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Cite This: ACS Synth. Biol. XXXX, XXX, XXX−XXX

Metabolic Engineering of the MEP Pathway in Bacillus subtilis for Increased Biosynthesis of Menaquinone‑7 Yanwei Ma,† Dale D. McClure,† Mark V. Somerville,§ Nicholas W. Proschogo,‡ Fariba Dehghani,† John M. Kavanagh,† and Nicholas V. Coleman*,§ †

School of Chemical and Biomolecular Engineering, The University of Sydney, Sydney, NSW 2006, Australia School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia § School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW 2006, Australia Downloaded via GUILFORD COLG on July 19, 2019 at 01:20:53 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Vitamin K is essential for blood coagulation and plays important roles in bone and cardiovascular health. Menaquinone-7 (MK-7) is one form of vitamin K that is especially useful due to its long half-life in the circulation. MK-7 is difficult to make via organic synthesis, and is thus commonly produced by fermentation. This study aimed to genetically modify Bacillus subtilis cultures to increase their MK-7 yield and reduce production costs. We constructed 12 different strains of B. subtilis 168 by overexpressing different combinations of the rate-limiting enzymes Dxs, Dxr, Idi, and MenA. We observed an 11-fold enhancement of production in the best-performing strain, resulting in 50 mg/L MK-7. Metabolite analysis revealed new bottlenecks in the pathway at IspG and IspH, which suggest avenues for further optimization. This work highlights the usefulness of Bacillus subtilis for industrial production of high value compounds. KEYWORDS: vitamin K, menaquinone-7, Bacillus subtilis, metabolic engineering, MEP pathway, biofilm formation

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it is for this reason a range of authors have examined MK-7 production using wild-type Bacillus subtilis natto, Bacillus licheniformis, and Bacillus amyloliquefaciens strains.15,16 Bacillus subtilis has a Generally Recognized as Safe (GRAS) status, it grows faster than eukaryotic hosts such as Saccharomyces cerevisiae, its genetics are well-characterized, specialized gene/protein/pathway/deletion libraries and databases have been established,17−19 a range of synthetic biology tools are available,20−22 and it is naturally competent for DNA uptake. Bacillus subtilis has been successfully used as a microbial cell factory for the production of molecules like poly-γ-glutamic acid,23 hyaluronic acid,24,25 surfactin,26 and Nacetyl-glucosamine.27 Therefore, there is considerable potential for the metabolic engineering of Bacillus subtilis to further improve MK-7 production.28,29 Due to the lack of efficient genetic tools to manipulate wild-type B. subtilis natto strains,30,31 B. subtilis 168 was chosen for metabolic engineering to enhance MK-7 production in this study. The biosynthesis of MK-7 is complex (Figure 1), requiring the methylerythritol phosphate (MEP) pathway (to assemble the C35 isoprenoid tail), the shikimic acid (SA) pathway (to supply the aromatic head),32 and the menaquinone pathway (to make the final product). The MEP pathway (also known as the isoprenoid pathway) is believed to be rate limiting for MK-

enaquinones are a class of compounds that are widely found in microorganisms where they play an important role in electron transport, oxidative phosphorylation, and sporulation.1−3 The menaquinones share a common chemical structure, characterized by a naphthoquinone “head” group and an isoprenoid “tail”. The length of this tail varies depending on the microorganism and is often expressed as MK-n; where n is the number of isoprenoid units in the tail (Figure 1).4 In humans, the menaquinones are known as Vitamin K2; they are essential cofactors involved in the carboxylation of glutamate residues of vitamin K dependent proteins. Menaquinones play an essential role in blood coagulation and are important for preventing osteoporosis,5 mitigating cardiovascular calcification,6 reducing cognitive diseases,7 suppressing inflammation,8 and preventing diabetes.9 Menaquinone-7 (MK-7) has a half-life of up to 72 h in the circulation,10 while that of MK-4 and vitamin K1 is only 1−2 h;11 this gives MK-7 particular advantages compared to other members of the vitamin K family. There is growing interest in using MK-7 as a supplement for the prevention and treatment of osteoporosis and atherosclerosis.12 The cost of MK-7 production is high ($200−$300 per gram).13 Chemical synthesis of MK-7 is challenging due to the need to stereoselectively synthesize the bioactive all-trans configuration of the molecule.14 Microbial production of MK-7 has the advantage of selectively producing the all-trans isomer; © XXXX American Chemical Society

Received: March 3, 2019 Published: June 19, 2019 A

DOI: 10.1021/acssynbio.9b00077 ACS Synth. Biol. XXXX, XXX, XXX−XXX

Research Article

ACS Synthetic Biology

Figure 1. Menaquinone-7 biosynthesis pathway in B. subtilis.

