Modular pathway engineering of Bacillus subtilis to promote de novo

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Modular pathway engineering of Bacillus subtilis to promote de novo biosynthesis of menaquinone-7 Shaomei Yang, Yingxiu Cao, Liming Sun, Congfa Li, Xue Lin, Zhigang Cai, Guoyin Zhang, and Hao Song ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.8b00258 • Publication Date (Web): 13 Dec 2018 Downloaded from http://pubs.acs.org on December 14, 2018

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ACS Synthetic Biology

Modular pathway engineering of Bacillus subtilis to promote de

novo biosynthesis of menaquinone-7 Shaomei Yang,† Yingxiu Cao,† Liming Sun,‡ Congfa Li,§ Xue Lin,§ Zhigang Cai,‖ Guoyin Zhang,‖ Hao Song†, *

† Key

Laboratory of Systems Bioengineering (Ministry of Education), School of Chemical Engineering and Technology,

and SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin University, Tianjin 300072, P. R. China ‡ Petrochemical

Research Institute, PetroChina Company Limited, Beijing 102206, China

§ College

of Food Science and Technology, Hainan University, Haikou 570228, China

‖ Chifeng

Pharmaceutical Company Limited, Chifeng, Inner Mongolia 024000, China

ABSTRACT Menaquinone-7 (MK-7), a valuable vitamin K2, plays an important role in the prevention of osteoporosis and cardiovascular calcification. We chose B. subtilis 168 as the chassis for the modular metabolic engineering design to promote the biosynthesis of MK-7. The biosynthetic pathway of MK-7 was categorized into four modules, namely, the MK-7 pathway (Module I), the shikimate (SA) pathway (Module II), the methylerythritol phosphate (MEP) pathway (Module III), and the glycerol metabolism pathway (Module IV). Overexpression of menA (Module I) resulted in 6.6 ± 0.1 mg/L of MK-7 after 120 hours’ fermentation, which was 2.1-fold that of the starting strain BS168NU (3.1 ± 0.2 mg/L). Overexpression of aroA, aroD and aroE (Module II) had a negative effect on the synthesis of MK-7. Simultaneous overexpression of dxs, dxr, yacM and yacN (Module III) enabled the yield of MK-7 to 12.0 ± 0.1 mg/L. Moreover, overexpression of glpD (Module IV) resulted in an increase of the yield of MK-7 to 13.7 ± 0.2 mg/L. Furthermore, deletion of dhbB reduced the consumption of the intermediate metabolite isochorismate, thus promoting the yield of MK-7 to 15.4 ± 0.6 mg/L. Taken together, the final resulting strain MK3-MEP123-Gly2-ΔdhbB with simultaneous overexpression of menA, dxs, dxr, yacM-yacN, glpD and deletion of dhbB enabled the yield of MK-7 to 69.5 ± 2.8 mg/L upon 144 hours’ fermentation in a 2-L baffled flask.

KEYWORDS: Menaquinone-7, Bacillus subtilis 168, modular pathway engineering, MK-7 biosynthesis pathway, shikimate (SA) pathway, methylerythritol phosphate (MEP) pathway, glycerol metabolism pathway

Naturally occurring vitamin K compounds (terperiod-quinones) comprise a plant form, phylloquinone (PK, vitamin K1), and a 1

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number of bacterial menaquinones (MKs, vitamin K2).1 There are 14 kinds of MK-n, where n stands for the number of isoprene units in the side chain.2, 3 Vitamin K is an essential cofactor for the posttranslational conversion of glutamic acid residues of specific proteins in the blood and bone into γ-carboxyglutamic acid (Gla), and a daily vitamin K intake of 1 μg per kg body weight is recommended as being adequate for all age groups beyond the neonatal phase.4 PK is more abundant in foods but less bioactive than MK-n (in particular MK-7).5 The effects of long chain MK-n such as MK-7 on normal blood coagulation are greater and longer lasting than that of PK and MK-4.6, 7 Due to its long half-life and good bioavailability, MK-7 is widely used as dietary supplements or drug treatments in the food, pharmaceutical and healthcare industries for prevention of osteoporosis,8 cardiovascular calcification9 and Parkinson’s disease10. The costs of treating osteoporosis and cardiovascular diseases are high and will likely increase with the aging of populations. The global osteoporosis drug market in 2009 was estimated to be $8-9.4 billion, and the market for cardiovascular drugs was estimated to be $110-140 billion.11 Thus, there is a large market for menaquinones and the biosynthesis of MK-7 has received much attention from academia and industry. Bacillus subtilis natto was used to produce MK-7 with traditional mutagenesis and optimization of fermentation processes. Song et al. screened a 1-hydroxy-2-naphthoic acid (HNA)-resistant B. subtilis natto mutant with a MK-7 yield of 3.6 mg/L.12 Luo et al. isolated a Bacillus natto strain from the traditional Japanese food natto, and the yield of MK-7 was up to 32.2 mg/L after fermentation for 72 h.13 Sato et al. screened a menadione-resistant mutant strain from B. subtilis isolated from natto, which produced 35.0 mg/L MK-7 after 4 days’ cultivation.14 Tsukamoto et al. screened a B. subtilis natto mutant OUV23481 with resistance to HNA, p-fluoro-D,L-phenyalanine, m-fluoro-D,L-phenyalanine and β-2-thienylalanine, which enabled a productivity of MK-7 1,719 μg/100 g natto.15 Bacillus amyloliquefaciens was also studied for the MK-7 biosynthesis. Xu et al. isolated six fibrinogenase-producing strains from Chinese douchi, and found that B. amyloliquefaciens Y-2 could produce 7.1 mg/L of MK-7, i.e., 141 μg/(g DCW (dry cell weight)). Overexpression of HepS in B. amyloliquefaciens Y-2 further led to an 93.62% increase in the MK-7 production.16 However, owing to the lack of clarified genome annotation and matured genomic editing tools for engineering B. subtilis natto and B. amyloliquefaciens, their rational engineering for further improvement of the yield of MK-7 was rather challenging at this stage. Herein, we chose Bacillus subtilis as the platform microorganism for rational pathway engineering design to increase the yield of MK-7. B. subtilis 168 is one of the best-characterized model microorganisms, which attracted wide attention for industrial use due to its rapid growth and it was ‘generally regarded as safe’ (GRAS).17,

18

The biosynthesis pathway of MK-7 in B.

subtilis was presented in Figure 1, which could be categorized into four modules, namely the MK-7 pathway (Module I), the shikimate (SA) pathway (Module II), the methylerythritol phosphate (MEP) pathway (Module III), and the glycerol metabolism pathway (Module IV). MK-7 is a non-protein component of the B. subtilis electron transport chain synthesized from chorismate through the MK-7 pathway (Module I). To promote the biosynthesis of MK-7, we increased the metabolic flux of the MK-7 pathway by in situ replacement of the native promoter that drives the menFDHBEC operon and the hepS-menG-hepT operon in the genome of B. subtilis with an artificial double promoter PlapS19. Meanwhile, the overexpression of a heterogenous gene menI and a native gene menA was implemented. Since the precursor for the MK-7 biosynthesis is chorismate, a branch point of the SA pathway (Module II).20, 21 Thus, we engineered the SA pathway to increase the supply of chorismate by overexpressing the genes aroA, aroD, aroK and aroE with the PAE expression cassette22, respectively. The side chain of MK-7 is provided by heptaprenyl diphosphate, an intermediate of the MEP pathway (Module III). We then engineered the MEP pathway to increase the supply of heptaprenyl diphosphate by overexpressing the genes dxs, dxr, yacM-yacN, ispE, yqiD and yqfP with a strong promoter P4323, respectively. Glycerol is the most suitable carbon source for the MK-7 synthesis.24 We further engineered the glycerol metabolism pathway to increase the glycerol utilization by overexpressing the genes glpF-glpK, glpD and tpi with the promoter P43, respectively. Lastly, the consumption of pyruvate, chorismate and isochorismate was reduced by knocking out the genes ldh, alsS-alsD, aroH-trpE, pabB-pabA and dhbB, respectively. We systematically studied the effect of the overexpression of the sixteen genes and two operons in the above four modules and the knockout of seven genes in B. subtilis on the biosynthesis of MK-7. Fermentation results of the constructed recombinant 2


