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Characterization of a novel serine hydroxymethyltransferase isolated from marine bacterium Arthrobacter sp. and its application on L-serine production

Characterization of a novel serine hydroxymethyltransferase isolated from marine bacterium... Ann Microbiol (2015) 65:1689–1698 DOI 10.1007/s13213-014-1008-7 ORIGINAL ARTICLE Characterization of a novel serine hydroxymethyltransferase isolated from marine bacterium Arthrobacter sp. and its application on L-serine production Junjie Huang & Lin Chen & Nan Hu & Wei Jiang & Gaobing Wu & Ziduo Liu Received: 27 June 2014 /Accepted: 21 November 2014 /Published online: 7 January 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract The current L-serine production relies mainly on Introduction cellular or enzymatic conversion from the precursor glycine plus a C1 compound. To date, only several reports have been Only several reports have been published on L-serine produc- published on L-serine production from glycine and methanol tion from glycine and methanol by methylotrophic bacteria by methylotrophic bacteria with the serine pathway. This work with the serine pathway (Hagishita et al. 1996;Shen et al. aimed to isolate a novel serine hydroxymethyltransferase 2010), and all the microorganisms used in the studies reported (SHMT) from the methanol-using Arthrobacter sp. and use were from the terrestrial environment. However, many other it for L-serine production with the enzymatic conversion bacteria also probably have the same function. The marine method. Here, A novel glyA gene was isolated from the realm covers 70 % of the earth’s surface, and provides the methanol-using Arthrobacter sp. by thermal asymmetric in- largest inhabitable space for living organisms, particularly terlaced PCR (TAIL-PCR), encoding a serine microbes (Das et al. 2006); thus isolating some novel and hydroxymethyltransferase (SHMT) with 440 amino acids, useful marine bacteria is feasible and important. belonging to the α-family of fold type I, and pyridoxal-5- Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is a phosphate (PLP) dependent enzymes. The enzyme was stable ubiquitous, highly conserved pyridoxal-5-phosphate (PLP)- in weakly alkali conditions, showing the optimal activity at dependent enzyme with tetrahydrofolate (THFA) as the C pH 7.8 and 45 °C, and a 2.75-fold increase in activity over the acceptor (Blakley 1955), and has been purified and extensive- corresponding enzyme of Escherichia coli. Two methods ly studied in animals (Jones and Priest 1976; Ulevitch and (resting cells reaction and enzymatic conversion) were Kallen 1977), plants (Rao and Appaji Rao 1982), and bacteria employed to produce serine. Using glycine (133 mM) and (Barra et al. 1983). SHMT plays an important role in the formaldehyde (13.3 mM) as substrates to produce serine by assimilation of C compounds, yielding the main L-serine enzymatic reaction, 93.6 mM L-serine was obtained with a intermediate. The enzymes involved in L-serine synthesis 70.4 % molar conversion rate from glycine to L-serine. Thus, are methanol dehydrogenase (EC 1.1.1.244) and SHMT, the the characteristics of this novel strain and its enzyme suggest latter of which is coded by the glyA gene. The former catalyzes that it has the potential for further research and industrial use. the oxidation of methanol to formaldehyde and the latter converts formaldehyde and glycine to L-serine by reaction. It is well-known that two molecules of glycine are needed for . . Keywords Arthrobacter sp. SHMT L-Serine enzymatic the synthesis of one molecule of L-serine, and the theoretical . . production Thermal asymmetric interlaced PCR RP-HPLC production yield is 50 % (mol/mol) (Izumi et al. 1993). If half of the C units are displaced by low-cost formaldehyde, the : : : : J. Huang L. Chen W. Jiang G. Wu Z. Liu (*) theoretical ratio of glycine conversion may be 100 % (Izumi State Key Laboratory of Agricultural Microbiology, College of Life et al. 1993). Therefore, isolating a strain with high methanol Science and Technology, Huazhong Agricultural University, dehydrogenase and SHMT activity is the key, and in this