7 production;33 this pathway has been intensively studied for production of hemiterpenes (isoprene and isopentenol), monoterpenes (limonene), sesquiterpenes (farnesene, bisabolene and amorphadiene), diterpenes (taxadiene), carotenoids, ubiquinone, and menaquinone.16,34−41 The enzymes Dxs and Dxr have been consistently identified as rate limiting enzymes in the MEP pathway,42,43 while there is evidence both for and against the Idi enzyme being rate-limiting.39,44 The enzyme UbiA/MenA, which joins the isoprenoid tail with the aromatic head, appears to be rate limiting for the downstream pathway of MK-7 synthesis.16,33,45,46 The first aim of this study was to improve MK-7 production in B. subtilis, using a variety of synthetic gene clusters with different gene contents and gene arrangements (Figure 2). The second aim was to examine the metabolite profile of the resultant recombinants to identify pathway bottlenecks for future iterations of the design-construction-evaluation-optimization (DCEO) cycle.

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RESULTS AND DISCUSSION Increasing MK-7 Production by Overexpression of MEP Pathway Genes. To investigate the effect of increasing carbon flux through the MEP pathway on MK-7 production, genes dxs, dxr, and idi were overexpressed either individually or together. The gene clusters were assembled under the control of the IPTG-inducible Pspac promoter, and integrated into the amyE locus in B. subtilis 168 chromosome via double crossover homologous recombination. Single gene overexpression of dxs, dxr, or idi resulted in 3.7 fold (p = 0.1), 2.1 fold (p < 0.001),

Figure 2. Schematic diagram of gene clusters inserted in pUS258 under the control of IPTG inducible promoter Pspac.

and 2.5 fold (p = 0.3) increases in MK-7 production compared to the empty plasmid control, respectively (Figure 3). When dxs and dxr were co-overexpressed, MK-7 production was increased by 4.6 fold compared to the control (p = 0.02), and B


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Figure 3. (A) Growth curve and (B) MK-7 production by B. subtilis 168 strains overexpressing the MEP pathway genes. MK-7 was quantified at 120 h. Mean values ± standard deviations are shown (n = 3). Two-tailed t test was used for statistical analysis. Statistical analysis on the figure shows comparison to the vector only control. ns (p > 0.05), * (0.01 < p < 0.05), ** (0.001 < p < 0.01), *** (p < 0.001).

Figure 4. (A) Growth curve and (B) MK-7 production by strains co-overexpressing the MEP pathway and downstream menA gene. MK-7 was quantified at 120 h. Mean values ± standard deviations are shown (n = 3). Two-tailed t test was used for statistical analysis. Statistical analysis on the figure shows comparison to the vector only control. ns (p > 0.05), * (0.01 < p < 0.05), ** (0.001 < p < 0.01), *** (p < 0.001).

was observed compared to cultures overexpressing dxs-dxr-idi only (p = 0.01). These findings suggest a “push and pull” dynamic48 between overexpressing the upstream MEP pathway genes and the downstream menA gene on MK-7 production. As the synthetic gene cluster was driven by a single promoter (Pspac), in theory, the closer a single gene in the cluster was located to the promoter, the stronger the relative expression of that gene.49 To investigate this effect, menA was inserted in front of the dxs-dxr-idi cassette (Figure 4). The construct menA-dxs-dxr-idi increased MK-7 production to 50 mg/L, a 1.7 fold-increase compared to the dxs-dxr-idi-menA gene cluster (p = 0.003), confirming that the gene order had an impact on the MK-7 yield, and that higher menA expression was beneficial for the pathway overall. Because the MK-7 yield from constructs expressing dxs, dxr, and idi was less than that of constructs expressing dxs and dxr (Figure 3), this suggested idi may have an inhibitory effect in some contexts. Therefore, a construct containing menA, dxs, and dxr was made, for comparison to menA-dxs-dxr-idi (Figure 4). The MK-7 production of menA-dxs-dxr-idi was 1.3 fold higher than menA-dxs-dxr (p = 0.05), suggesting that overexpressing idi was actually beneficial, at least in the presence of menA, dxs, and dxr. The IPP/DMAPP isomerase

by 1.2-fold and 2.3-fold compared to individual overexpression of dxs and dxr, respectively (p = 0.5 and p = 0.007). It is likely that overexpressing dxs alone resulted in the buildup of MEP, which required overexpression of dxr to convert in the subsequent step, and thus it is logical that overexpression of dxs and dxr together would result in more carbon flux through the MEP pathway. When dxs and dxr were co-overexpressed with idi, the MK-7 production dropped by 3-fold, compared to cultures overexpressing dxs and dxr only (p = 0.005). This was a surprising finding, as overexpression of idi is known to improve the flux through the MEP pathway.39,42,47 It is possible that overexpression of idi without the overexpression of the downstream pathway genes could result in an increased carbon flux into isoprene, decreasing the carbon available for the biosynthesis of MK-7. Increasing MK-7 Production by Overexpression of MEP Pathway Genes and menA. The biosynthesis of MK-7 not only relies on the biosynthesis of the isoprenoid tail via the MEP pathway, it also relies on joining the isoprenoid tail with the naphthoquinone ring via the MenA enzyme. Therefore, the menA gene was added to the end of the dxs-dxr-idi gene cluster and co-overexpressed (Figure 4). A 4.4 fold increase of MK-7 C


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Figure 5. Pellicle biofilm formation in stationary phase shake-flask cultures of B. subtilis strains. Numbers indicate MK-7 production by corresponding strains.

