indicate of fermentation supernatant. (b) the cell growth of BS19 and BS20. (c) The relative transcriptional levels of genes which were overexpressed. (d) The relative transcriptional levels of gene pyk (in strain BS19), uppS (in strain BS19), hepS/T (in strain 20), ispH (in strain 20), compared with that of the corresponding genes in the control strain BS18. The transcriptional levels of pyk, uppS, hepS/T, ispH were regarded as 1 in strain BS18, respectively.

Figure 8 | Production of MK-7 by BS20 in a 15-L bioreactor. (a) The production of MK-7 in fed-batch fermentation, including cell precipitate and fermentation supernatant. (b) Trends of cell growth and glucose concentration during fed-culture in a 15L bioreactor.

Table 1. strain and plasmids used in this study

Names

Characteristics

Reference

Strains

BS168

Lab stock

BS1

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB

This study

BS2

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE

This study

BS3

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB

This study

BS4

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE

This study

BS5

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB

This study

BS6

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt

This study

BS7

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG

This study

BS8

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 Phbs- aroA

This study

BS9

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK

This study

BS10

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA

This study

BS11

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T

This study

BS12

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG

This study

BS13

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs

This study

BS14

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni

This study

BS15

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA

This study

BS16

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA Pmena-menA :: thrC

This study

BS17

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA Pmena-menA :: thrC Pmena-menA :: dacA

This study

BS18

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA Pmena-menA :: thrC Pmena-menA :: dacA Phag- Rap60 Pnative- Phr60 :: hag

This study

BS19

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA Pmena-menA :: thrC Pmena-menA :: dacA Phag- Rap60 Pnative- Phr60 :: hag Pabrb-pyk::pyk Pabrb-uppS::uppS

This study

BS20

Bacillus subtilis 168 Pveg-kinA-ΔPAS-A ΔkinB ΔspoIIA Δspo0IIE, P43-menF P43-menB Phbs-menE P43-entC ΔdhbB Phbs-tkt P43-ppsA ΔptsG Phbs- aroGfbr :: lox72 P43-aroK Phbs-ispA P43- hepS/T Phbs-kdpG P43-dxr P43-dxs P43-fni Pmena-menA :: lacA Pmena-menA :: thrC Pmena-menA :: dacA Phag- Rap60 Pnative- Phr60 :: hag Pabrb-pyk::pyk Pabrb-uppS::uppS PspoiiA-ispH::spoIIE PspoiiA-HepS/T::spoIIA

This study

SP1

BS168 derivate, expressing of spo0A with Pgrac100 Bacillus subtilis 168 Pgrac100-spo0A :: spo0A

This study

SP2

BS168 derivate, expressing of Rap60 with Pgrac100 Bacillus subtilis 168 Pgrac100-Rap60 :: amye

This study

SP3

BS168 derivate, expressing of Rap60 and Phr60 with Phag Bacillus subtilis 168 Phag-rap60, Phag-phr60 :: hag

This study

plasmids

P7Z6

Pmd18-T containing lox71-zeo-lox66 cassette

PDG148

Amp, Km, E.coli-B.subtilis shuttle vector, containing cre under the control of Pspac

This study

pHT01

Amp, Cm, E.coli-B.subtilis shuttle vector

This study

pHT01-Pspoiia-gfp

pHT01 ferivate with GFP gene under the control of Pspoiia

This study

(Fig. 1)

(Fig. 2)

(Fig. 3)

(Fig. 4)

(Fig. 5)

(Fig. 6)

(Fig. 7)

(Fig.8)