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strains suggested that the genes menA (Module I), dxs, dxr, yacM-yacN (Module II) and glpD (Module IV) and dhbB were mostly crucial in affecting the MK-7 biosynthesis. The final resulting strain MK3-MEP123-Gly2-ΔdhbB that integrated the simultaneous overexpression of menA, dxs, dxr, yacM-yacN, glpD and the knockout of dhbB enabled the production of MK-7 up to 15.4 ± 0.6 mg/L after 120 hours’ fermentation in 250 mL flasks, which was 5.0-fold that of the starting strain BS168NU (3.1 ± 0.2 mg/L). Its production of MK-7 reached 38.9 ± 2.0 mg/L after 72 hours of fermentation in 2-L baffled flasks, which was 3.2-fold that of BS168NU (12.3 ± 1.8 mg/L). Upon supplement of 2% glycerol and 4% soy peptone, the yield of MK-7 could reach 69.5 ± 2.8 mg/L after 144 hours’ fermentation. Results and discussion Enhancing metabolic flux of the MK-7 pathway In B. subtilis, the MK-7 pathway (Module I, Figure 1) comprises nine enzymatic reactions. The encoding genes of the first six enzymes constitute the menFDHBEC operon, and the gene menG constitutes the hepS-menG-hepT operon along with hepS/hepT. To promote the biosynthesis of MK-7, we firstly tried to enhance the metabolic flux of the MK-7 pathway. The native promoters of the menFDHBEC operon and the hepS-menG-hepT operon in the genome of the starting strain BS168NU were replaced in situ with the promoter PlapS, respectively, which achieved the recombinant strains MK1 and MK4 (Figure 2a). Because the gene menI was not identified in the genome of B. subtilis, we used the respective gene menI from E. coli, resulting in the strain MK2. Finally, we chose the native gene menA from B. subtilis encoding the 1,4-dihydroxy-2-naphthoate (DHNA) heptaprenyltransferase, obtaining the strain MK3. Shake flask fermentation experiments were performed for these recombinant strains to analyze their growth, transcriptional levels of the overexpressed genes, and the MK-7 production. As shown in Figure S1a, the overexpression of these genes had an unobvious effect on the cell growth. Under control of the PlapS promoter, the transcriptional level of the menFDHBEC operon in MK1 was 3.4-fold that of this operon in the starting strain BS168NU, which showed that PlapS was effective in regulating the gene expression. However, the MK-7 production by MK1 had insignificant change in comparison to that by BS168NU (3.1 ± 0.2 mg/L) upon 96 and 120 hours’ fermentation (Figure 2b). A plausible rationale was that the six enzymes encoded by this operon were not a bottleneck limiting the biosynthesis of MK-7, whereas the overexpression of MenD in E. coli induced a 2-fold’ increase in the yield of menaquinone-8 (MK-8) over the control strain JM109 (60 μg MK-8/g WCW).25 E. coli is a facultative anaerobic bacterium that synthesizes mainly ubiquinone-8 (Q-8) under aerobic conditions but mainly produces MK-8 under anaerobic conditions.2 B. subtilis is an aerobic bacterium that can only synthesize MK-7. These two bacteria synthesize different quinones under different oxygen conditions to participate in the electron transport of the respiratory chain. Moreover, the overexpression of MenI from E. coli also did not improve the production of MK-7, which suggested that the hydrolysis of DHNA-CoA to DHNA (the seventh step) was also not a rate-limiting reaction for the MK-7 biosynthesis. However, MK3 could produce 6.6 ± 0.1 mg/L of MK-7, which was 2.1-fold that of BS168NU (Figure 2b), meanwhile, the transcriptional level of menA in MK3 was 6.2-fold that of menA in BS168NU (Figure S1b). It showed that the eighth reaction catalyzed by MenA, the prenylation of DHNA to 2-demethylmenaquinone (DMK), was a rate-limiting step in the MK-7 pathway. MK4 could produce 4.3 mg/L of MK-7 (Figure 2b), which was 1.4-fold that of BS168NU, and the transcriptional level of the hepS-menG-hepT operon was 3.7-fold of that in BS168NU (Figure S1b). HepS/HepT catalyzes the condensation of FPP and four molecules of IPP into heptaprenyl-PP, the direct precursor for DMK synthesis, and MenG catalyzes the last reaction, the methylation of DMK to MK-7.26 However, the combinatorial overexpression of the gene menA with the hepS-menG-hepT operon in MK34 (Figure 2c) resulted in a decrease in the cell growth (Figure S1c) and the MK-7 production (Figure 2d), in comparison to the overexpression of menA alone in MK3. As a result, MK3 was chosen for further strain engineering. Engineering SA pathway to improve the supply of chorismate The MK-7 pathway (Module I) initiated with chorismate, a key intermediate of the SA pathway. The SA pathway (Module II, Figure 1) is an ubiquitously existing pathway in plants, algae, fungi and bacteria, and its essential role for cellular metabolism is 3



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to provide precursors for the biosynthesis of three aromatic amino acids such as tyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp).27, 28 Through decades of efforts, the enzymatic reactions involved in the SA pathway of E. coli had been well elucidated and characterized.29 In E. coli, the condensation of PEP and E4P into 3-deoxy-arabino-heptulonate 7-phosphate (DAHP) was the fist step of the SA pathway, and three isoenzymes of DAHP synthase (AroG, AroF, and AroH) was subjected to feedback regulation by Phe, Tyr, and Trp, respectively,30, 31 whereas B. subtilis possessed only one DAHP synthase (AroA). SA kinase and 3-phosphoshikimate (S3P) 1-carboxyvinyltransferase were found to be the rate-limiting enzymes in the common aromatic amino acid pathway in E. coli.32,

33

However, AroD-driven reduction of 3-dehydroshikimate into SA was a

rate-limiting step for the biosynthesis of SA in B. subtilis, and simultaneous overexpression of aroA and aroD in B. subtilis resulted in the SA yield of 3.2 g/L in a batch fermentation.34 We chose to overexpress DAHP synthase (AroA), SA dehydrogenase (AroD), SA kinase (AroK) and S3P 1-carboxyvinyltransferase (AroE) to increase the supply of chorismate (Figure 3a). However, except that overexpression of AroK led to a slight increase (~5%) in the MK-7 production, overexpression of the other three enzymes inhibited the biosynthesis of MK-7 (Figure 3b). qRT-PCR showed that the transcriptional levels of these four genes were indeed significantly increased (>300-fold) compared with that of MK3 (Figure S2b). In addition, the cell growth was not affected by the overexpression of these enzymes (Figure S2a). A previous study investigated the feedback inhibition by aromatic amino acids on the biosynthesis of MK-7 in B. subtilis natto.15 It was found that the addition of three aromatic amino acids (Tyr, Phe and Try, each 100 mg/L) was strikingly decreased the yield of MK-7, suggesting that the three aromatic amino acids might participate in the feedback inhibition of the upstream of MK-7 biosynthetic pathway in B. subtilis natto. It was thus possible that the overexpression of AroA, AroD and AroE in B. subtilis would increase the synthesis of aromatic amino acids, which in turn resulted in the feedback inhibition on the SA pathway, and ultimately inhibited the biosynthesis of MK-7. Engineering MEP pathway to improve the supply of heptaprenyl-PP The study of Module I had demonstrated that MenA-driven prenylation of DHNA to DMK was the rate-limiting reaction for the synthesis of MK-7 in B. subtilis, therefore, it was also important to increase the supply of heptaprenyl-PP. The MEP pathway (Module III, Figure 1) is present in eubacteria, algae, cyanobacteria, and the chloroplasts of plants,35,

