TY - JOUR
AU1 - Kudo,, Fumitaka
AU2 - Mori,, Ayaka
AU3 - Koide,, Mai
AU4 - Yajima,, Ryo
AU5 - Takeishi,, Ryohei
AU6 - Miyanaga,, Akimasa
AU7 - Eguchi,, Tadashi
AB - Abstract 2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one) is a biosynthetic intermediate of 2-deoxystreptamine-containing aminoglycoside antibiotics, including butirosin, kanamycin, and neomycin. In producer microorganisms, 2DOI is constructed from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS) with the oxidized form of nicotinamide adenine dinucleotide (NAD+). 2DOI is also known as a sustainable biomaterial for production of aromatic compounds and a chiral cyclohexane synthon. In this study, a one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate was investigated. First, 3 polyphosphate glucokinases (PPGKs) were examined to produce G6P from d-glucose and polyphosphate. A PPGK derived from Corynebacterium glutamicum (cgPPGK) was found to be suitable for G6P production under ordinary enzymatic conditions. Next, 7 DOISs were examined for the one-pot enzymatic reaction. As a result, cgPPGK and BtrC, the latter of which is a DOIS derived from the butirosin producer Bacillus circulans, achieved nearly full conversion of d-glucose to 2DOI in the presence of polyphosphate. Graphical Abstract Open in new tabDownload slide Graphical Abstract Open in new tabDownload slide 2-deoxy-scyllo-inosose, 2-deoxy-scyllo-inosose synthase, polyphosphate glucokinase, biomaterial, enzymatic synthesis Abbreviations Abbreviations DHQS: 3-dehydroquinate synthase 2DOIA: 2-deoxy-scyllo-inosamine 2DOI: 2-deoxy-scyllo-inosose DOIS: 2-deoxy-scyllo-inosose synthase G6P: d-glucose 6-phosphate NAD+: the oxidized form of nicotinamide adenine dinucleotide NADH: the reduced form of nicotinamide adenine dinucleotide PPGK: polyphosphate glucokinase 2-Deoxy-scyllo-inosose (2DOI, [2S,3R,4S,5R]-2,3,4,5-tetrahydroxycyclohexan-1-one, Figure 1) is a 6-membered cyclitol and the initial biosynthetic intermediate in the biosynthesis of 2-deoxystreptamine-containing aminoglycoside antibiotics such as kanamycin, neomycin, butirosin, and gentamicin (Kudo and Eguchi 2016). 2DOI is biosynthesized from d-glucose 6-phosphate (G6P) by 2-deoxy-scyllo-inosose synthase (DOIS), which belongs to the 3-dehydroquinate synthase (DHQS) family in the shikimate pathway (Kudo et al. 1999; Nango et al. 2008, Wu et al. 2007, Osborn et al. 2017). The DHQS family of enzymes are classified according to their substrate specificities against phosphorylated sugars such as G6P (for DOIS), 3-deoxy-arabino-heptulosonate 7-phosphate (for DHQS), 3,4-dideoxy-4-amino-arabino-heptulosonate 7-phosphate (for aminoDHQS), and sedoheptulose 7-phosphate (for 2-epi-5-epi-valiolone synthase) (Figure S1). A representative DOIS is BtrC, which is encoded in the butirosin biosynthetic gene cluster derived from Bacillus circulans SANK 72073, and its reaction mechanism has been investigated extensively, including by crystal structure analysis (Figure S2) (Nango et al. 2008). DOIS recognizes G6P and oxidizes the C4-OH to trigger β-elimination of the C6-phosphate group by increasing the acidity at C5. The conserved glutamate residue among the DOISs works as a base to abstract H5 to prompt the β-elimination (Hirayama et al. 2007). After reduction of the C4 carbonyl group of the intermediate by NADH and ring opening of the hemiacetal moiety, an aldol-type condensation occurs between C1 and C6 to afford 2DOI. In the producer strains of aminoglycoside antibiotics, 2DOI is converted to 2-deoxy-scyllo-inosamine (2DOIA) by an aminotransferase (Figure 1) (Tamegai et al. 2002, Huang et al. 2002, Zachman-Brockmeyer et al. 2017, Popovic et al. 2006). Subsequently, 2DOIA is modified by a dehydrogenase (Kudo et al. 2005, Yokoyama et al. 2007) and the same aminotransferase to give 2-deoxystreptamine (4,6-diamino-1,2,3-trihydroxycyclohexane) (Yokoyama et al. 2005), which is decorated with various aminosugars to afford a variety of 2-deoxystreptamine-containing aminoglycoside antibiotics (Figure S3) (Kudo and Eguchi 2016). Figure 1. Open in new tabDownload slide Occurrence of 2-deoxy-scyllo-inosose (2DOI) in the biosynthesis of 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotics. Figure 1. Open in new tabDownload slide Occurrence of 2-deoxy-scyllo-inosose (2DOI) in the biosynthesis of 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotics. 