Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli

Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e9, 2017 www.elsevier.com/locate/jbiosc

Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli Yuki Soma,1, 3 Taiki Yamaji,1 Fumio Matsuda,2 and Taizo Hanai1, * Laboratory for Bioinformatics, Graduate School of Systems Lifesciences, Kyushu University, 804 Westwing, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan,1 Department of Bioinformatic Engineering, Graduate School of Information Science and Technology, Osaka University, 1-5 Yamadaoka, Suita 565-0871, Osaka, Japan,2 and Research Center for Transomics Medicine, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan3 Received 11 November 2016; accepted 15 December 2016 Available online xxx

Almost all synthetic pathways for biofuel production are designed to require endogenous metabolites in glycolysis, such as phosphoenolpyruvate, pyruvate, and acetyl-CoA. However, such metabolites are also required for bacterial cell growth. To reduce the metabolic imbalance between cell growth and target chemical production, we previously constructed a metabolic toggle switch (MTS) as a conditional flux redirection tool controlling metabolic flux of TCA cycle toward isopropanol production. This approach succeeded to improve the isopropanol production titer and yield while ensuring sufficient cell growth. However, excess accumulation of pyruvate, the precursor for acetyl-CoA synthesis, was also observed. In this study, for efficient conversation of pyruvate to acetyl-CoA (pyruvate oxidation), we designed a synthetic metabolic bypass composed of poxB and acs with the MTS for acetyl-CoA supply from the excess pyruvate. When this designed bypass was expressed at the appropriate expression level associated with the conditional metabolic flux redirection, pyruvate accumulation was prevented, and the isopropanol production titer and yield were improved. Final isopropanol production titer of strain harboring MTS with the synthetic metabolic bypass improved 4.4-fold compared with strain without metabolic flux regulation, and it was 1.3-fold higher than that of strain harboring the conventional MTS alone. Additionally, glucose consumption was also improved 1.7-fold compared with strain without metabolic flux regulation. On the other hand, introduction of the synthetic metabolic bypass alone showed no improvement in isopropanol production and glucose consumption. These results showed that the improvement in bioproduction process caused by synergy between the MTS and the synthetic metabolic bypass. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Synthetic metabolic bypass; Synthetic genetic circuit; Metabolic toggle switch; Biosynthetic pathway; Acetyl-CoA supply]

Microbial chemical production from renewable resources has attracted increasing attention as a potential alternative to petrochemical production because of the limited amounts of fossil oils and global concerns regarding sustainability and the environment (1e3). To expand the target chemicals that microorganisms can produce, many types of biosynthetic pathways have been constructed by combining heterogeneous metabolic pathways (4). Most synthetic pathways used for production of bulk chemical, such as bio-alcohols, biodiesels, and bioplastics, have been constructed based on endogenous metabolites in the glycolysis pathway, including phosphoenolpyruvate, pyruvate, and acetylCoA (5e8). These metabolites are also consumed in endogenous pathways that are responsible for bacterial cell growth and physiological activity (e.g., amino acid synthesis, fatty acid synthesis, the TCA cycle). Therefore, there is a conflicting relationship between endogenous metabolism and the biosynthetic pathway, termed metabolic imbalance, and this often limits microbial chemical product titers and yields (2,9,10). To achieve economic viability of microbial chemical production, it is necessary to overcome these

* Corresponding author. Tel.: þ81 92 642 6751; fax: þ81 92 642 6744. E-mail address: [email protected] (T. Hanai).

tradeoff relationships in order to ensure both efficient cell growth and target chemical productivity. In recent years, synthetic biological tools have been developed for metabolic flux regulation in order to facilitate reduction of the metabolic imbalance and improve productivity, titers, and yields in the production of several microbial chemicals (11). The first demonstration of regulation of the synthetic pathway was implemented in lycopene biosynthesis in Escherichia coli using the glnAP2 promoter as a dynamic controller that responds to accumulation of acetyl-phosphate as an indicator of excess glycolytic flux (12). A dynamic sensor-regulator system has been also developed for sensing the intracellular concentration of key intermediates for fatty acid ethyl ester production (13). These systems allow microbes to avoid the metabolic imbalance by inducing heterogeneous enzymes optimally in response to perturbation of intracellular metabolism, thereby significantly improving productivity, titers, and yields. In our previous study on isopropanol production by engineered E. coli, we demonstrated metabolic flux redirection using a synthetic genetic circuit, called a metabolic toggle switch (MTS) (14). Isopropanol is one of the simplest secondary alcohols that can be dehydrated to yield bio-propylene, which is currently derived from petroleum as a monomer for making polypropylene. Because polypropylene is currently used as a material for many industrial

1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

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products, it is expected that the world demand for propylene will grow in the future. To date, we have engineered a biosynthetic pathway and E. coli strains for the microbial production of isopropanol from inexpensive and renewable feedstock, such as biomass-derived saccharides (5,15,16). The biosynthetic pathway for isopropanol production uses acetyl-CoA as the precursor (Fig. 1A). Therefore, the production titer and yield of isopropanol are also decreased by the metabolic imbalance between aerobic bacterial growth depending on the TCA cycle and the biosynthetic pathway. To address this problem, we developed the MTS, which can redirect metabolic fluxes in the TCA cycle toward a biosynthetic