36

which provides

isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) for the biosynthesis of isoprenoids. However, the studies on the key enzymes in the MEP pathway in B. subtilis were rare. Xue et al. found that the yield of isoprene could be increased by 40% over that of the wild-type B. subtilis by overexpressing dxs.37 Julsing et al. observed that the knockout of yqfY and ypgA showed insignificant difference in the synthesis of isoprene in comparison to the wild-type strain.38 We chose to overexpress the dxs, dxr, yacM-yacN, ispE, yqfP and yqiD genes in the genome of MK3 using the promoter P43, respectively, which led to the strains MK3-MEP1, MK3-MEP2, MK3-MEP3, MK3-MEP4, MK3-MEP5, and MK3-MEP6 (Figure 4a). Dxs catalyzes the first reaction in the MEP pathway that condenses pyruvate and G3P to generate 1-deoxyxylulose-5-phosphate (DXP).39 In the strain MK3-MEP1 in which dxs was overexpressed, the production of MK-7 was 9.7 ± 0.2 mg/L after 120 hours’ fermentation, an increase by 47% over that of MK3 (Figure 4b). Under control of the P43 promoter, the transcriptional level of dxs in MK3-MEP1 was 2.4-folds that of dxs in MK3 (Figure S3b). In addition, in order to increase the transcriptional level of dxs, we also selected three other strong and constitutive promoters (PAE, PlapS, Pveg40, 41) to overexpress dxs, and found that the transcriptional levels of dxs among the three recombinant strains were 2.1-, 2.4- and 2.8-fold that of dxs in the control strain MK3, respectively. The effect of the promoter on the transcriptional level of dxs was PAE > PlapS (≈P43) > Pveg (Figure S3d). The MK-7 production by the strain MK3-PAE-dxs was 10.3 mg/L, an 56% increase compared with that of the control strain MK3, which was only 0.6 mg/L over that of the strain MK3-MEP1 (P43-dxs) (Figure S3e). The MK-7 production by the strains MK3-PlapS-dxs and MK3-Pveg-dxs were 9.9 ± 0.3 mg/L and 7.1 ± 0.7 mg/L, respectively. The other three recombinant strains had little difference in the MK-7 production, except for the strain with the overexpression of dxs using Pveg. 4


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Dxr catalyzes the reduction of DXP into MEP, which is the second step in the MEP pathway. For the strain MK3-MEP2 that overexpressed dxr, the yield of MK-7 was increased by 38% over that by the strain MK3, up to 9.1 ± 0.5 mg/L (Figure 4b). The genes yacM and yacN are close in the genome of B. subtilis. YacM catalyzes the reaction of MEP and cytosine triphosphate (CTP) to form 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME).42 YacN catalyzes the conversion of CDP-MEP to 2-C-methylerythritol 2,4-cyclo-diphosphate (MEC) with the concomitant release of cytidine 5’-diphospate (CMP).43 The strain MK3-MEP3 with the overexpression of yacM/yacN could produce 8.8 ± 0.7 mg/L of MK-7, increased by 33% over that by MK3 (Figure 4b). The above results showed that Dxs, Dxr, YacM and YacN were crucial enzymes that promoted the biosynthesis of MK-7. Therefore, we overexpressed dxs, dxr and yacM-yacN in a combinatorial way to explore whether a further increase in MK-7 synthesis could be obtained (Figure 4c). The recombinant strain MK3-MEP12 could produce 10.6 ± 0.4 mg/L of MK-7, while the MK-7 yield of MK3-MEP123 was 12.0 mg/L (Figure 4d). Overexpression of the other three genes (i.e., ispE, yqfP and yqiD) under the control of the P43 promoter in the strains MK3-MEP4, MK3-MEP5 and MK3-MEP6, respectively (Figure 4a) enabled a significant increase in the mRNA levels of these genes (Figure S3b), however, resulted in a decrease in the production of MK-7 (Figure 4b). However, the overexpression of IspA in E. coli, whose function is similar to YqiD in B. subtilis, could increase the yield of MK-8 in E. coli.25 FPP, formed by the catalysis of Yqid, is a substrate of HepS-MenG-HepT for the MK-7 biosynthesis (Figure 1), we thus overexpressed yqiD in the genome of the strain MK4 (with the overexpression of hepS-menG-hepT) and obtained the recombinant strain MK4-yqiD. However, the overexpression of yqiD led to a slight decrease in the MK-7 production (Figure S3f). Similarly, the overexpression of yqiD in the starting strain BS168NU also resulted in a minor decrease in the MK-7 production (Figure S3g). Engineering glycerol metabolism pathway to increase glycerol utilization Luo et al. (13) and Berenjian et al. (24) compared the effect of four carbon sources (i.e., soluble starch, sucrose, glucose and glycerol) on the MK-7 synthesis and the growth of B. subtilis natto, and found that the presence of glycerol in the media resulted in higher MK-7 production. In many bacteria, the uptake of glycerol is catalyzed in an energy-independent manner by a membrane channel protein, the glycerol facilitator GlpF.44 The main pathway of glycerol dissimilation involves a glycerol kinase GlpK that phosphorylates glycerol to glycerol-3-phosphate (Gly-3P), and a Gly-3P dehydrogenase GlpD that oxidizes Gly-3P to dihydroxyacetone phosphate (DHAP), an intermediate in glycolysis.45, 46 This is the only catabolism pathway for glycerol utilization known in B. subtilis. In B. subtilis, glpF and glpK are organized in an operon followed by glpD and preceded by glpP coding for an antiterminator regulating the expression of glpFK and glpD.45, 47 In addition, the glpFK operon is induced by Gly-3P and repressed by the rapidly metabolizable sugars.48 Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK.48 To avoid the sophisticated expression pattern of the glpFK operon under the control of termination/antitermination of transcription at an inverted repeat in the glpFK leader and the carbon catabolite repression, we chose to overexpress the glpFK operon by inserting it into the pksJ locus in the genome of B. subtilis using the constitutive promoter P43, obtaining the strain MK3-MEP123-Gly1. However, the MK-7 biosynthesis was not enhanced by this strategy (Figure 5b). On the other hand, since the expression of glpD was also controlled by termination/antitermination of transcription at an inverted repeat in the glpD leader, and mediated by the antiterminator protein GlpP in the presence of Gly-3P,47 we chose to overexpress glpD by inserting it into the pksJ locus in the genome of B. subtilis using the promoter P43 and obtained the strain MK3-MEP123-Gly2, resulting in ~14% increase in the yield of MK-7 (13.7 ± 0.2 mg/L) (Figure 5b). Also, the strain MK3-MEP123-Gly2 could deplete glycerol upon 96 hours’ fermentation, faster than that of the control strain MK3-MEP123 (Figure S4c). DHAP was catalyzed by triose phosphate isomerase (Tpi) and converted to G3P, the direct precursor for the biosynthesis of isoprenoids (Module III). However, the overexpression of tpi in the strain MK3-MEP123-Gly3 (Figure 5a) had insignificant effect on the cell growth, the synthesis of MK-7 and the glycerol consumption, suggesting that the conversion of DHAP to G3P was not a bottleneck in the MK-1 biosynthesis. 5