2DOI can be used as a chiral building block for organic synthesis as a cyclohexane scaffold because it contains 4 stereocenters on the cyclohexanone structure and a ketone to initiate a variety of chemical reactions. In fact, we synthesized carbaglucose 6-phosphate as an analog of G6P from a 2DOI derivative (Nango et al. 2004). Further treatment with hydrogen iodide converted 2DOI to catechol, which is a sustainable aromatic compound of carbohydrate origin (Kakinuma et al. 2000). Hansen and Frost used 0.5 M H3PO4 under reflux conditions to convert 2DOI to 1,2,4-trihydroxybenzene, which was deoxygenated with Rh/Al2O3 under reflux conditions to yield hydroquinone (Hansen and Frost 2002). 2DOI is expected to be a useful biomaterial. One of the bottlenecks in efficient production of 2DOI with DOIS is preparation of G6P as a substrate. In a previous study, we used hexokinase to prepare G6P from d-glucose and ATP for the DOIS reaction in situ, and produced 2DOI from d-glucose in a one-pot reaction (Kakinuma et al. 2000). Using this method, chiral deuterium labeled (6S)-[6-2H]G6P and (6R)-[6-2H]G6P were prepared to elucidate the stereochemical reaction mechanism of DOIS (Figure S2) (Nango et al. 2003). Genetically engineered Escherichia coli was constructed by Kogure and coworkers to supply G6P for the DOIS reaction in vivo leading to the efficient production of 2DOI by fermentation (29.5 g/L culture) (Kogure et al. 2007). Similar attempts have been made with Bacillus subtilis to enhance the production of 2DOI (38.0 g/L culture) (Lim et al. 2018). In addition, Saccharomyces cerevisiae has been examined as a host strain to produce 2DOI, although the knockouts of G6P isomerase and G6P dehydrogenase genes did not improve the production (titer not indicated) (Al-Fahad et al. 2020). The btrC gene has been introduced into the cyanobacterium Synechococcus elongatus to produce 2DOI without a carbon source in the culture medium (0.4 g/L culture) (Watanabe et al. 2018). Fermentation methodology is attractive to produce larger amounts of 2DOI, although the purification of neutral 2DOI from the culture takes several steps to remove the microorganism cells and the remaining medium (Takagi et al. 2006). Thus, chemical syntheses, including enzymatic syntheses, are advantageous because they produce relatively few contaminants and reduce the need for purification of 2DOI. In the previous enzymatic synthesis of 2DOI from d-glucose, we used ATP as a phosphorylation reagent to supply G6P in situ even though ATP is an expensive reagent for 2DOI production (Kakinuma et al. 2000). In the present study, we examined the use of polyphosphate glucokinases (PPGKs) with cheap polyphosphate to supply G6P in situ coupled with DOIS to produce 2DOI in one-pot enzymatic reaction. PPGK catalyzes the phosphorylation of d-glucose with polyphosphate, which is widely utilized as a food additive (EFSA Panel on Food Additives and Flavourings (FAF) 2019) and an environmentally and economically friendly biomaterial (Wang et al. 2018). Consequently, we achieved the efficient 2DOI production (0.73 g/L) in one-pot enzymatic reaction. Materials and methods Preparation of PPGK The codon-optimized artificial ppgk genes derived from Corynebacterium glutamicum (cgPPGK, UniProt Q8NPA4, A0A072Z821) (Lindner et al. 2010) and Thermobifida fusca (carbohydrate-binding module family 3 [CBM3]-tfPPGK, UniProt Q47NX5) (Liao et al. 2012) (Supplementary nucleotide sequences) were obtained from Operon Biotechnology. The cgppgk and CBM3-tfppgk gene fragments were inserted into the NdeI/XhoI restriction sites of pColdI to yield the expression plasmids cgppgk/pColdI and CBM3-tfppgk/pColdI. CBM3-tfppgk/pColdI was utilized as a PCR template to amplify the tfppgk gene with a primer set of tfppgk-F (5′-ACATATGGCATCTCGGGGAC-3′) and pColdI-R (5′-CGCATTCTCATTGCACCCAA-3′). The amplified tfppgk gene fragments were inserted into the NdeI/XhoI restriction sites of pColdI to yield the expression plasmid tfppgk/pColdI. The ppgk gene derived from Streptomyces coelicolor A3(2) (scPPGK, Sco5059, UniProt Q9ADE8) was cloned as scppgk/pColdI in the previous report (Koide et al. 2013). All of PPGKs were expressed as His-tag fusion protein at N-terminus. The expression plasmids cgppgk/pColdI and scppgk/pColdI were introduced into E. coli BL21(DE3) by electroporation. The resulting cgppgk/pColdI/BL21(DE3) and scppgk/pColdI/BL21(DE3) strains were cultured in lysogeny broth (LB) containing 50 µg/mL of ampicillin at 37 °C and 200 rpm until the OD600 reached 0.4-0.7, after which the culture was cooled in an ice-water bath. After cooling for 30 min, isopropyl 1-thio-β-d-galactopyranoside (final concentration 0.2 mM) was added and culture was continued at 15 °C overnight. The E. coli cells were harvested by centrifugation (5800 g, 4 °C, 20 min, HIMAC CENTRIFUGE, Hitachi, Ibaraki), and then washed with 50 mM Tris-HCl buffer containing 10% glycerol (pH 7.7, buffer A). After centrifugation (16 100 g, 4 °C, 20 min, Kubota 3700, Kubota), the cells were suspended in buffer A. The cell suspension was treated by sonication (QSonica Model Q55, Newtown, CT) to obtain a cell-free extract. After centrifugation (16 100 g, 4 °C, 20 min), the supernatant was loaded on a TALON Metal Affinity Resin® (Clontech) column and washed with buffer A containing 20 mM imidazole. The cgPPGK and scPPGK proteins were then eluted with buffer A containing 200 mM imidazole. The solutions were passed through a PD-10 column (GE Healthcare) to obtain purified cgPPGK and scPPGK (Figure S4). The enzyme solutions were stored in 1.5-mL plastic tubes and immediately used for enzymatic assays. To confirm the sizes of the recombinant proteins, SDS-PAGE (12.5%) was performed. The protein concentrations were estimated using a Nanodrop spectrophotometer (Thermo Scientific). The expression plasmid tfppgk/pColdI and pGro7 (TaKaRa Bio) were introduced to E. coli BL21(DE3) by electroporation to obtain the tfppgk/pColdI/pGro7/BL21(DE3) strain. The resulting tfppgk/pColdI/pGro7/BL21(DE3) was cultured in LB containing 50 µg/mL of ampicillin, 30 µg/mL of chloramphenicol, and 0.5 g/mL of l-arabinose for expression. The procedure to obtain tfPPGK was the same as that for cgPPGK and scPPGK (Figure S4). Assay of PPGK The enzymatic activities of cgPPGK, tfPPGK, and scPPGK were examined by detecting G6P formation using a continuous coupling enzymatic assay with G6P dehydrogenase and NAD+ from Sigma-Aldrich. The assay solution contained 22.5 mM d-glucose, 3 mM sodium hexametaphosphate (mainly [NaPO3]6, Kanto Chemical) or sodium polyphosphate EMPLURA® (Graham's salt; PolyP25, [NaPO3]n where n = ∼25, Merck), 5 mM MgCl2, 3 mM NAD+, 1 U/mL G6P dehydrogenase, and 1.9-7.6 µM cgPPGK, tfPPGK, or scPPGK in 200 µL of buffer A. The enzymatic reaction was initiated by adding the required PPGK at 28°C. NADH formation was monitored at 340 nm (ε340nm = 6220 M−1 cm−1) to estimate the specific activities of the PPGKs (µmol min−1 mg−1). The initial velocities were estimated from the reaction rate between 150 and 350 s. To find the optimum concentrations of MgCl2 and PolyP25 for the cgPPGK reaction, final concentrations of 0.2, 0.4, 0.6, and 1.0 mM MgCl2 and 