FIG. 1. Conditional boosting of acetyl-CoA supply for isopropanol production using the metabolic toggle switch (MTS) installed at the synthetic metabolic bypass. (A) Strategy to boost acetyl-CoA synthesis through metabolic flux regulation using the MTS installed at the synthetic metabolic bypass. Using the MTS, metabolic influx into the TCA cycle could be interrupted and redirected toward isopropanol production in response to the addition of IPTG, causing pyruvate accumulation. To prevent pyruvate accumulation, the synthetic metabolic bypass was induced, along with the metabolic flux redirection, which was composed of poxB and acs under control of the IPTGinducible promoter PLlacO1. (B) Design of the MTS installed at the synthetic metabolic bypass. The MTS was divided into three parts: (i) the repressor source (pTA216: PlacIq::lacI), (ii) the ON-OFF-module (under control of PLtetO1), and (iii) the OFF-ONmodule (under control of PLlacO1). The LacI repressor from pTA216 inhibits transcription from the PLlacO1 promoter. When transcription by the PLlacO1 promoter was induced by the addition of IPTG, expression of tetR and genes involved in the isopropanol production pathway (e.g., thlA, atoAD, adc, adhE) and p.a. bypass (e.g., poxB, acs) were upregulated. Thus, expression of gltA under the PLtetO1 promoter would be inhibited by the TetR repressor, resulting in interruption of the TCA cycle. Error bars show standard deviations (n ¼ 3).

J. BIOSCI. BIOENG., pathway in the response to the addition of isopropyl b-D-1thiogalactopyranoside (IPTG; Fig. 1A, B) (14). The MTS can interrupt the metabolic influx into the TCA cycle by regulating the expression of citrate synthase encoded by gltA (Fig. 1). Using this system, we succeeded in solving this trade-off relationship appropriately by redirecting excess flux caused by conditional interruption of the TCA cycle after ensuring that a sufficiently high cell density was obtained. The titers and yields of isopropanol production were improved by more than 3-fold (14). These designed regulatory processes for local metabolic flux have been shown to contribute to improvement of titers and yields. However, such regulation of metabolic flux may cause unexpected metabolic discord. In fact, conditional interruption of the TCA cycle by the MTS caused significant accumulation of pyruvate, a precursor of acetyl-CoA synthesis, in E. coli cells (14). For additional improvement of isopropanol production, the metabolic flux capacity of acetyl-CoA synthesis should be expanded to prevent the accumulation of pyruvate associated with conditional interruption of the TCA cycle. In the endogenous metabolism of E. coli, acetylCoA is mainly formed from pyruvate via pyruvate decarboxylation catalyzed by the pyruvate dehydrogenase complex (PDHc). The PDHc is a large complex composed of multiple copies of three subunits, i.e., E1 (pyruvate dehydrogenase; E.C. 1.2.4.1), E2 (dihydrolipoyl transacetylase; E.C. 2.3.1.12), and E3 (dihydrolipoyl dehydrogenase, E.C. 1.8.1.4) (17). The expression of PDHc is regulated by a complicated endogenous regulon, and its enzymatic activity is highly regulated by a variety of allosteric effectors and covalent modification (18). On the other hand, pyruvate can also be converted to acetate by pyruvate oxidase, encoded by poxB (E.C. 1.2.5.1), and acetate can be converted to acetyl-CoA by acetyl-CoA synthase, encoded by acs (E.C. 6.2.1.1). The sequential reactions of pyruvate oxidase and acetyl-CoA synthase can represent an alternative pathway for acetyl-CoA synthesis by PDHc. Thus, using poxB and acs, we designed a synthetic metabolic bypass to boost the acetylCoA supply independent from PDHc (Fig. 1A). In our previous work, MTS focused only competition in carbon flux between cell growth and isopropanol production. In this study, we redesigned the MTS controlling Acetyl-CoA supply from pyruvate by using the synthetic metabolic bypass, which is potentially resulting in a bottleneck associated with the metabolic flux redirection. For construction of the synthetic metabolic bypass, the poxB and acs genes were overexpressed under control of the IPTGinducible promoter PLlacO1. This expression module was integrated with the MTS (Fig. 1B). In this system, the addition of IPTG induced the expression of the tetR repressor under the PLlacO1 promoter. The induced tetR repressor inhibited the expression of genes under control of the PLtetO1 promoter. Using this system, the expression of gltA, encoding citrate synthase, could be inhibited during fermentation in response to the addition of IPTG. As a result, metabolic influx into the TCA cycle could be interrupted. At the same time, isopropanol synthesis and bypass were induced. We implemented this system in engineered E. coli to demonstrate isopropanol production and investigated the effects of the conditional enlargement of synthetic metabolic bypass on pyruvate accumulation and acetyl-CoA supply for isopropanol production. MATERIALS AND METHODS Chemicals and reagents All chemicals were purchased from Wako Pure Chemical Industry, Ltd. (Osaka, Japan) unless otherwise specified. Restriction enzymes and phosphatase were from New England Biolabs (Ipswich, MA, USA), ligase (rapid DNA ligation kit) was from Roche (Manheim, Germany), and KOD Plus Neo DNA polymerase was from Toyobo Co., Ltd. (Osaka, Japan). Oligonucleotides were synthesized by Life Technologies Japan Ltd. (Tokyo, Japan). Bacterial strains and plasmid construction Table 1 shows strains and plasmids used for this study. XL10-Gold (Agilent Technologies, Santa Clara, CA, USA) and DH5alphaZ1 (Expressys, Ruelzheim, Germany) were used to construct plasmids.