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Reducing the consumption of pyruvate, chorismate and isochorismate The lactate dehydrogenase Ldh is a cytoplasmic NADH-linked enzyme that converts pyruvate to lactate.49 Deletion of ldh had an unobvious effect on the cell growth and the biosynthesis of MK-7 (Figure 6a). Acetolactate synthase (AlsS) condenses two molecules of pyruvate to form acetolactate, which is converted to acetoin by spontaneous decarboxylation at low pH and by the action of acetolactate decarboxylase (AlsD).50 However, deletion of the alsS-alsD operon resulted in a dramatic decrease in the cell growth (Figure S5a). It had been reported that acetoin production in B. subtilis was a mechanism for maintaining the internal pH when cells entered the stationary phase.50 Thus, after entering the stationary phase, the internal pH of cells could not be maintained in the absence of acetoin, leading to the inhibited cell growth. As a result, the synthesis of MK-7 was greatly reduced (Figure 6a). As shown in Figure 1, the synthesis of MK-7 was originated from chorismate, however, chorismate also participated in the biosynthesis of three aromatic amino acids (Tyr, Phe and Trp) and folate. The wild-type B. subtilis 168 is a Trp auxotroph mutant, the deletion of aroH could block the competitive biosynthesis of Tyr and Phe. Knockout of aroH had a negligible effect on the cell growth (Figure S5a), but resulted in a substantial decrease in the MK-7 production (Figure 6a). We knocked out aroH in the genome of the strain MK1, which enabled the construction of the strain MK1-ΔaroH. MK-7 production by the strain MK1-ΔaroH was still slightly lower than that of the control strain MK1 after 120 hours’ fermentation (Figure S5c). It might be that the knockout of aroH resulted in the accumulation of chorismate and feedback inhibition on the SA pathway, thereby inhibiting the synthesis of MK-7. Deletion of pabB-pabA eliminated the biosynthesis of folic acid, which was found to have a slight effect on the MK-7 production (Figure 6a). B. subtilis uses the enterobactin precursor 2,3-dihydroxybenzoate (DHB) and its glycine derivative (DHBG) as siderophores, a low-molecular-mass iron chelators, in response to iron deprivation.51, 52 The dhbACEBF operon responsible for the synthesis of DHB from isochorismate, and isochorismate lyase (DhbB) catalyzed the first reaction, the turnover of isochorismate into (2S,3S)-2,3-dihydro-2,3-dihydroxybenzoate (DHDHB).53 Interestingly, deletion of dhbB could block the competitive biosynthesis of DHB, and drove isochorismate into the MK-7 pathway, resulting in the yield 13.4 ± 0.2 mg/L of MK-7, ~12% increase over that of the control strain (Figure 6a). Consequently, to improve the production of MK-7, the dhbB gene in the genome of strain MK3-MEP123-Gly2 was knocked out to obtain the strain MK3-MEP123-Gly2-ΔdhbB, enabling the yield of MK-7 up to 15.4 ± 0.6 mg/L (Figure 6b). Fermentation in 2-L baffled flasks The starting strain BS168NU and the final resulting recombinant strain MK3-MEP123-Gly2-ΔdhbB were fermented in 2-L baffled flasks to investigate the effect of the supply of oxygen on the cell growth and the production of MK-7. The cell growth could reach maximum after 48 hours’ fermentation in 2-L baffled flasks (Figure 7a). The yield of MK-7 produced by the strain MK3-MEP123-Gly2-ΔdhbB was 38.9 ± 2.0 mg/L after 72 hours of fermentation, which was 3.2-fold that of the strain BS168NU (12.3 ± 1.8 mg/L) (Figure 7a). These observations suggested that the supply of oxygen could accelerate the consumption of nutrients, promoting the cell growth and MK-7 synthesis. The fermentation broth at 48th hours was centrifuged at 13000 r/min for 8 minutes at 4°C. The concentrations of MK-7 in the resultant supernatant and the collected cell precipitation were measured, respectively. The MK-7 content in the supernatant of the starting strain BS168NU was 3.2 ± 0.1 mg/L, which accounted for 45% of the total MK-7 production (Figure S6a). However, the MK-7 content in the supernatant of the final strain MK3-MEP123-Gly2-ΔdhbB was 5.3 ± 0.1 mg/L and it accounted for 25% of the total MK-7 production. Although the content of MK-7 secreted extracellularly increased, the secretion ratio decreased from 45% to 25%. Previous studies showed that vitamin K2 produced by B. subtilis or B. subtilis natto was secreted extracellularly in the form of a soluble complex with a specific acidic binding factor during the culture process.54, 55 Structure analysis indicated that this vitamin K2-binding factor was an acidic glycoconjugate,55 which might limit the secretion of MK-7. The prolongation of the stationary period could favor the accumulation of metabolites. Thus, 1% glycerol and 2% soy peptone 6


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were supplemented to the fermentation broth at the 48th and the 96th hours of fermentation, which led to a prolongation of the stationary period of the cell growth of the final resulting strain MK3-MEP123-Gly2-ΔdhbB (Figure 7b). 69.5 ± 2.8 mg/L MK-7 was produced after 144 hours’ fermentation under this feed fermentation condition (Figure 7b). We also found that ~25% MK-7 was found in the supernatant at the 96th, 120th and 144th hours’ fermentation broth (Figure S6b). At this time, the feedstock cost of the entire fermentation process was only $4.85 per liter, and the final resulting strain enabled the production of MK-7 per liter for hundreds or even thousands persons per day. MK-7 is gaining adoption in the prevention and therapy of bone and vascular disease, with a growing market demand. Thus, metabolic engineering of B. subtilis for enhanced production of MK-7 would enable great economic benefits. Conclusions MK-7, as a major vitamin K2 (menaquinones), is widely used for supporting bone and cardiovascular health. MK-7 was previously produced by Bacillus subtilis natto, which was lack of clarified genome annotation and matured genomic editing tools and restricted further improvement in the MK-7 production. In this study, B. subtilis 168 was chosen as the platform microorganism for modular metabolic engineering design to enhance the yield of MK-7. We overexpressed sixteen genes and in situ replaced the native promoter of two operons in the four modules of MK-7 biosynthesis, and knocked out seven genes to block the synthesis of five metabolites. We found that the MK-7 pathway (Module I), the MEP pathway (Module III) and the glycerol metabolism pathway (Module IV) were crucial for the biosynthesis of MK-7. In the MK-7 pathway, the overexpression of menA resulted in 6.6 ± 0.1 mg/L of MK-7, which was 2.1-fold that of the starting strain BS168NU (Figure 2b). Therefore, MenA-driven transfer from heptaprenyl to DHNA was a rate-limiting step, which suggested that the supply of heptaprenyl was particularly important. Through overexpressing seven enzymes in the MEP pathway, respectively, to enhance the biosynthesis of heptaprenyl-PP, we found that Dxs, Dxr, YacM and YacN were key enzymes that improved the MK-7 production. Simultaneous overexpression of Dxs, Dxr, YacM and YacN could lead to a further increase of 82% in the MK-7 production (Figure 4d). The condensation of DHNA and heptaprenyl-PP into DMK, the direct precursor of MK-7, were found to play a crucial role in improving the biosynthesis of MK-7. Moreover, the overexpression of glpD in the glycerol metabolism pathway resulted in a ~14% increase in the MK-7 production. Furthermore, deletion of dhbB reduced the consumption of the intermediate isochorismate, which was found to promote the synthesis of MK-7 (Figure 6b). However, due to the feedback inhibition by chorismate, the increase of metabolic flux in the SA pathway (Module II) had a negative effect on the synthesis of MK-7. In all, we found that menA, dxs, dxr, yacM, yacN and glpD were the key genes that limited the MK-7 synthesis, and the knockout of dhbB could also enhance the production of MK-7. The final resulting strain MK3-MEP123-Gly2-ΔdhbB, with the overexpression of the genes menA, dxs, dxr, yacM, yacN and glpD and the knockout of dhbB, could produce 15.4 ± 0.6 mg/L after 120 hours of fermentation in 250 mL flasks, which was 5.0-fold that of the starting strain BS168NU (3.1 ± 0.2 mg/L). By adopting 2-L baffled flasks, its production of MK-7 reached 38.9 ± 2.0 mg/L after 72 hours of fermentation. Upon supplementing 2% glycerol and 4% soy peptone, the stationary period of the cell growth was extended and the yield of MK-7 reached 69.5 ± 2.8 mg/L. Development of high-density fermentation technology of B. subtilis would further enhance the yield of MK-7. Materials and methods Microorganisms, plasmids and cultivation conditions The strains and plasmids used in this study were listed in Table 1. All recombinant B. subtilis strains were derived from the laboratory stocked strain B. subtilis 168. Strains were grown at 37°C in Luria-Bertani (LB, 1% tryptone, 0.5% g/L yeast extract, and 1% NaCl) liquid medium. Solid media was obtained by adding 1.5% agar to the liquid media. Upon required, antibiotics were added to the growth media at the following concentrations: 100 μg/mL ampicillin (Amp) for the selection of Escherichia coli, 16 μg/mL neomycin (Nm) and 8 μg/mL chloramphenicol (Cm) for the selection of B. subtilis. The colonies that popped out the upp-cassette were selected on minimal medium (MM)56 plate containing 10 μM 5-fluorouracil (5-FU was purchased 7