2, 4, 8, and 10 mM PolyP25 were examined. For the enzymatic assay for cgPPGK, Tris-HCl buffer (50 mM, pH 7.7) with no glycerol was used because glycerol slightly inhibited the one-pot reaction with DOIS (see below). Preparation of 2DOIS The DOIS gene (BtrC, UniProt Q9S5E2) encoded in the butirosin biosynthetic gene cluster derived from Bacillus circulans SANK 72073 (Kudo et al. 1999, Ota et al. 2000) was originally cloned in the pET30 vector. The BtrC/pET30 plasmid was digested with NdeI/HindIII and introduced into the same restriction site of pET28 vector to yield btrC/pET28. Another btrC gene (btrC21558) encoded in the genome of the butirosin producer strain B. circulans ATCC 21558 (Tsukiura et al. 1973, Takeishi et al. 2015) (DDBJ Accession LC571042) was amplified by PCR with the genomic DNA as the template and a primer set of btrC21558-F (5′-ACATATGACGACTAAACAAATTTGTTTCG-3′) and btrC21558-R (5′-CAAGCTTGTCCCGGTTCATCC-3′). The resultant btrC21558 gene fragment was introduced into the NdeI/HindIII restriction site of the pET28 vector to yield btrC21558/pET28. The codon-optimized artificial DOIS genes with the NdeI site at the 5′-terminus and HindIII site at the 3′-terminus for GntB (UniProt Q70KD0) encoded in the gentamicin biosynthetic gene cluster (Unwin et al. 2004), IstC (UniProt Q2UZD2) encoded in the istamycin biosynthetic gene cluster (Piepersberg 2006), and DOIS derived from the Rickettsia endosymbiont of Ixodes scapularis (REIS_1500, Rick_DOIS, UniProt C4YVL4) [Gillespie et al. 2012] were obtained from Operon Biotechnology (Supplementary nucleotide sequences). The gntB, istC, and rick_dois gene fragments were digested with NdeI and HindIII. The resultant DNA fragments were inserted into the same restriction site of the pET28 vector to yield gntB/pET28, istC/pET28, and rick_dois/pET28. The genes for NeoC (UniProt Q53U19) encoded in the neomycin biosynthetic gene cluster (Kudo et al. 2005) and AlloH (UniProt Q0X0F4) encoded in a putative tobramycin biosynthetic gene cluster derived from Streptoalloteichus hindustanus JCM 3268 (Hirayama et al. 2006) were prepared according to previous reports (Kudo et al. 2005, Hirayama et al. 2006). The neoC and alloH genes, which were originally cloned in the pET30 vector, were digested with NdeI and HindIII. The resultant DNA fragments were introduced into the same restriction sites of the pET28 vector to obtain neoC/pET28 and alloH/pET28. The original size of the NeoC protein seemed to be unstable, presumably because the C-terminus 39 amino acid of NeoC (431 aa) was long compared with the other DOIS homologs (data not shown). Thus, a gene for short NeoC (NeoC-short, 392 aa) was amplified with a primer set of NeoC-F (5′-GCCATATGCAGACCACCCGC-3′) and NeoC-short-R (5′-CAAGCTTCAGTCCGGTACGGGGCCCGTG-3′) using the neoC/pET28 plasmid as a template. The resultant DNA fragments were introduced into the NdeI/HindIII site of pET28 to yield neoC-short/pET28. All of the expression plasmids (btrC/pET28, btrC21558/pET28, gntB/pET28, istC/pET28, rick_dois/pET28, alloH/pET28, and neoC-short/pET28) were introduced into E. coli BL21(DE3) to express as His-tag fusion protein at N-terminus. The culture conditions and purification methods were the same as those for the PPGKs except for the antibiotics used during the culture and the elution buffer used during the purification (Figure S4). To maintain the pET28-based plasmids, 50 µg/mL of kanamycin was added to the LB. For purification of the DOISs, 0.2 mM CoCl2 containing Tris-HCl buffer (pH 7.7) was used to wash and elute proteins from the TALON Metal Affinity Resin. Assay of 2DOIS The enzymatic activities of the DOISs were evaluated by detecting the release of inorganic phosphate during the enzymatic reaction. A continuous coupling enzymatic assay was conducted with purine nucleotide phosphorylase (Sigma–Aldrich) and 