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

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TABLE 1. List of bacterial strains and plasmids used in this study. Strain/plasmid E. coli strain BW25113 JW0336 JW0710 JW2294 TA1015 TA2261 TA2043 TA2044 TA2579 TA2620 TA2149 TA2690 TA3447 Plasmid pZE22-MCS pZS4Int-lacI pZS*32-MCS pTA147 pTA216 pTA669 pTA695 pTA802 pTA959 pTA965 pTA982 pTA1065 pTA1242 pTA1250 pTA1251

Relevant genotype Wild type BW25113 DlacI::kan BW25113 DgltA::kanR BW25113 Dpta::kanR Same as JW0336 but was removed kanR BW25113 DlacI/pTA147, pTA1065, pTA216 BW25113 DlacI DgltA/pTA147, pTA965, pTA216 BW25113 DlacI DgltA/pTA147, pTA982, pTA216 BW25113 DlacI DgltA/pTA147, pTA965, pTA1251 BW25113 DlacI DgltA Dpta/pTA147, pTA965, pTA1251 BW25113 DlacI DgltA Dpta/pTA147, pTA982, pTA216 BW25113 DlacI DgltA Dpta/pTA147, pTA959, pTA216 BW25113 DlacI/pTA147, pTA959, pTA1264 R

PLlacO1::MCS, ColE1, kanR PlacIq::lacI, pSC101, specR PLlacO1::MCS, pSC101*, cmR PLlacO1::thlA, atoAD, adc, Cbadh, ColE1, ampR PlacIq::lacI, pSC101*, cmR PLlacO1::tetR, PLtetO1::gltA.LAA, p15A, kanR PLlacO1::tetR, PLtetO1::MCS, p15A, kanR PLlacO1::tetR, poxB, PLtetO1::gltA.LAA, p15A, kanR PLlacO1::tetR, poxB, PLtetO1::gltA.LAA, p15A, specR PLlacO1::tetR, PLtetO1::gltA.LAA, p15A, specR PLlacO1::tetR, poxB, acs, PLtetO1::gltA.LAA, p15A, specR PLlacO1::tetR, PLtetO1::MCS, p15A, specR PLlacO1::poxB, pSC101*, cmR PLlacO1::poxB, acs, pSC101*, cmR PLlacO1::poxB, acs, PlacIq::lacI, pSC101*, cmR

Reference/source Datsenko and Wanner (19) Baba et al. (20) Baba et al. (20) Baba et al. (20) Soma et al. (14) This study This study This study This study This study This study This study This study

Expressys Expressys This study Soma et al. (14) Soma et al. (14) Soma et al. (14) Soma et al. (14) This study This study This study This study This This This This

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To introduce the isopropanol production pathway, we used the plasmid pTA147, harboring thalA, atoAD, adc, and adhE under the control of PLlacO1, which was constructed in a previous work (15). The low-copy plasmid pTA216 was used as a constitutive source of the LacI repressor, as reported previously (14). For construction of the redesigned MTS, the medium-copy plasmids pTA669 and pTA695 were used (14). The pyruvate decarboxylase gene poxB was amplified from the genome of BW25113 (19) by KOD polymerase chain reaction (PCR) using following primer set: T1364 (50 -GCCATCGGGCCCATTAAAGAGGAGAAAGGTACCATGAAACAAACGGTTGCAGCTTATATCG-30 ) and T1365 (50 -GCCATCAAGCTTTTACCTTAGCCAGTTTGTTT-TCGCCA30 ). The PCR product was digested using the restriction enzymes ApaI and HindIII and was inserted into the ApaI and HindIII sites of pTA669 (14), yielding the plasmid pTA802. The antibiotic-resistance marker of pTA669, pTA695, and pTA802 were converted from the kanamycin-resistance gene to the chloramphenicol-resistance gene by insertion of the chloramphenicol-resistance gene into the AatII and SacI sites, yielding plasmids pTA965, pTA1065, and pTA959, respectively. The chloramphenicol-resistance gene was cut out from pZS4Int-lacI (Expressys) using the restriction enzymes SacI and AatII. The acetyl-CoA synthase gene acs was amplified from the genome of BW25113 by KOD PCR using the following primer set: T1629 (50 GCCATCAAGCTTATTAAAGAGGAGAAAGGTACCATGAGCCA-30 ) and T1630 (50 GCCATCAAGCTTTTACGATGGCATCGCGATAGCC-30 ). The PCR product was digested by the restriction enzyme HindIII and inserted into the HindIII site of pTA959, yielding the plasmid pTA982. The plasmid pZS*32-MCS was constructed by replacing the gene cassette of the antibiotic-resistance gene and replication origin in pZE22-MCS with the DNA fragment cmR-pSC101, which was obtained by digestion of pTA216 by restriction enzymes AatII and AvrII. pTA802 was digested by Acc65I and HindIII to obtain the DNA fragment of poxB, which was then inserted into the Acc65I and HindIII sites of pZS*32-MCS, yielding the plasmid pTA1242. The acs gene was amplified from pTA982 by KOD PCR using primers T1629 (50 GCCATCAAGCTTATTAAAGAGGAGAAAGGTACCATGAGCCA-30 ) and T2004 (50 -