Idi mediates the interconversion of IPP and DMAPP.50 It appears that overexpressing idi resulted in a more-optimum ratio between IPP and DMAPP for the downstream pathway only when overexpressing menA, and not when menA was absent. Further evidence supporting the importance of menA was seen when comparing cultures expressing menA-dxs-dxr to those expressing only dxs-dxr, with the former construct producing 1.8 fold more MK-7 (p = 0.01). Interestingly, the effect of placing menA in front of dxs-dxr was more beneficial than placing both idi and menA at the end of dxs-dxr. The effect of relative positioning of dxr and dxs was also examined, but there was no significant difference in MK-7 production between dxs-dxr-idi-menA and dxr-dxs-idi-menA. Compared to the vector-only control, the best overexpression combination menA-dxs-dxr-idi increased MK-7 production by 11-fold to 50 mg/L (p = 0.002). This is the highest fold-increase in MK-7 production in any recombinant Bacillus system to date.16,51 A recently published study reported a 5-fold increase in MK-7 production (15 mg/L) via overexpression of menA, dxs, dxr, yacM-yacN (equivalent to ispD-ispF), glpD, and deletion of dhbB.51 The authors further increased the MK-7 titer to 70 mg/L via other process engineering strategies, such as increasing aeration and scaling up the culture volume to 2 L baffled flasks. We believe that the MK-7 production from our best performing strain can be further improved by applying similar strategies. It is worth noting that Yang et al.51 overexpressed menA, dxs, and dxr by inserting these genes into three different loci: yxlA, yjoB, and ydeO, respectively. However, their resulting strain MK3-MEP12 only produced 3.4-fold more MK-7 than the parental strain. In comparison, we achieved an 8.4-fold increase (p = 0.01) by placing the gene cluster menA-dxs-dxr under the control of one promoter in the amyE locus. Even though Yang et al.51 selected their overexpression loci based on the published Bacillus minimum genome,52,53 it is likely that the integration sites had an impact on the effectiveness of gene overexpression. The leakiness of the IPTG-inducible expression system was tested using the strain B. subtilis 168(pUS258ASRI); this showed that without IPTG induction, this strain produced the same level of MK-7 as the vector-only control B. subtilis(-

pUS258) (Supporting Figure S2). The confirmed tight control of expression is important for minimizing the metabolic burden on the cells when they are not actively required to make MK-7, thus contributing to the genetic stability of the recombinant. Enhanced MK-7 Production Induces Pellicle Formation in B. subtilis. The growth curves in Figure 3A and Figure 4A showed that there were no significant differences in initial growth rates between the control and the engineered strains. However, the higher MK-7 producing strains all showed substantial decreases in OD600 in stationary phase (Figure 3A and Figure 4A), which correlated with formation of large flocs of biofilm (i.e., pellicle) at the air/liquid interface (Figure 5). It was notable that the extent of pellicle formation in the engineered strains correlated to the levels of MK-7 in the cultures. Pellicle formation is commonly observed at the air/ liquid interface in static B. subtilis cultures,54 but does not usually occur in shaken cultures, such as those used in the present study (orbital shaking at 200 rpm). Pellicles were never seen in shaken cultures of the vector-only strain B. subtilis 168(pUS258) or wild-type B. subtilis natto. The pellicles were clearly hydrophobic, supporting droplets of liquid with large contact angles (Supporting Figure S3), although this was not quantified. It is possible that the hydrophobicity is directly related to the high MK-7 content of the recombinant cells, since this is a fat-soluble vitamin, and only poorly soluble in aqueous solution. Pellicle formation in the MK-7-overproducing strains may simply be due to increased hydrophobicity of MK-7-rich cells, but another possibility is an impact via the regulatory systems controlling biofilm formation. Because menaquinone is an important component of the aerobic electron transport chain, increased menaquinone may result in an increased demand for oxygen. When this increased demand is not satisfied, the KinB regulators may be activated,55 triggering biofilm matrix formation via a regulatory cascade involving SpoA, SinR, and AbrB.56,57 Evidence to support this idea also comes from the work of Pelchovich et al., who found that menaquinone was essential for complex colony development (CCD) in B. subtilis.58 Our data confirm the important role of MK-7 in biofilm formation in B. subtilis. This phenomenon is important to consider for future scale-up in industrial bioreactors, where D


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Figure 6. (A) Cell density, (B) isoprene production, (C) MK-7 production, and (D) MK-7/isoprene production ratio by B. subtilis 168 strains. Mean values ± standard deviations are shown (n = 3). Two-tailed t test was used for statistical analysis. Statistical analysis on the figure shows comparison to the vector only control. ns (p > 0.05), * (0.01 < p < 0.05), ** (0.001 < p < 0.01), *** (p < 0.001).