2-amino-6-mercapto-7-methylpurine ribonucleoside (methyl thioguanosine, Santa Cruz Biotechnology) according to the method reported by Webb (Webb 1992). The assay solution (600 µL in a micro quartz cuvette) contained 0.1 mM methyl thioguanosine (60 µL of a 1 mM solution), 1 U of purine nucleotide phosphorylase (6 µL of a 100 U/mL solution), 5 mM NAD+ (6 µL of a 0.5 M solution), 0.2 mM CoCl2, 10-400 µM G6P, and approximately 0.5-7.3 µM DOIS in 50 mM Tris-HCl buffer (pH 7.7). The reaction was initiated by addition of the DOIS and then incubation was carried out at 28°C. The increase in the absorbance at 360 nm, attributable to the formation of 2-amino-6-mercapto-7-methylpurine, per second was monitored using a UV-2450 spectrophotometer (Shimadzu). The initial velocity was determined from the linear portion of the optical density profile (150-250 s, ε360nm = 11 000 M−1 cm−1). Steady-state kinetic parameters were estimated by fitting the obtained data to the Michaelis–Menten equation. One-pot enzymatic synthesis of 2DOI For the one-pot enzymatic synthesis of 2DOI, an enzymatic solution containing 5 mM d-glucose, 3 mM PolyP25, 0.3 mM NAD+, 0.5 mM MgCl2, 0.2 mM CoCl2, 7.5 µM cgPPGK, and 7.5 µM BtrC in 50 mM Tris-HCl buffer (pH 7.7) was placed in a 5-mL plastic tube. The solution was incubated at 28 °C and 15 rpm for 24 h. The reaction was monitored by TLC developed with CHCl3: CH3OH: H2O (10:5:1, v/v/v) and stained with anisaldehyde–sulfuric acid dip. The stained colors of the spots for d-glucose (Rf = 0.3) and 2DOI (slightly higher Rf) were reddish and greenish, respectively. The enzyme reaction was quenched by adding a half volume of CH3OH and the precipitated protein was removed by centrifugation (16,100 g, 10 min). The obtained supernatant (100 µL) was mixed with 10 µL of O-(4-nitrobenzyl)hydroxylamine hydrochloride (NBHA, Acros) pyridine solution (5 mg of NBHA in 1 mL of pyridine) and incubated at 60 °C for 1 h. After removal of the solvent by a centrifugal concentrator (VC-36N, TAITEC), the residue was suspended in 300 µL of CHCl3. The suspension was loaded onto Sep Pak Plus Silica (690 mg/cartridge, Waters) that was conditioned with CHCl3 and washed with 7 mL of CHCl3: CH3OH (30:1, v/v). The 4-nitrobenzyloxime derivative of 2DOI was eluted with 5 mL of CHCl3: CH3OH (5:1, v/v). After removal of the solvent by the centrifugal concentrator, the residue was dissolved in 100 µL of CH3OH. An aliquot (5 µL) of this solution was analyzed by HPLC (Hitachi L-6000 pump, Hitachi L-4000 UV-detector, and Chromato-PRO) with a TSKgel ODS 80TM column (4.6 mm × 7.5 cm, 5 µm particle size, TOSOH). The mobile phase was 20% aqueous CH3OH with a flow rate of 1.0 mL/min and the elution mode was isocratic. The eluent was monitored at 273 nm. Another aliquot (2 µL) of the solution was analyzed by LC-ESI-MS (LC-MS-2020, Shimadzu) with a L-column2 ODS column (2.1 mm × 150 mm, 3 µm particle size, Chemical Evaluation and Research Institute, Japan). The mobile phase was CH3CN (solvent A) and water (solvent B) with a flow rate of 0.1 mL/min. A linear gradient of 20%-60% A from 0-30 min and 60%-80% A from 30-40 min was used for elution. One-pot enzymatic synthesis of 2DOIA The gene for the Gln:2DOI aminotransferase IstS (UniProt Q2UZC8) encoded in the istamycin biosynthetic gene cluster (Accession code: AJ845083) (Piepersberg 2006) was amplified by PCR with the genomic DNA derived from Streptomyces tenjimariensis JCM 8368 as the template and a primer set of istS-F (5′-ACATATGAGCGAACTCGCGATACTC-3′) and istS-R (5′-ACTGCAGCTCAGCGCAGC-3′). The resultant istS gene fragment was digested with NdeI/PstI and introduced into the same restriction sites of the pColdI vector to yield istS/pColdI. The istS/pColdI plasmid was introduced into E. coli BL21(DE3) to express the IstS protein. The culture conditions and purification methods were the same as those