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GCCATCGGATCCTTACGATGGCATCGCGATAGCC-30 ). The PCR product was digested by the restriction enzymes HindIII and BamHI and was inserted between the HindIII site and the BamHI site of pTA1242, yielding pTA1250. The DNA fragment of Ttrp-PlacIq::lacI was amplified from pTA216 by KDO PCR using primers T1866 (50 -GCCATCGGATCCAGCCCGCCTAATGAGCGGGCTTTTTTTTTCTAGACGTTGACACCATCGAATGGTGC-30 ) and T330 (50 -GCCATCGGATCCTCACTGCCCGCTTTCCAGTCG-30 ). The PCR product was digested by BamHI and inserted into the BamHI site of pTA1250, yielding pTA1251. BW25113 was used as the base strain for all E. coli variants engineered in this study. For construction of the conventional isopropanol production strain TA2261 (harboring the pathway for isopropanol production, but without the MTS), three plasmids pTA147 (PLlacO1::thlA, atoAD, adc, adhE), pTA216 (PlacIq::lacI), and pTA1065 (PLlacO1::tetR, PLtetO1::MCS) were introduced into TA1015 (BW25113 DlacI) (14,20), which was constructed as described previously (14). The control strain TA2043 (harboring the isopropanol production pathway and the conventional MTS) was obtained by introducing three plasmids, i.e., pTA147, pTA216, and pTA965 (PLlacO1::tetR, PLtetO1::gltA.LAA), into E. coli strain TA1184 (BW25113 DlacI DgltA), which was constructed as described previously (14). The strain with an enhanced synthetic metabolic bypass system (TA2044) was obtained by introducing three plasmids, i.e., pTA147, pTA216, and pTA982 (PLlacO1::tetR, poxB, acs, PLtetO1::gltA.LAA), into E. coli strain TA1184 (BW25113 DlacI DgltA). TA2579 (harboring the isopropanol production pathway and the MTS installed with the redesigned synthetic metabolic bypass) was obtained by introducing three plasmids pTA147, pTA965, and pTA1251 (PLlacO1::poxB, acs, PlacIq::lacI) into TA1184 (BW25113 DlacI DgltA). TA3447 (harboring the isopropanol production pathway and the synthetic metabolic bypass, but without the MTS) was obtained by introducing three plasmids, i.e., pTA147, pTA1065, and pTA1251, into TA1015 (BW25113 DlacI). Because the double-knockout of gltA and pta was lethal for E. coli strains, the deletion of pta was performed to DgltA strains which was complemented the gltA by the plasmid harboring gltA.LAA under control of PLtetO1 promoter (pTA959, pTA965 or pTA982). For this end, at first, TA1979, TA2593, TA1980 and TA2618 strains were obtained by introducing each two types of plasmids (for the constitutive lacI expression and the gltA under control of PLtetO1) into TA1184, as described in Table 1. Next, TA1988, TA2604, TA2128 and TA2622 strains were obtained by deletion of pta gene of TA1979, TA2593, TA1980 and TA2618 respectively through the P1 transduction by using JW2294 (BW25113 Dpta::kanR) as the donor strain (14,20). Thus, TA2012, TA2613, TA2145 and TA2625 strains were obtained by removal of kanamycin resistance gene in TA1988, TA2604, TA2128 and TA2622 strains through the FLPpromoted recombination by using pCP20 (19). At last, TA2027, TA2620, TA2149 and TA2690 were obtained by introducing the pTA147 harboring the isopropanol production pathway into TA2012, TA2613, TA2145 and TA2625 respectively. Culture medium and conditions For construction of strains and plasmids, E. coli strains were cultured using 3 mL LuriaeBertani (LB) medium in test tubes by incubation at 37 C on a rotary shaker (250 rpm). For production of isopropanol, we used M9 medium (6.0 g/L Na2HPO4, 3.0 g/L KH2PO4, 1.0 g/L NH4Cl, 0.50 g/L NaCl, 0.17 mg/L FeCl3, 1.0 mM MgSO4, and 0.10 mM CaCl2) containing 0.1% (v/v) A5 trace metals mix, 10 ppm thiamin hydrochloride, 100 mg/mL ampicillin, 40 mg/mL chloramphenicol, and 100 mg/mL spectinomycin. The A5 trace metals mix contained the following (in 5 M HCl): 2.9 g/L H3BO3, 1.8 g/L MnCl2$4H2O, 0.22 g/L ZnSO4$7H2O, 0.39 g/L Na2MoO4$2H2O, 0.079 g/L CuSO4$5H2O, and 0.05 g/L CO(NO3)2$6H2O. Precultures were cultured with 3 mL M9 medium containing 10 g/L glucose at 37 C on a rotary shaker (250 rpm). Overnight cultures were passaged at 1% (v/v) into 20 mL fresh M9 medium containing 20 g/L glucose and cultured at 30 C on a rotary shaker (250 rpm). For induction of the target gene expression, 0.1 mM IPTG was added. Analytical methods for extracellular metabolites The optical density was measured using a 96-well plate reader (Infinity 200 Pro; Tecan, Männedorf, Switzerland). Alcohol compounds were quantified using a GC-2010 Plus gas chromatograph (Shimadzu, Kyoto, Japan) equipped with a flame ionization detector and an AOC-20 automatic injector and sampler (Shimadzu). The separation of alcohol compounds was carried out using a DB-WAX capillary column (30 m; 0.32 mm inside diameter; 0.50 mm film thickness; Agilent Technologies). The gas chromatograph oven temperature was initially held at 40 C for 5 min, increased at 15 C/min to 100 C, and then increased at 100 C/min to 230 C, where it was maintained for 1.4 min. Helium was used as the carrier gas with a column flow rate of 48 mL/min. The injector and detector temperatures were maintained at 225 C and 235 C, respectively. For each measurement, 0.5 mL of culture supernatant was injected in the split-injection mode (1:12 split ratio). 1-Propanol was used as the internal standard. For measurement of glucose, 5 mL of filtered supernatant was analyzed using a high-performance liquid chromatography system (LC-20AD; Shimadzu) equipped with an autosampler (SIL-20AC HT; Shimadzu), a guard column (Shodex SP-G; Showa Denko K.K., Tokyo, Japan), ligand exchange chromatography column (Shodex SP0810; Showa Denko K.K.), and a differential refractive index detector (RID-10A; Shimadzu). The mobile phase was MilliQ water, the flow rate was 1.0 mL/min, and the column was kept at 80 C. For measurement of the fermentation by-products (aketoglutarate, citrate, pyruvate, malate, succinate, lactate, fumarate, formate, and acetate), 1 mL of filtered supernatant was analyzed using a high-performance liquid chromatography system (LC-20AD; Shimadzu) equipped with an autosampler (SIL-20AC HT; Shimadzu), a guard column (RSpak KC-G; Showa Denko K.K.), ion-exclusion chromatography column (Shodex RSpak KC-811; Showa