Page 8 of 26

from Sigma-Aldrich Corporation (Sigma-Aldrich, USA)). These recombinant B. subtilis strains were cultured in test tubes with 5 mL LB broth at 37°C for 14 h with shaking at 200 rpm. 300 μL cells cultured in exponential growth phase were inoculated into 250 mL flasks with 30 mL fresh fermentation culture media (3% (v/v) glycero, 6% soy peptone, 0.5% yeast extract, 0.3% K2HPO4, 0.05% MgSO4·7H2O, pH 7.3) and incubated at 37oC for 144 h at 200 rpm. All fermentations were done in the absence of light to avoid the degradation of MK-7. The biomass of the recombinant strains over the course of the experiment was detected using a spectrophotometer at 600 nm after an appropriate dilution. The fermentation liquor at 96th and 120th hours were used for the high performance liquid chromatograph (HPLC) analysis to determine the concentration of MK-7. Measuring the fermentation broth at two time points was to further verify the reliability of the results. DNA manipulation techniques The transformation of E. coli was performed according to the standard procedures.57 Plasmid DNA was extracted from E. coli using the TIANprep Mini Plasmid Kit (TIANGEN, Beijing, China). The restriction enzymes and the T4 DNA ligase (Fermentas) were used as recommended by the manufacturer.57 The transformation of B. subtilis was performed by using the competent cells, as described by Anagnostopoulos and Spizizen.58 Genome DNA was extracted from B. subtilis using the TIANamp Bacteria DNA Kit (TIANGEN). DNA polymerases (TransFast Taq DNA Polymerase and TransTaq DNA Polymerase High Fidelity, TransGen Biotech) were used as recommended by the manufacturers.57 Overlap extension by polymerase chain reaction (OE-PCR) was carried out as described.59,

60

Mutated DNA fragments are generated and ligated

through the overlapping ends, in which the overlapping ends anneal, allowing the 3’ overlap of each strand to serve as the primer for the extension of the complementary strand. The primers were synthetized, and the DNAs were sequenced by Genewiz Biotech (Suzhou, China). All primers used in this study were listed in Table S1 (Supporting Information, SI). The fragments containing promoter and the heterologous gene were synthetized by Genscript Biotech (Nanjing, China). The method of marker-free gene modification was derived from Liu et al. (61, 62), which was used here for the gene overexpression and knockout. Under the control of the promoter of the arabinose operon (ara), the expression of the Nm-resistance gene (neo) was repressed by the transcriptional repressor AraR. Firstly, the chromosomal araR locus was replaced with the counter-selective marker cassette (Para-neo) by a double-crossover homologous recombination method, and the colonies were selected on a Nm-resistance plate. Secondly, the selective marker cassette (CR) containing the Cm-resistance gene (cat) and the araR gene was integrated into the upstream region of the target locus by a double-crossover event (upstream recombination fragment U and downstream recombination fragment G) and the colonies were selected on a Cm-resistance plate. Finally, the eviction of the selective marker cassette together with the target locus was achieved by an intra-genomic single-crossover event (homologous recombination fragment D) and the colonies were selected on a Nm-resistance plate. There was another markerless mutation delivery system in B. subtilis stimulated by a double-strand break in the chromosome,63 which was also used here to in situ replacement of the promoter, especially for essential genes. Firstly, the chromosomal upp gene, which encoded the uracil phosphoribosyltransferase, was knocked out. Secondly, the upstream and downstream sequences of the promoter to be replaced and new promoter sequence were ligated to the integrated plasmid pSS by enzyme digestion, obtaining pSS’, which was then integrated into the target locus by a double-crossover event (upstream recombination fragment U and downstream recombination fragment D) and a positive selection for the Cm-resistance. Finally, the eviction of the upp-cassette was achieved by exogenous endonuclease I-SceI and a positive selection for the 5-FU resistance. B. subtilis strains containing the upp gene could not grow in the minimal medium containing 10 μM 5-FU, however, the upp-deficient B. subtilis strain could. Construction of the starting strain BS168NU In order to carry out the markerless gene modification for B. subtilis 168, we constructed the starting strain with the Para-neo and the defect of Upp. Firstly, we constructed and integrated the counter-selective marker cassette Para-neo in the chromosome. The 1.000 kb UaraR, 0.210 kb Para and 1.002 kb DaraR fragments were amplified from the B. subtilis genome using the primers 8


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araR-neo-U1/araR-neo-U2q, Para1/Para2 and araR-neo-D1q/araR-neo-D2, respectively. The 0.781 kb neo fragment was amplified from plasmid pUB110 by using the primers araR-neo-1q/araR-neo-2. These four PCR fragments were then ligated in the order of U-P-neo-D by splicing by OE-PCR using the primers araR-neo-U1/araR-neo-D2. Then, this UPneoD fragment was used to transform into the competent cells of B. subtilis 168 to obtain the strain BS168N. The DNA sequencing was performed by using the primers CX-neo1/CX-neo2. Secondly, we defected the native upp gene on the chromosome. The 1.195 kb Uupp, 1.168 kb Rupp, 0.845 kb Dupp and 0.507 kb Gupp fragments were amplified from the B. subtilis genome using the primers upp-U1/upp-U2q, upp-R1q/CR2, upp-D1/upp-D2 and upp-G1q/upp-G2, respectively. The 0.901 kb cat fragment was amplified from plasmid pC194 by using the primers upp-CR1q/upp-C2. These five PCR fragments were then ligated in the order U-D-C-R-G by splicing by OE-PCR using the primers upp-U1/upp-G2. Then, this UDCRG fragment was used to transform into the competent cells of BS168N to obtain the strain BS168NU. In situ replacement of the promoter in the B. subtilis chromosome The promoter of menFDHBEC operon was in situ replaced by the constitutive promoter PlapS. The specific method was described as below. The U fragment, amplified from B. subtilis 168 with primers men-U1m/men-U2qm, was inserted into pSS vector63 using the BglII and XhoI restriction locus, yielding the recombinant plasmid pSS-men-U. The P (promoter PlapS) fragment, synthesized by Genscript Biotech and ligated to plasmid pUC57-1.8k-P1, was amplified from pUC57-1.8k-P1 by using the primers PlapS1/PlapS2. The D fragment was amplified from B. subtilis 168 with primers men-D1q/men-D2. These two PCR fragments were ligated in the order of P-D by splicing by OE-PCR using the primers PlapS1qm/men-D2m, and then was inserted into the pSS-men-U vector using the BamHI and KpnI restriction locus, yielding the recombinant plasmid pSS-men-UPD. Finally, this recombinant plasmid was used to transform into the competent cells of B. subtilis and the recombinant strain MK1 was obtained by two-step screening. DNA sequencing was done at Genewiz Biotech by using primer CX-men. In situ replacement of the promoter of the hepS-menG-hepT operon was similar to that of the menFDHBEC operon. Gene overexpression The selection of the overexpression locus of all genes on the chromosome was based on the study of Bacillus minimum genome. Genome engineering revealed large dispensable regions in B. subtilis, and the removal of dispensable genomic regions showed that this genome minimization affected neither cell viability nor the key physiological and developmental processes of B. subtilis.64, 65 The specific method of gene overexpression was derived from Liu et al.61, 62 For example, the modified menA gene fragment was constructed as follows. The 1.115 kb UmenA, 1.057 kb AmenA, 1.053 kb DmenA and 0.897 kb GmenA fragments were

amplified

from

the

B.

subtilis

168

genome

using

the

primers

yxlA-menA-U1/yxlA-menA-U2q,

yxlA-menA-1q/yxlA-menA-2, yxlA-menA-D1q/yxlA-menA-D2 and yxlA-menA-G1q/yxlA-menA-G2, respectively. The 0.442 kb

P

(promoter

PlapS)

fragment

was

amplified

from

plasmid

pUC57-1.8k-P1

by

using

the

primers

yxlA-menA-P1/yxlA-menA-P2. The 2.069 kb CR (cat-araR) fragment was amplified from BS168NUm by using the primers yxlA-menA-CR1q/CR2. These six PCR fragments were then ligated in the order of U-P-A-D-CR-G by splicing by OE-PCR using the primers yxlA-menA-U1/yxlA-menA-G2. Finally, this UPADCRG fragment was used to transform the competent cells of BS168NU. And the recombinant strain MK3 was obtained by the two-step screening process, as mentioned in Section DNA

manipulation

techniques.