pellicle formation may pose challenges for monitoring, aeration, and mixing. Alternatively, pellicle formation may actually be beneficial for product recovery, since it enables facile separation of biomass from culture supernatant. A previous study from our group59 showed a linear relationship between biofilm formation and MK-7 production in static cultures, but this did not hold true for shaken cultures. Our finding of a link between MK-7 and biofilm formation in B. subtilis has important implications beyond biotechnological applications, especially for work on the medical significance of biofilms in Gram positive bacteria; e.g., it may be possible to target MK-7 biosynthesis to inhibit biofilms. Isoprene Production as a Measurement of MEP Pathway Carbon Flux. Isoprene is hypothesized to be an ideal reporter of MEP pathway flux60,61 since it is directly produced from DMAPP, and it has a high volatility and thus partitions into the headspace, where it can be analyzed with high sensitivity by GC−MS. Isoprene was tested as a potential indicator of the MEP pathway flux in our recombinant B. subtilis strains (Figure 6). This experiment revealed that the growth yield of B. subtilis was very sensitive to the culture conditions. In the previous shake flask experiments (50 mL culture in 250 mL flask, loosely capped), the optical density of the empty vector control B. subtilis 168(pUS258) continued to increase until 72 h and stayed at the same level from 72 h to 120 h (Figure 3A and Figure 4A), while in the isoprene production experiments (1 mL culture in 20 mL gas-tight bottles) optical density readings of the empty vector control were similar to the large-flask cultures at 24 h but declined dramatically at 48 h (Figure 6A). The mechanism underlying this effect remains to be

determined, but is likely to be related to differences in oxygen and/or CO2 flux between the liquid and gas phases of cultures. The isoprene concentration remained unchanged between 24 h and 48 h for all strains, while over the same interval, the MK-7 concentration increased significantly (p < 0.001 for strain 168(pUS258SRIA)). Similar to the shake flask results (Figure 3B), the strain overexpressing dxs-dxr-idi produced less MK-7 than the strain overexpressing dxs-dxr only at 48 h (p < 0.001), but the isoprene concentration produced by these two strains were similar, and the MK-7/isoprene ratio was decreased by the overexpression of idi (p < 0.001) (Figure 6D). An additional overexpression of menA increased both isoprene (p = 0.04) and MK-7 production at 48h (p < 0.001) compared to the strain overexpressing dxs-dxr-idi, resulting in an increased MK-7/isoprene ratio (p < 0.001). Overall, the differences in isoprene production by different strains were relatively minor, and therefore, it was concluded that isoprene production was not a good indication of MEP pathway carbon flux, at least under these experimental conditions. While many isoprene synthases from plants have been expressed in microbes,62 a native microbial isoprene synthase has not yet been identified. Sivy et al.61 partially purified an isoprene synthase activity from B. subtilis but the enzyme activity was very labile, and they could not confirm the identity of the enzyme. More recently, Ge et al.63 demonstrated a promiscuous activity of IspH that led to isoprene formation from HMBPP in Bacillus cells; it is possible that IspH is the “missing” bacterial isoprene synthase, but further work is needed to confirm this. The level of isoprene production in E


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B. subtilis. We succeeded in improving MK-7 production in B. subtilis 168 by overexpressing rate-limiting MEP pathway genes and menA, and by optimizing the gene order in the recombinant gene cluster. The best performing strain produced MK-7 concentrations of 50 mg/L; this was 11-fold higher than the parent strain. Our strain B. subtilis 168(pUS258ASRI) is the best-performing MK-7 producing recombinant microbe reported to date in terms of fold-change vs wild type, and we believe that with further optimization of culture conditions, this strain will also give substantially higher volumetric yields of MK-7. Our approach here was quite different to that of a recent study with the same aims and bacterial host;51 in that work, menA, dxs, dxr, yacM, yacN, and glpD were modified individually, while in our work, a complete additional synthetic gene cluster was integrated. We argue that our approach is conceptually and technically simpler and offers more flexibility since the components of the synthetic gene cluster can be easily rearranged using plasmid modifications in E. coli, before adding the new synthetic gene cluster back into the original B. subtilis 168 strain. It would be interesting to add the other stimulatory genes (yacM, yacN, glpD) detected in the other study to our construct to enable a better side-by-side comparison of these alternative approaches to genome engineering. We showed that headspace isoprene was a poor reporter of MEP pathway flux in B. subtilis, but that the intracellular metabolite profile was a very useful way to identify pathway bottlenecks for targeting in future work. We are the first to show a dramatic and unexpected effect of MEP pathway gene overexpression on the overall behavior of B. subtilis cultures, with massive pellicle formation seen in the highest MK-7 producing strains. The mechanism underlying this effect requires further investigation. The results from this work demonstrate the potential of B. subtilis for producing MK-7 and other valuable isoprenoids using metabolic engineering.

Bacillus cells may depend primarily on IspH activity rather than on overall MEP pathway flux. Intermediate Metabolite Profiling To Identify MEP Pathway Bottlenecks. To more accurately investigate the MEP pathway carbon flux and identify pathway bottlenecks, mass spectrometry was used to measure intracellular intermediate metabolites. The metabolite analysis was performed at two time points in B. subtilis 168 cultures containing either the empty vector pUS258 or the best MK-7 producing strain carrying plasmid pUS258ASRI. The levels of intermediate metabolites were similar between the control and the engineered strain at the early time-point in the exponential growth phase (3 h) (Figure 7), but dramatic differences

Figure 7. Intracellular MEP pathway intermediate metabolites profiles in B. subtilis 168(pUS258) and B. subtilis 168(pUS258ASRI) during exponential growth phase (3 h) and stationary phase (120 h). Mean values ± standard deviations are shown (n = 3).