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

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Denko K.K.), and conductivity detector (CCD-10A; Shimadzu). The mobile phase was 5.0 mM p-nitrophenylphosphate, the flow rate was 1.0 mL/min, and the column was kept at 40 C. Analytical methods for intracellular metabolites To monitor the intracellular metabolite concentration, liquid chromatography triple quadrupole mass spectrometry (LC-QqQ-MS) and gas chromatography quadrupole mass spectrometry (GC-Q-MS) analyses were performed according to the method described by Kato et al. (21). TA2043, TA2579, TA2027, and TA2620 strains were cultured, and cultures were induced with 0.1 mM IPTG at 9 h. Cells were collected at 3 and 23 h to measure intracellular metabolites. Intracellular metabolites were extracted from collected cells according to the chloroform-methanol method (22). Cells were collected from culture broth by filtration through a 0.45-mm membrane filter and were quickly frozen in liquid nitrogen and stored at 80 C until further operation. The cells were resuspended in 2.0 mL precooled (80 C) methanol solution containing 804 mg/L adipic acid and 9 mg/L (þ)-10-camphorsulfonic acid by vigorously mixing with 0.25 g of 0.6-mm zirconia silica beads (ZS06-0001; Biomedical Science). After vigorously mixing, the sample was incubated at 20 C for 1 h. The suspension was then centrifuged at 15,000 g at 9 C for 5 min, after which 500 mL of the supernatant was transferred to a new 2.0-mL microcentrifuge tube. The collected supernatant was vigorously mixed with 700 mL chloroform and 500 mL MilliQ water and was then centrifuged at 15,000 g at 9 C for 5 min. The water phase of the extract (600 mL) was transferred to a new 2.0-mL microcentrifuge tube, dried under vacuum, and stored at 80 C until further operation. LC-QqQ-MS and GC-Q-MS analyses were then performed as described by Kato et al. (21). For LC-QqQ-MS analysis, dried extracts were dissolved in 50 mL MilliQ water and analyzed using an LC-QqQ-MS system (high-performance liquid chromatography, Agilent 1290 Infinity; MS, Agilent 6460 with Jet Stream Technology; Agilent Technologies, Waldbronn, Germany) controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies). For GC-Q-MS analysis performed

J. BIOSCI. BIOENG., using the GCMSQP-2010 system (Shimadzu, Kyoto, Japan), dried extracts were derivatized at 30 C for 90 min with 100 mL of 20 mg/mL methoxyamine hydrochloride in pyridine and at 37 C for 30 min after the addition of 50 mL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (GL Science, Tokyo, Japan).