Wuhan 430070, China e-mail: lzd@mail.hazu.edu.cn study, one methylotrophic strain Arthrobacter sp. with high SHMT activity was isolated. N. Hu The study of enzymatic properties can contribute to a better College of Biotechnology and Pharmaceutical Engineering, Nanjing use of SHMT. In previous reports, many glyA genes were Tech University, Nanjing 211800, People’s Republic of China 1690 Ann Microbiol (2015) 65:1689–1698 Table 1 Primersused in thisstudy obtained by shotgun technology (Hamilton et al. 1985), direct PCR amplification based on the known genome information Primer 5′ to 3′ Reference (Schirch et al. 1985; Vidal et al. 2005), cDNA library (Byrne et al. 1992;Garrowetal. 1993), southern-blot hybridization DP-F CTSACCAAYAARTACGC This work CGAGGGYT (Miyata et al. 1993), and a genomic library constructed with DP-R ACCATSGGCGGSCKSGG This work the application of a probe (Shen et al. 2010). PCR amplifica- RTCGAAGG tion would be convenient only with known genome informa- SP1-F GGTCGAAGATCTTGGCT This work tion. Otherwise the aforementioned methods would be time- GAGCGTTTG SP2-F GAAGACGTTGTTGCCAA This work consuming, and thus libraries such as EMBL and GenBank GGGCATC with information about various genomes and nucleic acids SP3-F CGCAGGTGGAGATCACT This work would facilitate the search for information about relevant GTGAATCG SP1-R AACAATCATCTTCGGCT This work genes. In this work, the partial glyA (PglyA)sequence was GGTGCTCG amplified with degenerate primers designed based on the SP2-R TGGTAGGTTTCCTCATC This work result of multiple sequence alignment before the flanking CACGCCGTA sequences of PglyA were obtained by thermal asymmetric SP3-R AGCTTCATGCCGTGGGT This work CAAATGG interlaced PCR (TAIL-PCR). AD1 TGWGNAGSANCASAGA (Liu and Whittier 1995) The current L-serine production relies mainly on cellular or AD2 AGWGNAGWANCAWAGG (Liu and Whittier 1995) enzymatic conversion from the precursor glycine plus a C AD3 CAWCGICNGAIASGAA (Liu and Whittier 1995) compound (Peters-Wendisch et al. 2005). Because there is no AD4 TCSTIGNCITWGGA (Liu and Whittier 1995) need to add any coenzymes PLP and tetrahydrofolate (THFA) AD5 NGTCGASWGANAWGAA (Liu and Huang 1998) into the reaction system, a resting cells system is a useful AD6 NTCGASTWTSGWGTT (Liu and Huang 1998) method to produce L-serine from glycine and methanol by AD7 WGTGNAGWANCANAGA (Liu and Huang 1998) methylotrophic bacteria with the serine pathway (Izumi et al. AD8 WCAGNTGWTNGTNCTG (Emelyanov et al. 2006) 1993; Hagishita et al. 1996). Another useful method is the enzymatic reaction, manufacturing L-serine by constructing AD9 WGCNAGTNAGWANAAG (Amedeo et al. 2000) engineering bacteria and adding coenzymes (Hsiao et al. AD10 AWGCANGNCWGANATA (Amedeo et al. 2000) 1988), which has been rarely reported. In China, the enzymat- glyA-F CGGGATCCATGAGCAACC This work ic reaction method is mainly used for L-serine production with AGACTTTTGAA glyA-R CCGCTCGAGCTACTCGGA This work the enzyme primarily from Escherichia coli SHMT (Sun AACCTTTGGCA 2000; Zuo et al. 2007) In this paper, the two methods (resting cells reaction and IUPAC ambiguity codes, M=A/C, R=A/G, W=A/T, S=G/C, Y=C/T, enzymatic conversion) have been tried for L-serine produc- K=G/T, and N=A/G/C/T. I indicate inosine. F, forward; R, reverse. Restriction sites BamH1 and Xho1in primers glyA-F/R are underlined, tion, and the latter method was used to evaluate the difference respectively; Start and stop codons are in bold, respectively between the Arthrobacter sp. SHMT engineering bacterium and the Escherichia coli SHMT engineering bacterium in L- serine production. Media and growth conditions Materials and methods Escherichia coli strains were incubated in Luria-Bertani (LB) Materials medium, supplemented with ampicillin (100 μg/ml) if neces- sary, and other bacteria were cultured in agar 2216 medium. Restriction enzymes, pMD18-T vectors, T4 DNA ligases, Taq Screening media I and II were used for the screening of strains DNA polymerases, and DNA markers were purchased from with SHMT activity. Screening medium I consisted of meth- TaKaRa Company (Dalian, China). Kits for plasmid extrac- anol 1.2 % (v/v), (NH ) SO 37.84 mM, KH PO 7.34 