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MATERIALS AND METHODS Chemicals and Reagents. Isoprene (Cat. No. I19551), tributylamine (Cat. No. 90780), glyceraldehyde-3-phosphate (Cat. No. G5251), dihydroxyacetone-phosphate (Cat. No. D7137), sodium pyruvate (Cat. No. P2256), 3′-Azido-3′deoxythymidine (AZT, Cat. No. A2169), phylloquinone (K1, Cat. No. 47773) were purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Menaquinone-7 (Cat. No. sc-218691) and menaquinone-9 (Cat. No. sc-211788) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). 1-DeoxyD-xylulose 5-phosphate (DXP), 2-C-methyl-D-erythritol-4phosphate (MEP), 4-diphosphocytidyl-2-C-methylerythritol (CDPME), 2-C-methyld-erythritol-2,4-cyclodiphosphate (MEcPP), (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP) and farnesyl pyrophosphate (FPP) were purchased from Echelon Biosciences (Salt Lake City, UT, USA). Isopropyl-β-D-1-thiogalactopyranoside (IPTG, Cat. No. BIO-37036), 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (X-gal, BIO-37035), ISOLATE II Plasmid Mini Kit (Cat. No. BIO-52056), and ISOLATE II PCR and Gel Kit (Cat. No. BIO-52059) were purchased from Bioline (Eveleigh, NSW, Australia). HPLC grade methanol, dichloromethane, acetonitrile, acetic acid, isopropanol, and hexane were purchased from Merck (Bayswater, VIC, Australia). Synthetic

became apparent in stationary phase (120 h), when DXP, MEP, and MEcPP accumulated to high levels in the engineered strain, but not in the vector-only control. The accumulation of DXP, MEP, and MEcPP but not HMBPP, IPP/DMAPP, or FPP suggests that the enzyme IspG was rate limiting for the pathway in the strain overexpressing menA-dxs-dxr-idi. Since the upstream metabolites DXP and MEP also accumulated (i.e., not just MEcPP), it may also be useful to further enhance dxs and dxr gene expression to maximize pathway flux. Li et al.64 found that a balanced overexpression of ispG and ispH was required to optimize the carbon flux through the MEP pathway to increase the production of β-carotene, and that overexpression of ispG without ispH resulted in toxic accumulation of HMBPP. Similarly, Kirby et al.65 also found that IspG and IspH were the critical steps when introducing a functioning MEP pathway into yeast to replace the MVA pathway. Therefore, the next logical steps in improving MK-7 production in our bestperforming strain would be co-overexpression of IspG and IspH.

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CONCLUSIONS The aim of this work was to develop metabolic engineering strategies for the improvement of MK-7 production in F


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ACS Synthetic Biology oligonucleotides and gBlock gene fragments were purchased from Integrated DNA Technologies (Singapore). Restriction endonucleases (various), Deoxynucleotide (dNTP) Solution Mix (Cat. No. N0447S), Shrimp Alkaline Phosphatase (rSAP) (Cat. No. M0371S), T4 DNA ligase (Cat. No. M0202S), T4 DNA polymerase (Cat. No. M0203S), Q5 High-Fidelity 2× Master Mix (Cat. No. M0492S), and Taq DNA polymerase with ThermoPol buffer (Cat. No. M0267S) were purchased from New England Biolabs (Ipswich, MA, USA). GelRed 10 000× in water (Cat. No. 41003) and GelGreen 10 000× in Water (Cat. No. 41005) were purchased from Biotium (Fremont, CA, USA). Bacterial Growth Media and Conditions. All cultures were grown aerobically with shaking at 200 rpm and 37 °C unless otherwise specified. For molecular biology work, Escherichia coli and Bacillus strains were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract and 10 g/L NaCl), with additions, where required, as follows: Ampicillin (Ap) at 100 μg/mL, chloramphenicol (Cm) at 25 μg/mL, spectinomycin (Sp) at 100 μg/mL, IPTG at 1 mM, X-gal at 80 μg/mL, agar at 1.5 wt %/vol, and starch at 1 wt %/vol. For MK-7 production, SYG medium was used. This medium was formulated in our lab to enable high density growth of B. subtilis, and contained 50 g/L soy peptone, 20 g/L yeast extract, 50 g/L glycerol, 3.86 g/L K2HPO4, 1.62 g/L KH2PO4 and 2 mL/L trace element solution. The trace element solution (added after autoclaving) contained 6.37 g/L Na2EDTA· 2H2O, 1.0 g/L ZnSO4·7H2O, 0.5 g/L CaCl2·2H2O, 2.5 g/L FeSO4·7H2O, 0.1 g/L NaMoO4·2H2O, 0.1 g/L CuSO4·5H2O, 0.2 g/L CoCl2·6H2O, 0.52 g/L MnSO4·H2O, 60.0 g/L MgSO4·7H2O. Additions to the SYG medium were the same as above for LB medium. In MK-7 production experiments, B. subtilis cultures grown in SYG broths were incubated at 40 °C and 200 rpm. Construction of E. coli/Bacillus Integrative Shuttle Vector pUS258 Containing MEP Pathway Genes. All strains and plasmids used in this study are summarized in Table 1. All primers used in this study are summarized in Supporting Table S1. The sequences of gBlocks are given in Supporting Note S1. To construct the E. coli/Bacillus integrative shuttle vector pUS258 (Figure 8) a gBlock fragment containing a multiple cloning site (MCS) and lacZα gene was digested with SalI and EcoRI, and ligated to SalI/EcoRI-digested plasmid pAPNC21366 to yield plasmid pAPNCblue. The section of pAPNCblue containing the spectinomycin resistance gene SpecR, lacI gene and lacZα-MCS fragment was PCR amplified, which added flanking EcoRI sites. This PCR product was digested with EcoRI, and ligated to an EcoRI-digested gBlock fragment containing the ColE1 high copy number plasmid origin of replication and the Bacillus subtilis amyE gene front section (500 bp) and back section (500 bp) to yield plasmid pUS258. Plasmid pUS258 integrates into the chromosomal amylase gene of B. subtilis via homologous recombination (recombinants can be detected by loss of amylase activity) and this plasmid provides an improved multiple cloning site (including blue/white selection), an improved selection marker (SpecR), and a more compact size than the original plasmid pAPNC213 or similar Bacillus integrative vectors. Eleven different pUS258-derived plasmids (pUS258S, pUS258R, pUS258I, pUS258A, pUS258SR, pUS258SRI, pUS258SIA, pUS258ASR, pUS258RSIA, pUS258SRIA, pUS258ASRI) were constructed by inserting different combina-