RESULTS Construction of an MTS installed at the synthetic metabolic bypass To boost acetyl-CoA supply by the synthetic metabolic bypass associated with the conditional flux redirection, poxB and acs were cloned downstream of tetR under control of the PLlacO1 promoter in plasmid pTA982. The synthetic metabolic bypass and the MTS (pTA982) were introduced into E. coli strains TA1184 (DlacIDgltA) with the isopropanol production pathway (pTA147), and the resulting strain was renamed TA2044. Using TA2261 (control strain; isopropanol productive but without the MTS), TA2043 (conventional MTS stain), and TA2044, we investigated the effects of conditional enhancement of the synthetic metabolic bypass on isopropanol production. Fig. 2 shows the results of isopropanol production in flask fermentation. The conventional MTS strain TA2043 produced 3.5-fold higher isopropanol concentrations (53.1 mM) than the control strain TA2261 (14.4 mM) during a 70-h fermentation (Fig. 2C). Additionally, the glucose consumption rate of TA2043 improved by 35% compared with that of TA2261 (Fig. 2B). These results reproduced our

FIG. 2. Effects of the conditional enlargement of the synthetic metabolic bypass associated with the metabolic flux redirection. (A) Time course of cell growth (OD600). (B) Time course of extracellular glucose concentration. (C) Time course of extracellular isopropanol concentration. (D) Time course of extracellular acetate concentration. Symbols: open circles, TA2261 (control strain, ON-OFF: none, OFF-ON: isopropanol production); open triangles, TA2043 (conventional MTS strain, ON-OFF: TCA cycle, OFF-ON: isopropanol production); open squares, TA2044 (ON-OFF: TCA cycle, OFF-ON: isopropanol production and synthetic metabolic bypass). Error bars show standard deviations (n ¼ 3).

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

VOL. xx, 2017 previous findings on the construction of MTS (14). TA2044 showed significant growth inhibition (w31%) at the logarithmic growth phase (Fig. 2A). Despite this significant growth inhibition, the isopropanol production titer and the glucose consumption of TA2044 were almost the same as those of TA2043 (Fig. 2B, C). Thus, the specific isopropanol production rate at the logarithmic growth phase of TA2044 was 1.8-fold higher than that of TA2043. These results indicated that the synthetic metabolic bypass could improve isopropanol production if it was possible to reduce growth inhibition. To mitigate this growth inhibition, we focused on the enzyme expression burden of the synthetic metabolic bypass. TA2044 expressed the synthetic metabolic bypass genes from a mediumcopy plasmid harboring the p15A replication origin. To reduce the cost of the expression of the synthetic metabolic bypass components, we downregulated its expression level by replacement of poxB and acs from the medium-copy plasmid (pTA982) with the low-copy plasmid (pTA1251). This downregulated synthetic metabolic bypass (pTA1251) was introduced into E. coli strain TA1184 (DlacIDgltA) with the MTS (pTA965) and the isopropanol production pathway (pTA147), and the resulting strain was designated TA2579. Using TA2261 (control strain; isopropanol productive but without the MTS), TA2043 (conventional MTS stain), and T2579, we investigated the effects of the expression level of synthetic

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metabolic bypass components on isopropanol production and cell growth. As shown in Fig. 3A, the cell growth rates of all three strains were almost the same at the logarithmic growth phase. As a result, TA2579 produced 26% more isopropanol (63.6 mM) than TA2043 within 55 h (Fig. 3C), and the isopropanol production rate of TA2579 (1.16 mM1) was 61% higher than that of TA2043 (0.72 mM h1; Fig. 3C). The isopropanol production yield of TA2579 also was enhanced by 17% compared with that of TA2043. Furthermore, the glucose consumption rate of TA2597 (2.08 mM h1) was also increased by 12% compared with that of TA2043 (1.85 mM h1; Fig. 3B). The final isopropanol production titer of TA2579 was 4.4-fold higher than that of the conventional isopropanol production strain TA2261. When the downregulated synthetic metabolic bypass without the MTS were co-expressed with the isopropanol production pathway, there were no improvements in isopropanol production or glucose consumption, although the growth inhibition was clearly resolved (Fig. 6) For the further improvement, we performed pta (2.3.1.8) deletion and changing of the copy number for poxB and acs, however, no improvement in isopropanol production and glucose consumption was achieved (Fig. S1). However, acetate and pyruvate accumulation were reasonable to the copy number ratio between pta and acs. These results indicate the poxB and acs amount were controlled by the redesigned MTS in E. coli.

FIG. 3. Effects of the downregulation of synthetic metabolic bypass. (A) Time course of cell growth (OD600). (B) Time course of extracellular glucose concentration. (C) Time course of extracellular isopropanol concentration. (D) Time course of extracellular acetate concentration. Symbols: open circles, TA2261 (control strain, ON-OFF: none, OFF-ON: isopropanol production); open triangles, TA2043 (conventional MTS strain, ON-OFF: TCA cycle, OFF-ON: isopropanol production); closed squares, TA2579 (ON-OFF: TCA cycle, OFF-ON: isopropanol production and the synthetic metabolic bypass). Error bar show standard deviations (n ¼ 3).