for the PPGKs except for the elution buffer used during purification (Figure S4). For the one-pot enzymatic synthesis of 2DOIA, 50 mM HEPES–NaOH buffers (pH 7.7) were used for purification of the proteins. An enzymatic solution containing 20 mM d-glucose, 3 mM PolyP25, 40 mM l-glutamine, 0.9 mM NAD+, 0.4 mM pyridoxal 5′-phosphate (PLP), 0.5 mM MgCl2, 0.2 mM CoCl2, 1 µM cgPPGK, 1 µM BtrC, and 2 µM IstS in 50 mM HEPES–NaOH buffer (pH 7.7) was placed in a 5-mL plastic tube. The reaction was monitored by TLC developed with CHCl3: CH3OH:1 M aqueous NH3 (1:3:2, v/v/v) and stained with the ninhydrin dip. The enzymatic solution was incubated at 28 °C for 35 h until the reaction did not proceed. The reaction was then quenched by adding an equal volume of ethanol, and the precipitated proteins were removed by centrifugation (16 100 g, 10 min). The supernatant was loaded onto Dowex AG1-X8 (OH− form) to remove anionic compounds. The flow-through solution was then loaded onto Amberlite CG-50 (NH4+ form) and washed with distilled water. The adsorbed 2DOIA was eluted with 2 M aqueous NH4+. After evaporation, the residue was dissolved in water and loaded onto Dowex AG1-X8 (SO42− form) to obtain 2DOIA sulfate (0.34 mmol, 72 mg) from 400 mg of d-glucose (2.2 mmol, in twenty-two 5 mL tubes, 15% yield). The chemical structure of 2DOIA was confirmed by NMR (Bruker DRX500) in D2O. Results and discussion We have previously characterized a PPGK derived from S. coelicolor A3(2) (scPPGK) (Koide et al. 2013). Therefore, we compared the catalytic efficiency of scPPGK with those of 2 well-characterized stable PPGKs derived from C. glutamicum (cgPPGK) (Lindner et al. 2010) and T. fusca (tfPPGK) (Liao et al. 2012) to select an appropriate enzyme for a one-pot reaction with DOIS. The specific activities (µmol·min−1·mg−1) of the PPGKs were evaluated in the presence of d-glucose (22.5 mM), polyphosphate (n = 6 or 25, 3 mM), MgCl2 (5 mM), G6P dehydrogenase, and NAD+ at 28°C by detecting NADH formation (Table 1). Among the PPGKs, that produced from C. glutamicum (cgPPGK) with PolyP25 showed the highest activity (0.76 µmol min−1 mg−1). The optimum concentrations of MgCl2 and PolyP25 in the cgPPGK reaction were 0.5 mM and up to 4 mM, respectively (Figures S5 and S6). Table 1. Specific activities (µmol min–1 mg–1) of polyphosphate glucokinases (PPGKs) with PolyP6 or PolyP25 at 28 °C . PolyP6 . PolyP25 . scPPGK 0.039 0.050 cgPPGK 0.066 0.76 tfPPGK 0.062 0.41 . PolyP6 . PolyP25 . scPPGK 0.039 0.050 cgPPGK 0.066 0.76 tfPPGK 0.062 0.41 Open in new tab Table 1. Specific activities (µmol min–1 mg–1) of polyphosphate glucokinases (PPGKs) with PolyP6 or PolyP25 at 28 °C . PolyP6 . PolyP25 . scPPGK 0.039 0.050 cgPPGK 0.066 0.76 tfPPGK 0.062 0.41 . PolyP6 . PolyP25 . scPPGK 0.039 0.050 cgPPGK 0.066 0.76 tfPPGK 0.062 0.41 Open in new tab Next, we investigated the catalytic efficiencies of DOISs from several microbial origins that were deposited in public DNA data banks (Figures S7 and S8). We expected that highly active DOISs might exist because of amino acid sequence diversity. The kinetic properties of the following 7 DOIS homologs were examined: 2 BtrCs from the butirosin producers Bacillus circulans SANK 72073 (Kudo et al. 1999) and ATCC 21 558 (Accession code: LC571042), GntB from the gentamicin producer Micromonospora echinospora (Unwin et al. 2004), IstC from the istamycin producer S. tenjimariensis (Piepersberg 2006), NeoC (NeoC-short with C-terminus 39 aa trimmed from the original NeoC) from the neomycin producer Streptomyces fradiae (Kudo et al. 2005), AlloH from the apramycin/tobramycin producer Streptoalloteichus hindustanus (Hirayama et al. 2006), and Rick-DOIS (REIS_1500) from the Rickettsia endosymbiont of Ixodes scapularis (Gillespie et al. 2012). Although it has not been reported that the Rickettsia endosymbiont of Ixodes scapularis