Table 1. Strains and Plasmids Used in This Study Strains E. coli TOP10

E. coli ER2925

B. subtilis natto strain B17 B. subtilis 168 Plasmids pAPNC213 pAPNCblue pSB1C3 pSBS pSBSR pSBSRI pSBSRIA pSBSIA pUS258 pUS258S pUS258R pUS258I pUS258A pUS258SR pUS258SRI pUS258SIA pUS258ASR pUS258RSIA pUS258SRIA pUS258ASRI

genotype/characteristic

reference

F− mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(ara-leu) 7697 galU galK λ− rpsL(StrR) endA1 nupG ara-14 leuB6 f huA31 lacY1 tsx78 gln V44 galK2 galT22 mcrA dcm-6 hisG4 rf bD1 R(zgb210::Tn10)TetS endA1 rpsL136 dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2 Wild-type, isolated from natto

Invitrogen

NEB

trpC2

BGSC

E. coli/B. subtilis shuttle vector, integrates at aprE locus, Pspac promoter, lacIR, AmpR, SpecR pAPNC213, lacZα-MCS High copy BioBrick assembly plasmid pSB1C3 containing dxs; CmR pSB1C3 containing dxs and dxr; CmR pSB1C3 containing dxs, dxr and idi; CmR pSB1C3 containing dxs, dxr, idi and menA; CmR pSB1C3 containing dxs, idi and menA; CmR E. coli/B. subtilis shuttle vector, integrates at amyE locus, Pspac-lacZα-MCS, lacIR, SpecR pUS258, Pspac::dxs pUS258, Pspac::dxr pUS258, Pspac::idi pUS258, Pspac::menA pUS258, Pspac::dxs-dxr pUS258, Pspac::dxs-dxr-idi pUS258, Pspac::dxs-idi-menA pUS258, Pspac::menA-dxs-dxr pUS258, Pspac::dxr-dxs-idi-menA pUS258, Pspac::dxs-dxr-idi-menA pUS258, Pspac::menA-dxs-dxr-idi

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tions of dxs, dxr, idi, and menA genes into the multiple cloning site (Figure 8) These genes were obtained by PCR amplification from chromosomal DNA of B. subtilis 168. In each case, mutations were introduced in the forward primer to give a strong ribosome binding site (AAGGAGG). Details of the construction of these plasmids are in Supporting Note S2. The protocols for routine recombinant DNA work are given in Supporting Note S3. Integration of Synthetic Gene Clusters into Bacillus subtilis. Naturally competent cells of B. subtilis were prepared following an existing protocol.68 The competent cells were divided into 500 μL aliquots, then each aliquot was mixed with 125 μL sterile glycerol, and stored at −80 °C until required. Cells were transformed by adding plasmid DNA (1 μg) and incubating at 37 °C for 30 min. Cells were then plated on LBSp agar plates and grown overnight at 37 °C. Individual colonies were patched on fresh LB-Sp plates and onto LB-Spstarch agar plates, and incubated overnight at 37 °C. The starch plates were overlaid with iodine solution (0.33 wt %/vol iodine, 0.66 wt %/vol potassium iodide), and recombinants without clearing zones were identified, and the corresponding patches on the LB-Sp plate were used for colony PCR to confirm double crossover integration. To perform colony PCR, approximately 1 mm3 of cells was picked and resuspended in 100 μL TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and incubated at 95 °C for 10 min, then 1 μL of the lysate was used as the PCR template. Primer pairs used to confirm double crossover are listed in the Supporting Table S2. Glycerol stocks G