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

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Effects of the synthetic metabolic bypass on pyruvate accumulation The synthetic metabolic bypass succeeded in improving the titer and yield of isopropanol production associated with the conditional redirection of metabolic flux by the MTS. To confirm the effects of the synthetic metabolic bypass and the MTS on the metabolic flux distribution around pyruvate and acetyl-CoA, the intracellular metabolite levels of TA2661, TA2043, and TA2579 were monitored by LC-QqQ-MS analysis. As shown in Fig. 4, pyruvate accumulated along with the progression of fermentation for all three strains (Fig. 4A). The intracellular pyruvate level of TA2043 reached 1.9-fold higher than that of control strain TA2261 at 43 h (Fig. 4A). Although still higher than the control strain, pyruvate accumulation in TA2579 harboring the MTS and the synthetic metabolic bypass was reduced by 33% compared with that of TA2043. Similar tendency was shown in the intracellular phosphoenolpyruvate level among the three strains (Fig. 4B). On the other hand, enhancement of intracellular acetyl-CoA levels was observed only in strains harboring the MTS, i.e., TA2043 and TA2579 (Fig. 4C). The citrate levels of these two strains were 63% lower than that of TA2261 (Fig. 4D). These results demonstrated that the MTS contributed to enhancement of the acetyl-CoA supply through interruption of the TCA cycle. As a result, the intracellular acetyl-CoA level of TA2579 was enhanced 6-fold

J. BIOSCI. BIOENG., compared with that of TA2043 at 23 h. These results verified that the synthetic metabolic bypass succeeded in conversion of the excess pyruvate toward acetyl-CoA, resulting in improvement of isopropanol production. Optimization of the induction time for dynamic metabolic flux regulation Conditional metabolic flux redirection requires optimal timing for improvement of isopropanol production since conditional interruption of the TCA cycle suppresses cell growth. We had already investigated the optimal timing for the conventional MTS strain TA2043 (IPTG addition at 6 h), but had not yet determined this parameter for strain TA2579, which harbored the synthetic metabolic bypass. To investigate the optimal timing for the induction of the conditional flux redirection with enhancement of the synthetic metabolic bypass, the chemical inducer IPTG was added to TA2579 at various times during the logarithmic growth phase (Fig. 5). Isopropanol production titer was highest when IPTG was added at 6 or 9 h (OD600 ¼ 0.3e0.8) from the start of batchfermentation (Fig. 5A). The optimal timing for the induction of the metabolic flux redirection was the same for the conventional MTS strain TA2043 and TA2579. In these cases, the specific isopropanol and specific glucose consumption rates were also highest, and the specific growth rate (m) was similar to that of the conventional MTS strain TA2043 (m ¼ 0.23 h1; Fig. 5B). When IPTG was added

FIG. 4. Monitoring of the intracellular metabolism around the MTS and the synthetic metabolic bypass. (A) Time course of intracellular pyruvate concentration. (B) Time course of intracellular phosphoenolpyruvate (PEP) concentration. (C) Time course of intracellular acetyl-CoA concentration. (D) Time course of intracellular citrate concentration. Symbols: open circles, TA2261 (control strain, ON-OFF: none, OFF-ON: isopropanol production); open triangles, TA2043 (conventional MTS strain, ON-OFF: TCA cycle, OFF-ON: isopropanol production); closed squares, TA2579 (ON-OFF: TCA cycle, OFF-ON: isopropanol production and the synthetic metabolic bypass). Error bars show standard deviations (n ¼ 3).

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FIG. 5. Optimization of the induction time of the metabolic flux redirection by the addition of IPTG. (A) Comparison of the total isopropanol production titers between the MTS strains TA2043 (open bar) and TA2579 (closed bars). For TA2579, 0.1 mM IPTG was added at various times after induction (0, 6, 9, and 12 h). For TA2043, 0.1 mM IPTG was added at 9 h, i.e., the optimized conditions described in a previous work (14). Each plot shows the cell density at the induction time. (B) Correlation between the cell density at the induction of metabolic flux redirection and specific cell growth rate (circles), specific glucose consumption rate (squares), and specific isopropanol production rate (triangles) for TA2579 (n ¼ 3).

at other time points, the isopropanol production titer decreased by 21e37% compared with that of the conventional MTS strain TA2043 (Fig. 5A). When IPTG was added at 0 h (OD600 ¼ 0.1), specific growth rate of TA2579 decreased by 70% as compared with that of TA2043. Although growth inhibition was not observed when IPTG was added at 12 h, isopropanol production titer and specific isopropanol production rate decreased by 37% and 59%, respectively, as compared with that of TA2043. DISCUSSION Metabolic imbalance is the cause of the decrease in productivity and yield of microbial chemical production in conventional static metabolic engineering, e.g., permanent deletion of competing pathways and overexpression of exogenous enzymes depending on static control of transcription and translation. Therefore, there is a growing interest in the development of synthetic biological tools for dynamic and/or conditional flux regulation, e.g., metabolite sensor-regulators (12,13) and other synthetic genetic circuits (14,23,24). We had constructed an MTS for conditional redirection