produces aminoglycoside antibiotics, its genome sequence shows that the DOIS gene Rick-DOIS (REIS_1500) is associated with the paromamine/neamine-type pseudodisaccharides of aminoglycoside biosynthetic genes (REIS_1492, 1493, 1496, and 1501∼1505) (Figure S8) (Gillespie et al. 2012). All of the tested DOISs, including Rick-DOIS, showed the expected enzymatic activity (Figure S9) and the steady-state kinetic parameters were determined (Table 2). These results suggest that Rick-DOIS could function as a DOIS, which might produce unidentified 2DOI-derived aminoglycoside antibiotics. Among the tested DOISs, the 2 BtrCs and IstC showed high kcat/KM values in the range of 4200-6300 M−1·s−1. From these, the biochemically well-characterized DOIS (BtrC) derived from B. circulans SANK 72073 (Kudo et al. 1999) was selected as the DOIS for the one-pot enzymatic synthesis of 2DOI from d-glucose and polyphosphate. Table 2. Steady-state kinetic parameters of the 2-deoxy-scyllo-inosose synthases (DOISs) at 28 °C . BtrC . BtrC21558 . GntB . IstC . Rick-DOIS . Km [µM] 474 ± 80 680 ± 62 993 ± 47 93 ± 17 432 ± 90 kcat [s−1] 0.20 ± 0.04 0.43 ± 0.14 0.077 ± 0.003 0.058 ± 0.007 0.040 ± 0.006 kcat/Km [M−1•s−1] 4240 6320 773 6240 933 . BtrC . BtrC21558 . GntB . IstC . Rick-DOIS . Km [µM] 474 ± 80 680 ± 62 993 ± 47 93 ± 17 432 ± 90 kcat [s−1] 0.20 ± 0.04 0.43 ± 0.14 0.077 ± 0.003 0.058 ± 0.007 0.040 ± 0.006 kcat/Km [M−1•s−1] 4240 6320 773 6240 933 . NeoC-short . AlloH . Km [µM] 401 ± 17 278 ± 11 kcat [s−1] 0.0022 ± 0.0002 0.040 ± 0.006 kcat/Km [M−1•s−1] 54.6 1440 . NeoC-short . AlloH . Km [µM] 401 ± 17 278 ± 11 kcat [s−1] 0.0022 ± 0.0002 0.040 ± 0.006 kcat/Km [M−1•s−1] 54.6 1440 Open in new tab Table 2. Steady-state kinetic parameters of the 2-deoxy-scyllo-inosose synthases (DOISs) at 28 °C . BtrC . BtrC21558 . GntB . IstC . Rick-DOIS . Km [µM] 474 ± 80 680 ± 62 993 ± 47 93 ± 17 432 ± 90 kcat [s−1] 0.20 ± 0.04 0.43 ± 0.14 0.077 ± 0.003 0.058 ± 0.007 0.040 ± 0.006 kcat/Km [M−1•s−1] 4240 6320 773 6240 933 . BtrC . BtrC21558 . GntB . IstC . Rick-DOIS . Km [µM] 474 ± 80 680 ± 62 993 ± 47 93 ± 17 432 ± 90 kcat [s−1] 0.20 ± 0.04 0.43 ± 0.14 0.077 ± 0.003 0.058 ± 0.007 0.040 ± 0.006 kcat/Km [M−1•s−1] 4240 6320 773 6240 933 . NeoC-short . AlloH . Km [µM] 401 ± 17 278 ± 11 kcat [s−1] 0.0022 ± 0.0002 0.040 ± 0.006 kcat/Km [M−1•s−1] 54.6 1440 . NeoC-short . AlloH . Km [µM] 401 ± 17 278 ± 11 kcat [s−1] 0.0022 ± 0.0002 0.040 ± 0.006 kcat/Km [M−1•s−1] 54.6 1440 Open in new tab 2DOI production with BtrC (3, 4, 5, or 7.5 µM) and cgPPGK (3, 4, 5, or 7.5 µM) was investigated with several concentrations of d-glucose (3, 5, or 7 mM) and PolyP25 (1.8, 3.0, or 4.2 mM) in the presence of 0.5 mM MgCl2 and 0.3 mM NAD+ at 28 °C for 24 h. The TLC results indicated that all tested reaction conditions afforded 2DOI (Figure S10). However, when high concentrations of d-glucose (> 7 mM) were utilized, approximately half of the d-glucose was not consumed. For a mixture containing d-glucose (5 mM), PolyP25 (3 mM), MgCl2 (0.5 mM), NAD+ (0.3 mM), BtrC (7.5 µM), and cgPPGK (7.5 µM), all of d-glucose was consumed within 24 h at 28 °C. To estimate the production yield of 2DOI under these conditions, quantitative HPLC analysis was conducted after the 4-nitrobenzyloxime derivatization of 2DOI with NBHA (Figure 2). The HPLC results indicated that 22.5 µmol of 2DOI was produced from 25 µmol of d-glucose in 5 mL of the reaction solution (0.73 g/L reaction). Thus, the production yield of 2DOI from d-glucose was estimated to be 90%. When the reaction volume was increased to 15 mL, d-glucose was fully consumed. This production yield is remarkable by comparison with previously reported enzymatic synthesis of DOI with hexokinase (38% yield) (Kakinuma et al. 2000). We also investigated the conversion of d-glucose to 2DOIA via 2DOI in a one-pot reaction. We selected l-glutamine:2DOI