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Figure 8. Structure of E. coli/Bacillus integrative shuttle vector pUS258.

acetic acid (0.30 g/L). A flow rate of 0.3 mL/min was used with a constant column oven temperature of 40 °C. The calibration curve was determined using five standard concentrations between 78.125 ng/mL and 1250 ng/mL (Supporting Figure S1, linear regression, R2 = 0.9998). Headspace GC−MS Analysis of Isoprene Production. B. subtilis cultures for isoprene analysis were grown in 1 mL SYG broths in 20 mL gastight amber vials. Broths were inoculated with an overnight SYG culture to an initial OD600 of 0.05, IPTG was added, and the cultures were grown for 24 or 48 h. At the end of the incubation period, cultures were heated in a sand bath at 45 °C for 10 min, then 1 mL of the headspace was sampled manually and injected into the GC. The GC−MS analysis (using a protocol modified from Vickers et al.69) was conducted using a Clarus 680 GC system connected to a Clarus SQ 8C GC−MS system (PerkinElmer, USA). The samples were injected in a split mode (4:1) with a spit flow rate of 6.5 mL/min at 250 °C with helium as a carrier gas and at a constant total flow of 1.3 mL/min. The oven temperature was programed to hold at 30 °C for 2 min, then increased to 120 °C at a rate of 40 °C/min and held for 2 min, while maintaining the ion source and transfer line at 150 and 250 °C respectively. Isoprene was detected in both total ion current (TIC) and selected ion monitoring (SIM) mode using mass to charge (m/z) ratios 67, 68, and 69 with an interchannel delay of 0.1 s. The OD600 and MK-7 titer of each culture were also measured, as described previously. Seven isoprene calibration standards were prepared by dissolving pure isoprene in acetone on ice and serially diluting the acetone in sterile SYG broth to cover a range between 0.0195 mg/L and 1.25 mg/L. The calibration standard (1 mL)

having a final concentration of 10% (wt/vol) glycerol were prepared and stored at −80 °C. MK-7 Production, Extraction, and HPLC Analysis. B. subtilis cultures for MK-7 production were grown in 50 mL SYG medium in 250 mL shake flasks. Broths were inoculated with an overnight SYG culture to an initial OD600 of 0.05, IPTG (1 mM) was added immediately, and the cultures were grown for 120 h at 40 °C. The cell density was monitored throughout growth using a UV−vis spectrophotometer at 600 nm. MK-7 was quantified at the end of growth (120 h). For MK-7 extraction and analysis, an internal standard (IS) consisting of phylloquinone (PK, 2600 μg/mL in isopropanol) was added to the culture (100 μL of IS into 50 mL culture to give a final concentration of 5200 ng/mL). The whole culture (50 mL) was then lysed by bead-beating for 2 min (BioSpec Mini-BeadBeater 16) to obtain a homogeneous suspension. Five mL of the suspension was mixed with 10 mL isopropanol and 25 mL n-hexane, vigorously vortexed, and centrifuged to facilitate phase separation. 100 μL of the upper organic layer was diluted in 900 μL methanol/dichloromethane (9:1 v/v) for HPLC analysis. A Shimadzu Prominence-i LC-2030 HPLC system coupled with a fluorescence detector (Shimadzu RF-20Axs, 35 °C cell temperature, excitation wavelength 243 nm and emission wavelength 430 nm) was used for analysis of MK-7. Separation of metabolites was achieved using a C18 column (150 mm × 2.0 mm × 3 μm particle size, 12 nm pore size, YMC-Pack ODS-AM), and a postcolumn zinc column (to fully reduce MK-7 to form the corresponding fluorescent hydroquinone). The mobile phase contained methanol:dichloromethane (9:1, v/v), ZnCl2 (1.37 g/L), sodium acetate (0.41 g/L), and glacial H