of the metabolic flux of the TCA cycle toward the target isopropanol production, resulting in dramatic improvements in titers and yields (14). However, we found that conditional flux redirection caused excessive accumulation of pyruvate, an important intermediate for both isopropanol production and cell growth. Therefore, in this study, we focused the design of a strain for conditional enhancement of the capacity of metabolic flux to prevent stagnation of the metabolic influx toward the acetyl-CoA supply via pyruvate decarboxylation. For this purpose, we designed a synthetic metabolic bypass as a system to conditionally boost acetyl-CoA supply in the engineered E. coli harboring the MTS. This enhancement of conditional bypass succeeded in reducing pyruvate accumulation and improved isopropanol productivity, titer, and yield when its expression level was preferred. This result demonstrated that the synthetic metabolic bypass functioned as an alternative pyruvate decarboxylation pathway, boosting acetyl-CoA supply. As a result of resolving this bottle neck at the stage of pyruvate decarboxylation, glucose consumption was also improved in TA2579. Since aerobic or micro-aerobic conditions are suitable for isopropanol by E. coli, we employed the synthetic

FIG. 6. Effects of co-expression of the downregulated synthetic metabolic bypass and isopropanol production pathways. (A) Time course of cell growth (OD600). (B) Time course of extracellular glucose concentration. (C) Time course of extracellular isopropanol concentration. Symbols: open circles, TA2261 (control strain, ON-OFF: none, OFF-ON: isopropanol production); open triangle, TA2043 (conventional MTS strain, ON-OFF: TCA cycle, OFF-ON: isopropanol production); closed circle, TA3447 (ON-OFF: none, OFF-ON: isopropanol production and the synthetic metabolic bypass). Error bars show standard deviations (n ¼ 3).

Please cite this article in press as: Soma, Y., et al., Synthetic metabolic bypass for a metabolic toggle switch enhances acetyl-CoA supply for isopropanol production by Escherichia coli, J. Biosci. Bioeng., (2017), http://dx.doi.org/10.1016/j.jbiosc.2016.12.009

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metabolic bypass using poxB and acs. For anaerobic fermentation, pflB (2.3.1.54, oxygen sensitive) also could be used for acetyl-CoA forming bypass from pyruvate. However, the synthetic metabolic bypass could not improve glucose consumption and isopropanol production when it was coexpressed with only the isopropanol production pathway. The advantages of the synthetic metabolic bypass may be enhanced synergistically through the functions of the MTS. The key enzymes in the glycolytic pathway, i.e., phosphofructokinase I and pyruvate kinase II, can enhance its enzymatic activity by decreasing the ATP/ ADP ratio (25e27). Therefore, glucose consumption can be enhanced by the induction of ATP shortage associated with the conditional interruption of the TCA cycle by the MTS. The synthetic metabolic bypass also consumes one molecule of ATP for the conversion of one molecule of pyruvate to one molecule of acetyl-CoA. The combination of conditional interruption of the TCA cycle and the synthetic metabolic bypass may enhance glucose consumption by accelerating the ATP shortage. Moreover, some recent studies have shown that manipulation of ATP utilization can enhance glucose consumption and target chemical production (28e30). We also showed the importance of the balanced carbon flow between pyruvate, acetate, and acetyl-CoA in order to achieve efficient production of isopropanol via the synthetic metabolic bypass (Fig. S1). Although it was difficult to achieve the finest balance of such carbon flow by the modification of copy number ratio alone, several regulatory factors for gene expression level have been developed and evaluated [e.g., promoter (31), RBS (32), 50 -UTR (33,34), ssrA protein degradation tag sequence (35)]. These advanced engineering tools should facilitate fine-tuning of key carbon flow and efficient functionalization of the genetic circuit in living cell. Finally, we optimized the induction time required for conditional flux regulation to balance the distribution of the carbon source between bacterial cell growth and isopropanol production. Depending on the induction time, the specific growth rate and specific isopropanol production rate were dramatically altered. The final isopropanol production titer was maximized when the product of the specific growth rate and the specific isopropanol production rate was maximized. These results demonstrated that the MTS could switch between two different E. coli phenotypes and that optimization of the induction time was important for the metabolic flux redirection. Dynamic and/or conditional regulation of gene expression using the synthetic genetic circuit has become a promising strategy in metabolic engineering for regulation of metabolic flux (11,36). However, there are still room for improvement of both the synthetic metabolic bypass and the MTS in order to reduce temporal accumulation of pyruvate and acetate. Thus, current efforts in synthetic biology to achieve quantitative control of gene expression would contribute to optimization of the expression levels of target genes controlled by the synthetic genetic circuit (37). Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2016.12.009. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (23119002) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) and a Grant-in-Aid for Creative Scientific Research (14J10450) from Japan Society for the Promotion of Science (JSPS). References 1. Prather, K. L. J. and Martin, C. H.: De novo biosynthetic pathways: rational design of microbial chemical factories, Curr. Opin. Biotechnol., 19, 468e474 (2008).

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