The

subsequent

DNA

sequencing

was

performed

by

using

the

primers

yxlA-menA-P1/yxlA-menA-2. The overexpression of other genes was similar to that of menA. Gene knockout The method of gene knockout was similar to that of gene overexpressing,61, 62 for example, the modified ldh gene fragment was constructed as follows. The 1.132 kb Uldh, 1.009 kb Dldh and 0.663 kb Gldh fragments were amplified from the B. subtilis 168 genome using the primers ldh-U1/ldh-U2, ldh-D1q/ldh-D2 and ldh-G1q/ldh-G2, respectively. The 2.069 kb CR fragment was 9



Page 10 of 26

amplified from BS168NUm by using the primers ldh-CR1q/CR2. These four PCR fragments were then ligated in the order U-D-CR-G by splicing by OE-PCR using the primers ldh-U1/ldh-G2. Finally, this UDCRG fragment was used to transform the competent cells of MK3-MEP123 and the strain MK3-MEP123-Δldh was obtained by two-step screening. The knockout method of other genes was similar to that of ldh. MK-7 extraction and HPLC analysis MK-7 was extracted from the fermentation media by using a mixture of 2-propanol and n-hexane with the ratio of 3/4/8 (fermentation liquor/2-propanol/n-hexane, v/v/v). In each experiment after the addition of organic solution, the sample was vigorously shaken with a vortex mixer for 2 minutes, and then centrifuged at 3000 r/min for 10 minutes. The organic layer was then collected from the aqueous layer and evaporated under vacuum to recover the extracted MK-7. We then used methanol to dissolve MK-7. The concentrations of MK-7 in methanol were analyzed by HPLC using a Waters 2695 HPLC system composed of an autosampler and an UV-detector. The separation was achieved on a Symmetry® C18 column (4.6 ×250 mm, 5.0 μm) (Waters, USA). The column temperature was 50°C. The mobile phase was methanol with a flow rate of 1.0 mL/min. The absorption of UV light was monitored at the wavelengths of 270 nm. The standard sample of MK-7 was purchased from ChromaDex Co. (USA). The concentration of glycerol in the culture was analyzed by HPLC using a Waters 2695 HPLC system composed of an autosampler and a refractive index detector. The samples were separated on an Aminex® HPX-87H column (Bio-Rad, USA) at 65°C, using 5 mM H2SO4 as the mobile phase with a flow rate of 0.6 mL/min. Quantitative RT-PCR analysis The total RNA of B. subtilis was extracted with RNAprep Pure Cell/Bacteria Kit (TIANGEN) as recommended by the supplier and was reverse-transcribed in cDNA using the FastQuant RT Kit (with gDNase) (TIANGEN) according to the manufacturer’s instructions. The qRT-PCR was carried out by LightCycler 480 (Roche Diagnostics GmbH, Mannheim, Germany) using the SuperReal PreMix Plus (SYBR Green) (TIANGEN). The ccpA gene served as the internal control of the qRT-PCR. The relative transcriptional levels were calculated according to Pfaffl.66 Microbial fermentation in 2-L flasks The B. subtilis strain was firstly cultured in the test tube with 5 mL LB broth at 37°C for 14 h with shaking at 200 rpm. 100 μL cells were then inoculated into 500 mL flasks with 100 mL fresh fermentation culture media and incubated at 37°C for 12 h at 200 rpm. At last, 25 mL cells were inoculated into 2-L baffled flasks with 225 mL fresh fermentation culture media and incubated at 37 °C for 144 h at 200 rpm. All fermentations were performed in the absence of light to avoid the degradation of MK-7. At the 48th hour and 96th hour during the feed-batch fermentation process, 1% (v/v) glycerol and 2% (w/v) soy peptone were added simultaneously.

ASSOCIATED CONTENT Supporting Information Table shows primers used in this study. Figures show the growth of the starting strain and all the recombination strains, the relative transcriptional levels of gene overexpressed, glycerol consumption of some recombinant strains, secretion and intracellular accumulation of MK-7 produced by B. subtilis. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. 10


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Author Contributions S.Y. and H.S. designed the experiments; S.Y. performed the experiments; Y.C. helped in revising the manuscript; Y.C., L.S., C.L., X.L., Z.C., G.Z. helped with some experiments; and S.Y. and H.S. wrote the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by Chifeng Pharmaceutical Company Limited, the Innovation and Development Project of Oceanic Economics (Project number: BHSF2017-06) funded by the State Oceanic Administration and Binhai New District of Tianjin, and the National Young Overseas High-Level Talents Introduction Plan.

ABBREVIATIONS Gly, glycerol; Gly-3P, glycerol-3-phosphate; DHAP, dihydroxyacetone phosphate; 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; DXP, 1-deoxyxylulose-5-phosphate; MEP, methyl-erythritol-4-diphosphate; CDP-ME, 4-(cytidine 5'-diphospho)-2-C-methylerythritol; CDP-MEP, 2-phospho-4-(cytidine 5'-diphospho)-2-C-methylerythritol; MEC, 2-C-Methylerythritol 2,4-cyclodiphosphate; HMBPP, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate; DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GPP, Geranyl diphosphate; FPP, farnesyl diphosphate; Hepta-PP, heptaprenyl diphosphate; CHA, chorismate;

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; PPA, prephenate;

PABA,

para-aminobenzoic

acid;

ADC,

4-amino-4-deoxychorismate;

DHDHB,

(2S,3S)-2,3-dihydro-2,3-dihydroxybenzoate.

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25. Kong, M. K., and Lee, P. C. (2011) Metabolic engineering of menaquinone-8 pathway of Escherichia coli as a microbial platform for vitamin K production. Biotechnol. Bioeng. 108, 1997-2002. 26. Meganathan, R. (2001) Biosynthesis of menaquinone (vitamin K2) and ubiquinone (coenzyme Q): a perspective on enzymatic mechanisms. Vitam. Horm. (London, U. K.) 61, 173-218. 27. Jiang, M., and Zhang, H. (2016) Engineering the shikimate pathway for biosynthesis of molecules with pharmaceutical activities in E. coli. Curr. Opin. Biotechnol. 42, 1-6. 28. Krämer, M., Bongaerts, J., Bovenberg, R., Kremer, S., Müller, U., Orf, S., Wubbolts, M., and Raeven, L. (2003) Metabolic engineering for microbial production of shikimic acid. Metab. Eng. 5, 277-283. 29. Herrmann, K. M., and Weaver, L. M. (1999) The shikimate pathway. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 473-503. 30. Davies, W., and Davidson, B. (1982) The nucleotide sequence of aroG, the gene for 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase (phe) in Escherichia coli K12. Nucleic Acids Res. 10, 4045-4058. 31. Zurawski, G., Gunsalus, R. P., Brown, K. D., and Yanofsky, C. (1981) Structure and regulation of aroH, the structural gene for the tryptophan-repressible 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase of Escherichia coli. J. Mol. Biol. 145, 47-73. 32. Oldiges, M., Kunze, M., Degenring, D., Sprenger, G. A., and Takors, R. (2010) Stimulation, monitoring, and analysis of pathway dynamics by metabolic profiling in the aromatic amino acid pathway. Biotechnol. Prog. 20, 1623-1633. 33. Lutke-Eversloh, T., and Stephanopoulos, G. (2008) Combinatorial pathway analysis for improved L-tyrosine production in Escherichia coli: identification of enzymatic bottlenecks by systematic gene overexpression. Metab. Eng. 10, 69-77. 34. Liu, D. F., Ai, G. M., Zheng, Q. X., Liu, C., Jiang, C. Y., Liu, L. X., Zhang, B., Liu, Y. M., Yang, C., and Liu, S. J. (2014) Metabolic flux responses to genetic modification for shikimic acid production by Bacillus subtilis strains. Microb. Cell Fact. 13, 1-11. 35. Wagner, W. P., Helmig, D., and Fall, R. (2000) Isoprene biosynthesis in Bacillus subtilis via the methylerythritol phosphate pathway. J. Nat. Prod. 63, 37-40. 36. Eisenreich, W., Bacher, A., Arigoni, D., and Rohdich, F. (2004) Biosynthesis of isoprenoids via the non-mevalonate pathway. Cell. Mol. Life Sci. 61, 1401-1426. 37. Xue, J., and Ahring, B. K. (2011) Enhancing isoprene production by genetic modification of the 1-deoxy-d-xylulose-5-phosphate pathway in Bacillus subtilis. Appl. Environ. Microbiol. 77, 2399-2405. 38. Julsing, M. K., Rijpkema, M., Woerdenbag, H. J., Quax, W. J., and Kayser, O. (2007) Functional analysis of genes involved in biosynthesis of isoprene in Bacillus subtilis. Appl. Microbiol. Biotechnol. 75, 1377-1384. 39. Hess, B. M., Xue, J., Markillie, L. M., Taylor, R. C., Wiley, H. S., Ahring, B. K., and Linggi, B. (2013) Coregulation of terpenoid pathway genes and prediction of isoprene production in Bacillus subtilis using transcriptomics. PLoS One 8, e66104. 40. Wang, J. P., Yeh, C. M., and Tsai, Y. C. (2006) Improved subtilisin YaB production in Bacillus subtilis using engineered synthetic expression control sequences. J. Agric. Food Chem. 54, 9405-9410. 41. Willenbacher, J., Mohr, T., Henkel, M., Gebhard, S., Mascher, T., Syldatk, C., and Hausmann, R. (2016) Substitution of the native srfA promoter by constitutive Pveg in two B. subtilis strains and evaluation of the effect on Surfactin production. J. Biotechnol. 224, 14-17.