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ACS Synthetic Biology was added into a 20 mL amber headspace vial, which was immediately sealed, and heated in a sand bath at 45 °C for 10 min. Headspace samples (1 mL) was sampled and analyzed by GC−MS as above. The signal area generated by the headspace isoprene was converted to its liquid concentration (assuming all isoprene was dissolved in the liquid phase) to construct a calibration curve (Supporting Figure S1, linear regression, R2 = 0.9953). LC−MS/MS Analysis of MEP Pathway Intracellular Metabolites. The intracellular intermediate metabolite measurement was carried out following protocol modified from Bongers et al.60 Cultures (50 mL) were grown in SYG medium containing IPTG as described above. At time 3 and 120 h, samples (5 mL) were centrifuged (2300g, 15 min), the supernatant discarded, and the cell pellet resuspended in 1 mL of ice cold 80% (v/v) methanol in water. The sample was spiked with 25 μL of AZT (250 μM) to act as an internal standard. The samples were then lysed by bead beating (BioSpec Mini-Beadbeater 16) for 1 min and centrifuged (20 000g, 1 min). Supernatants (800 μL) were transferred into amber HPLC vials and dried under nitrogen gas until the volume was less than 10 μL, then Milli-Q water (100 μL) was added. The LC−MS/MS analysis was performed on an Agilent HPLC HP1100 (Hewlett-Packard, USA) coupled with an amaZon SL quadrupole ion trap mass spectrometer with atmospheric pressure chemical ionization (APCI) ion source (Bruker, USA). An injection volume of 10 μL intracellular metabolite extract was separated on a Sunfire C18 column (150 mm × 3 mm ID × 5 μm particle size, column oven 55 °C). The mobile phase consisted of 7.5 mM aqueous tributylamine adjusted to pH 5.0 with glacial acetic acid (eluent A) and acetonitrile (eluent B). The flow rate of the mobile phase was controlled at 0.3 mL/min with a gradient profile as follows: over the first 10 min, decreased eluent A from 100% to 85%; over the next 25 min, decreased eluent A from 85% to 70%; then over the next 10 min, decreased eluent A from 70% to 30%; then over the next 2 min, decreased eluent A to 0%; held at 0% eluent A for a further 2 min, then increased eluent A to 100% over the next 1 min; held at 100% eluent A over the next 10 min. The metabolite standards were prepared in Milli-Q water and serially diluted to 7 levels between 0.78125 μM and 50 μM. Calibration curves were fit and linear regression was performed (Supporting Figure S1). Isomers IPP and DMAPP could not be separated on the chromatogram using this method thus were expressed as a sum of both metabolites. The MS acquisition parameters are listed in Supporting Table S3. Statistical Analysis. Experiments were performed in triplicates (n = 3) and the data were presented as mean values ± standard deviations. Two tailed t tests were used for statistical analysis of data, with significance levels denoted as ns (p > 0.05), * (0.01 < p < 0.05), ** (0.001 < p < 0.01), *** (p < 0.001).

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double crossover confirmation PCR; Supporting Table S3. LC−MS/MS acquisition parameters for detection of MEP pathway intracellular intermediate metabolites and standards; Supporting Figure S1. Calibration curve of (A) MK-7, (B) PK, (C) isoprene, (D) DXP, (E) MEP, (F) CDP-ME, (G) MEcPP, (H) HMBPP, (I) IPP/ DMAPP, and (J) FPP; Supporting Figure S2. MK-7 production by strains 168/pUS258ASRI with or without 1 mM IPTG induction; Supporting Figure S3. Evidence for hydrophobicity of pellicle biofilm; Supporting Note S1. gBlock sequences; Supporting Note S2. Construction of the MEP Pathway gene clusters; Supporting Note S3. Routine methods for recombinant DNA work (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Fariba Dehghani: 0000-0002-7805-8101 Nicholas V. Coleman: 0000-0002-3594-1754 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the Australian Research Council (Industrial Transformation Training Centre Scheme, grant number: IC140100026). Yanwei Ma was supported by an Australian Postgraduate Award scholarship.

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ABBREVIATIONS PYR, pyruvate; G3P, glyceraldehyde-3-phosphate; DXP, 1deoxy-D-xylulose-5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; CDP-ME, 4-diphosphocytidyl-2-C-methylerythritol; CDP-MEP, 4-diphosphocytidyl-2-C-methylerythritol 2-phosphate; MEcPP, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate; HMBPP, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate; DMAPP, demethylallyl diphosphate; IPP, isopentenyl diphosphate; HPP, heptaprenyl diphosphate; E4P, erythrose-4-phosphate; PEP, phosphoenolpyruvate; SA, shikimic acid; CHA, chorismate; IsoCHA, isochorismate; DHNA, 1,4-dihydroxy-2-naphthoate; DMK-7, 2-demethylmenaquinone-7; DXS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; IspD, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; IspE, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; IspF, 2-Cmethyl-D-erythritol 2,4-cyclodiphosphate synthase; IspG, 2-Cmethyl-D-erythritol-2,4-cyclodiphosphate reductase; IspH, 4hydroxyl-3-methylbut-2-enyl diphosphate reductase; IspA, farnesyl diphosphate synthase; HepS, heptaprenyl diphosphate synthase component I; HepT, heptaprenyl diphosphate synthase component II; MenA, 1,4-dihydroxy-2-naphthoate octaprenyltransferase; AroA, 3-phosphoshikimate 1-carboxyvinyltransferase; AroB, 3-dehydroquinate synthase; AroC, 3dehydroquinate dehydratase; AroD, shikimate 5-dehydrogenase; AroK, shikimate kinase; AroE, 5-enolpyruvoylshikimate-3phosphate synthase; AroF, chorismate synthase; MenF, menaquinone-specific isochorismate synthase; DhbC, isochorismate synthase; MenD, 2-succinyl-5-enolpyruvyl-6-hydroxy-3cyclohexene-1-carboxylate synthase; MenC, o-succinylbenzoate synthase; MenE, 2-succinylbenzoate-CoA ligase; MenB, 1,4-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssynbio.9b00077. Supporting Table S1. List of primers used in this study; Supporting Table S2. List of primer pairs used for I


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dihydroxy-2-naphthoyl-CoA synthase; MenH/MenG, demethylmenaquinone methyltransferase.

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Bacillus subtilis - ACS Publications - American Chemical Society

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