aminotransferase IstS, which is derived from the istamycin biosynthetic pathway (Piepersberg 2006), because we have the IstS protein in our hand to investigate whether IstS is involved in another transamination to construct the 1,4-diaminocyclitol moiety in istamycin biosynthesis (Figure S3). As a result, an acceptable yield of 2DOIA (15% isolated yield as the sulfate salt) was obtained from d-glucose, l-glutamine, and PolyP25 (Figure S11). Figure 2. Open in new tabDownload slide HPLC analysis of a 4-nitrobenzyloxime derivative of 2DOI from a one-pot enzymatic reaction of d-glucose and PolyP25 with cgPPGK and BtrC. Solid arrow, 4-nitrobenzyloxime of 2DOI; and broken arrow, 4-nitrobenzyloxime of d-glucose. Figure 2. Open in new tabDownload slide HPLC analysis of a 4-nitrobenzyloxime derivative of 2DOI from a one-pot enzymatic reaction of d-glucose and PolyP25 with cgPPGK and BtrC. Solid arrow, 4-nitrobenzyloxime of 2DOI; and broken arrow, 4-nitrobenzyloxime of d-glucose. Frost and coworkers have demonstrated benzenoid production from d-glucose with enzymes in the shikimate pathway (Draths and Frost 1995, Li and Frost 1998, Kambourakis et al. 2000, Draths and Frost 1994). They introduced dehydroshikimate biosynthetic genes and the dehydroshikimate dehydratase gene into E. coli to produce protocatechuic acid, which was then converted to catechol by protocatechuic acid decarboxylase (Draths and Frost 1995). They produced 18.5 mM catechol from 56 mM d-glucose on a 1-L scale after culturing the genetically engineered E. coli for 48 h. Although this methodology is remarkable because it involves benzene-free production of aromatic compounds, it requires many enzymatic genes and careful control of the metabolism of E. coli. By comparison, the enzymatic synthesis of 2DOI established in the present study is a desirable alternative methodology to produce useful biomaterials from d-glucose. In summary, efficient enzymatic production of 2DOI from d-glucose and polyphosphate was achieved with DOIS and PPGK. Furthermore, 2DOIA was prepared from d-glucose, polyphosphate, and l-glutamine in a one-pot reaction. Development of immobilized DOIS and PPGK for improving the efficiency of 2DOI production will be investigated in future research. Author contribution F.K. and T.E. designed the research; A. Mori, M.K., R.Y., and R.T. performed the experiments; F.K., A. Mori, M.K., R.Y., R.T., A. Miyanaga, and T.E. analyzed data; F.K. and A. Miyanaga wrote the manuscript; all authors approved the final version of the manuscript. Funding This work was supported in part by the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (B) under Grant 19H02895 to F.K. and Grant 24350078 to T.E., and the Ministry of Education, Culture, Sports, Science and Technology (MEXT) through a Grant-in-Aid for Scientific Research on Innovative Areas under Grant 16H06451 to T.E. Disclosure statement The authors declare no conflict of interest. References EFSA Panel on Food Additives and Flavourings (FAF) . Re-evaluation of phosphoric acid–phosphates – di-, tri- and polyphosphates (E 338–341, E 343, E 450–452) as food additives and the safety of proposed extension of use . EFSA J 2019 ; 17 : e05674 . PubMed OpenURL Placeholder Text WorldCat Al-Fahad AJ , Al-Fageeh MB, Kharbatia NM et al. Metabolically engineered recombinant Saccharomyces cerevisiae for the production of 2-deoxy-scyllo-inosose (2-DOI) . Metab Eng Commun 2020 ; 11 : e00134 . 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TI - One-pot enzymatic synthesis of 2-deoxy-scyllo-inosose from d-glucose and polyphosphate
JF - Bioscience Biotechnology and Biochemistry
DO - 10.1093/bbb/zbaa025
DA - 2021-01-07
UR - https://www.deepdyve.com/lp/oxford-university-press/one-pot-enzymatic-synthesis-of-2-deoxy-scyllo-inosose-from-d-glucose-HLfwIe12dd
SP - 108
EP - 114
VL - 85
IS - 1
DP - DeepDyve
ER -