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42. Jin, Y., Liu, Z., Li, Y., Liu, W., Tao, Y., and Wang, G. (2016) A structural and functional study on the 2-C-methyl-d-erythritol-4-phosphate cytidyltransferase (IspD) from Bacillus subtilis. Sci. Rep. 6, 1-11. 43. Liu, Z., Jin, Y., Liu, W., Tao, Y., and Wang, G. (2018) Crystal structure of IspF from Bacillus subtilis and absence of protein complex assembly among IspD/IspE/IspF enzymes in the MEP pathway. Biosci. Rep. 38, 1-14. 44. Yeh, J. I., Kettering, R., Saxl, R., Bourand, A., Darbon, E., Joly, N., Briozzo, P., and Deutscher, J. (2009) Structural characterizations of glycerol kinase: unraveling phosphorylation-induced long-range activation. Biochemistry 48, 346-356. 45. Holmberg, C., Beijer, L., Rutberg, B., and Rutberg, L. (1990) Glycerol catabolism in Bacillus subtilis: nucleotide sequence of the genes encoding glycerol kinase (glpK) and glycerol-3-phosphate dehydrogenase (glpD). J. Gen. Microbiol. 136, 2367-2375. 46. Beijer, L., Nilsson, R. P., Holmberg, C., and Rutberg, L. (1993) The glpP and glpF genes of the glycerol regulon in Bacillus subtilis. J. Gen. Microbiol. 139, 349-359. 47. Glatz, E., Persson, M., and Rutberg, B. (1998) Antiterminator protein GlpP of Bacillus subtilis binds to glpD leader mRNA. Microbiology 144, 449-456. 48. Darbon, E., Servant, P., Poncet, S., and Deutscher, J. (2002) Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P~GlpK dephosphorylation control Bacillus subtilis glpFK expression. Mol. Microbiol. 43, 1039-1052. 49. Larsson, J. T., Rogstam, A., and Von, W. C. (2005) Coordinated patterns of cytochrome bd and lactate dehydrogenase expression in Bacillus subtilis. Microbiology (London, U. K.) 151, 3323-3335. 50. Renna, M. C., Najimudin, N., Winik, L. R., and Zahler, S. A. (1993) Regulation of the Bacillus subtilis alsS, alsD, and alsR genes involved in post-exponential-phase production of acetoin. J. Bacteriol. 175, 3863-3875. 51. Pi, H., and Helmann, J. D. (2017) Sequential induction of Fur-regulated genes in response to iron limitation in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 114, 12785-12790. 52. Rowland, B. M., Grossman, T. H., Osburne, M. S., and Taber, H. W. (1996) Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes. Gene 178, 119-123. 53. May, J. J., Wendrich, T. M., and Marahiel, M. A. (2001) The dhb operon of Bacillus subtilis encodes the biosynthetic template for the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin. J. Biol. Chem. 276, 7209-7217. 54. Yanagisawa, Y., and Sumi, H. (2005) Natto Bacillus contains a large amount of water-soluble vitamin K (menaquinone-7). J. Food Biochem. 29, 267-277. 55. Ikeda, H., and Doi, Y. (1990) A vitamin-K2-binding factor secreted from Bacillus subtilis. FEBS J. 192, 219-224. 56. Fabret, C., Ehrlich, S. D., and Noirot, P. (2010) A new mutation delivery system for genome-scale approaches in Bacillus subtilis. Mol. Microbiol. 46, 25-36. 57. Sambrook, J. F., and Russell, D. W. (2001) Molecular cloning: a laboratory manual (3rd edition). Immunology 49, 895-909. 58. Anagnostopoulos, C., and Spizizen, J. (1961) Requirements for transformation in Bacillus subtilis. J. Bacteriol. 81, 741-746. 59. Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51-59.

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60. Shevchuk, N. A., Bryksin, A. V., Nusinovich, Y. A., Cabello, F. C., Sutherland, M., and Ladisch, S. (2004) Construction of long DNA molecules using long PCR-based fusion of several fragments simultaneously. Nucleic Acids Res. 32, e19. 61. Liu, S., Endo, K., Ara, K., Ozaki, K., and Ogasawara, N. (2008) Introduction of marker-free deletions in Bacillus subtilis using the AraR repressor and the ara promoter. Microbiology (London, U. K.) 154, 2562-2570. 62. Dong, H., and Zhang, D. (2014) Current development in genetic engineering strategies of Bacillus species. Microb. Cell Fact. 13, 63. 63. Shi, T., Wang, G., Wang, Z., Fu, J., Chen, T., and Zhao, X. (2013) Establishment of a markerless mutation delivery system in Bacillus subtilis stimulated by a double-strand break in the chromosome. PLoS One 8, e81370. 64. Westers, H., Dorenbos, R., van Dijl, J. M., Kabel, J., Flanagan, T., Devine, K. M., Jude, F., Seror, S. J., Beekman, A. C., and Darmon, E. (2003) Genome engineering reveals large dispensable regions in Bacillus subtilis. Mol. Biol. Evol. 20, 2076-2090. 65. Ara, K., Ozaki, K., Nakamura, K., Yamane, K., Sekiguchi, J., and Ogasawara, N. (2011) Bacillus minimum genome factory: effective utilization of microbial genome information. Biotechnol. Appl. Biochem. 46, 169-178. 66. Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, e45.

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Table 1. Strains and plasmids used in this study. Name

Sources

Relevant genotype

reference

Strains Laboratory

B. subtilis 168

trpC2

BS168NU

trpC2, ΔaraR::neoR, Δupp

This work

BS168NUm

trpC2, ΔaraR::neoR, Δupp::cat-araR

This work

MK1

BS168NU, PlapS-menFDHBEC

This work

MK2

BS168NU, ΔpksL::PlapS-menI

This work

MK3

BS168NU, ΔyxlA::PlapS-menA

This work

MK4

BS168NU, PlapS-hepS-menG-hepT

This work

MK34

BS168NU, ΔyxlA::PlapS-menA, PlapS-hepS-menG-hepT

This work

BS168NU-yqiD

trpC2, ΔaraR::neoR, Δupp, ΔpksJ::P43-yqiD

This work

MK4-yqiD

BS168NU, PlapS-hepS-menG-hepT, ΔpksJ::P43-yqiD

This work

MK1-ΔaroH

BS168NU, PlapS-menFDHBEC, ΔaroH

This work

MK3-SA1

BS168NU, ΔyxlA::PlapS-menA, ΔxlyB::PAE-aroA

This work

MK3-SA2

BS168NU, ΔyxlA::PlapS-menA, ΔpksF::PAE-aroD

This work

MK3-SA3

BS168NU, ΔyxlA::PlapS-menA, ΔyokG::PAE-aroK

This work

MK3-SA4

BS168NU, ΔyxlA::PlapS-menA, Δhom::PAE-aroE

This work

MK3-MEP1

BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::P43-dxs

This work

MK3-PAE-dxs

BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::PAE-dxs

This work

MK3-Pveg-dxs

BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::Pveg-dxs

This work

MK3-PlapS-dxs

BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::PlapS-dxs

This work

MK3-MEP2

BS168NU, ΔyxlA::PlapS-menA, ΔydeO::P43-dxr

This work

MK3-MEP3

BS168NU, ΔyxlA::PlapS-menA, ΔyqaL::P43-yacM-yacN

This work

MK3-MEP4

BS168NU, ΔyxlA::PlapS-menA, ΔyqaQ::P43-ispE

This work

stock

16


or

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


MK3-MEP5

BS168NU, ΔyxlA::PlapS-menA, ΔpksJ::P43-yqfP

This work

MK3-MEP6

BS168NU, ΔyxlA::PlapS-menA, ΔpksJ::P43-yqiD

This work

BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::P43-dxs,

MK3-MEP12

ΔydeO::P43-dxr BS168NU, ΔyxlA::PlapS-menA, ΔyjoB::P43-dxs,

MK3-MEP123

ΔydeO::P43-dxr, ΔyqaL::P43-yacM-yacN

This work

This work

MK3-MEP123-Gly1

MK3-MEP123, ΔpksJ::P43-glpF-glpK

This work

MK3-MEP123-Gly2

MK3-MEP123, ΔpksJ::P43-glpD

This work

MK3-MEP123-Gly3

MK3-MEP123, ΔpksL::P43-tpiA

This work

MK3-MEP123-Δldh

MK3-MEP123, Δldh

This work

MK3-MEP123-ΔalsSD

MK3-MEP123, ΔalsS-alsD

This work

MK3-MEP123-ΔaroH

MK3-MEP123, ΔaroH

This work

MK3-MEP123-ΔpabBA

MK3-MEP123, ΔpabB-pabA

This work

MK3-MEP123-ΔdhbB

MK3-MEP123, ΔdhbB

This work

MK3-MEP123-Gly2-ΔdhbB

MK3-MEP123, ΔpksJ::P43-glpD, ΔdhbB

This work

Plasmids Laboratory

pUB110

NmR

pC194

CmR

pSS

Integrate plasmid, AmpR, CmR

pSS-men-UPD

pSS-SGT-UPD

stock Laboratory stock (63)

AmpR, CmR, Umen-upp cassette-PlapS-Dmen, used for the in situ replacement of the native promoter of the menFDHBEC operon AmpR, CmR, USGT-upp cassette-PlapS-DSGT, used for the in situ replacement of the native promoter of the hepS-menG-hepT operon

This work

This work

pUC57-1.8k-P1

AmpR, containing promoter PlapS, synthetized by Genscript Biotech

This work

pUC57-1.8k-P2

AmpR, containing promoter PAE, synthetized by Genscript Biotech

This work

pUC57-1.8k-P3

AmpR, containing promoter P43, synthetized by Genscript Biotech

This work

17



pUC57-1.8k-menI

AmpR, containing the menI gene from E. coli, synthetized by Genscript Biotech

NmR, neomycin resistance; CmR, chloramphenicol resistance; AmpR, ampicillin resistance.

18


Page 18 of 26

This work

Page 19 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. The de novo biosynthesis pathway from glycerol to MK-7 in B. subtilis 168, which was categorized into four

modules

I-IV.

Enzymes:

Module

I:

MenF,

isochorismate

2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate

synthase;

synthase;

MenD, 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 of E. coli; MenA, 1,4-dihydroxy-2-naphthoate heptaprenyltransferase; MenG, demethylmenaquinone methyltransferase; HepS/HepT, heptaprenyl diphosphate synthase component I/II. Module II: AroA, 3-deoxy-7-phosphoheptulonate synthase; AroB, 3-dehydroquinate synthase; AroC, 3-dehydroquinate 3-phosphoshikimate

dehydratase;

AroD,

shikimate

1-carboxyvinyltransferase;

1-deoxyxylulose-5-phosphate

synthase;

Dxr,

dehydrogenase;

AroF,

chorismate

AroK,

shikimate

synthase.

1-deoxyxylulose-5-phosphate

kinase;

Module

III:

reductoisomerase;

AroE, Dxs, YacM,

2-C-methylerythritol 4-phosphate cytidylyltransferase; IspE, 4-diphosphocytidyl-2-C-methylerythritol kinase; YacN, 2-C-methylerythritol 2,4-cyclodiphosphate synthase; YgfY, 4-hydroxy-3-methylbut-2-enyl diphosphate synthase;

YqfP,

4-hydroxy-3-methylbut-2-enyl

diphosphate

reductase;

YpgA,

isopentenyl-diphosphate

δ-isomerase; YqiD, farnesyl diphosphate synthase. Module IV: GlpF, glycerol uptake facilitator; GlpK, glycerol kinase; GlpD, glycerol-3-phosphate dehydrogenase; Tpi, triosephosphate isomerase. Other related pathways: 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.

19



Figure 2. The native promoter of the menFDHBEC operon and the hepS-menG-hepT operon in the genome of the BS168NU strain were replaced in situ by the PlapS promoter, respectively, achieving the recombinant strains MK1 and MK4. The menI gene from E. coli and the native menA gene from B. subtilis were overexpressed by inserting into the pksL and yxlA locus in the genome of BS168NU under the control of PlapS, respectively, obtaining the strains MK2 and MK3. Finally, the strain MK34 was obtained by the co-overexpression of menA and hepS-menG-hepT. (a)-(c) Schematic of the gene overexpression in the genome, denoting the corresponding strains. (b)-(d) MK-7 production by the starting strain and the recombinant strains from Module I after 96 and 120 hours’ fermentation. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

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Figure 3. The genes aroA, aroD, aroK and aroE of the SA pathway (Module II) were overexpressed at the xlyB, pksF, yokG and hom locus in the genome of MK3 with the PAE promoter, respectively, achieving the strains MK3-SA1, MK3-SA2, MK3-SA3 and MK3-SA4. (a) Schematic of the gene overexpression in the genome of MK3, denoting the corresponding strains. (b) MK-7 production by the control strain and the recombinant strains from Module II after 96 and 120 hours’ fermentation. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

21



Figure 4. By overexpressing the genes dxs, dxr, yacM-yacN, ispE, yqfP and yqiD of the MEP pathway (Module III) at the yjoB, ydeO, yqaL, yqaQ and pksJ locus in the genome of MK3 using the P43 promoter, respectively, leading to the recombinant strains MK3-MEP1, MK3-MEP2, MK3-MEP3, MK3-MEP4, MK3-MEP5 and MK3-MEP6. The strain MK3-MEP12 was achieved by co-overexpressing dxs and dxr, and the strain MK3-MEP123 was achieved by overexpressing dxs, dxr and yacM-yacN simultaneously. (a)-(c) Schematic of the gene overexpression in the genome of MK3, denoting the corresponding strains. (b)-(d) MK-7 production by the control strain and the recombinant strains from Module III after 96 and 120 hours’ fermentation. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

22


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Figure 5. The operon glpF-glpK, the genes glpD and tpi of the glycerol metabolism pathway (Module IV) were overexpressed by inserting into the pksJ and pksL locus in the genome of MK3-MEP123, respectively, which led to the strains MK3-MEP123-Gly1, MK3-MEP123-Gly2, and MK3-MEP123-Gly3. (a) Schematic of genes overexpressed in the genome of MK3-MEP123, denoting the corresponding strains. (b) MK-7 production by the control strain and the recombinant strains from Module IV after 96 and 120 hours’ fermentation. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

23



Figure 6. By knocking out the genes ldh, alsS-alsD, aroH, pabB-pabA and dhbB in the genome of the strain MK3-MEP123, the corresponding strains MK3-MEP123-Δldh, MK3-MEP123-ΔalsSD, MK3-MEP123-ΔaroH, MK3-MEP123-ΔpabBA and MK3-MEP123-ΔdhbB were achieved, respectively. Deletion of dhbB in the genome of MK3-MEP123-Gly2 led to the strain MK3-MEP123-Gly2-ΔdhbB. (a) MK-7 production of the control strain and the corresponding knockout strains from MK3-MEP123 after 96 and 120 hours’ fermentation. (b) MK-7 production by the strain MK3-MEP123-Gly2-ΔdhbB. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

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Figure 7. Fermentation in 2-L baffled flasks. (a) The growth curves of the starting strain BS168NU (▲) and the final resulting strain MK3-MEP123-Gly2-ΔdhbB (■), and the MK-7 production by the strains BS168NU (▲) and MK3-MEP123-Gly2-ΔdhbB (■) in 2-L flasks. The fermentation conditions were the same as that in the 250 mL flasks. (b) The growth curve (■) and the MK-7 production (■) of the strain MK3-MEP123-Gly2-ΔdhbB. 1% glycerol and 2% soy peptone were supplemented to the fermentation medium at the 48th and 96th hours during fermentation, respectively. Results were represented by the mean values of three replicates and the error bar by the standard deviation.

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Insert Table of Contents Graphic and Synopsis Here

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Modular pathway engineering of Bacillus subtilis to promote de novo

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