mM, 4 2 4 2 4 tion and DNA purification were obtained from Qiagen Com- K HPO ·3H O 13.15 mM, MgSO ·7H O0.81 mM, MnSO · 2 4 2 4 2 4 −3 pany (Germany). All oligonucleotide primers (Table 1)were H O 0.012 mM, FeSO ·7H O7.19×10 mM, biotin 2.05× 2 4 2 −4 −4 synthesized and all DNA fragments were sequenced by 10 mM, thiamine hydrochloride 3.32×10 mM, NaNO GenScript Company (Nanjing, China). All the chemicals were 35.30 mM, and NaCl 34.22 mM, with the pH of the mixture of HPLC grade or biotechnology grade and purchased from being adjusted to 7.5. The components listed above were also Sigma unless specially noted. The THFA used in the enzy- used in the mixture of screening medium II except that the matic reaction to produce L-serine was independently synthe- concentration of methanol was increased to 5 % (w/v), and sized by our laboratory (Sun et al. 2000). 1.5 % (w/v) agar and 0.27 M glycine were added. Ann Microbiol (2015) 65:1689–1698 1691 Microorganism isolation at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min; and one final additional cycle at 72 °C for 10 min. The PCR product The bacteria were cultured by enrichment culture in screening was gel-purified, digested with BamH I and Xho I, and then culture medium I at 28 °C, and 1.2 % (v/v) methanol was ligated into the pGEX-6p-1 vector (Amersham Biosciences) added once every 2 days. After 7–10 days of growth, bacterial (Lin et al. 2009). The recombinant plasmids were verified by suspension was diluted to 10,000 times, 100,000 times, and DNA sequencing and designated as pGEX-6p-AmglyA. one million times with sterile water. Then these samples were Using the same method described above, the glyA gene spread onto plates filled with medium II, followed by 4–5days (Gene ID: 947022) from E. coli was also ligated into the of incubation at 28 °C. Finally, Arthrobacter sp. was isolated pGEX-6p-1 vector and designated as pGEX-6p-EcglyA. and preserved for further use for its high SHMT activity. Expression and purification of SHMT Gene cloning To optimize the expression of SHMT, the pGEX-6p-AmglyA To amplify the PglyA gene, degenerate primers DP-F and DP- plasmids were transformed into E. coli BL21 (DE3) compe- R(Table 1) were designed based on the result of multiple tent cells (Stratagene, USA). The transformants were cultured sequence alignment (Fig. 1a)of glyA gene sequences which in Luria–Bertani broth at 37 °C until the cells reached an belong to the genus of Arthrobacter (Arthrobacter sp. FB24 optical density of 0.6–0.8 at 600 nm. Then, protein expression and Arthrobacter aurescens TC1) and were obtained from the was induced by adding IPTG to a final concentration of 1 mM. NCBI database. After 6–8 h, the cells were harvested and disrupted with High Flanking fragments of PglyAwere amplified by TAIL-PCR Pressure Homogenizer (NS100IL 2 K, Niro Soavi, Germany). (Liu and Huang 1998) using ten arbitrary degenerate primers, SHMT of Arthrobacter sp. (AmSHMT) was purified using AD1-10 (Table 1), and six nested specific primers, SP1-3 F glutathione-S-transferase (GST)-free affinity purification and SP1-3 R (Table 1), designed according to the sequencing method (Lin et al. 2009). SHMT of E. coli (EcSHMT) was also purified with the same method. result of PglyA. The cycling conditions for TAIL-PCR were listed in Table 2. The full ORF of glyA was obtained from the chromosome Enzyme assay DNA of A. mysorens by PCR with the forward and reverse primers (glyA-F and glyA-R, Table 1). The PCR reaction was Standard enzyme activity assay was conducted as described performed as follows: one cycle at 94 °C for 4 min; 30 cycles below. Moderate enzyme was added into the reaction system Fig. 1 Design of degenerate primers and SDS-PAGE analysis of the (glutathione-S-transferase) and fusion protein (AmSHMT and GST). purified AmSHMT. (a)The glyA gene sequences belonged to the genus of Lane1: protein marker. Lane 2: purified AmSHMT without GST. Lane Arthrobacter and were obtained from NCBI database (http://www.ncbi. 3: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by nlm.nih.gov/). Arthrobacter sp. FB24 (GI: 116668568), 1: 830533– IPTG. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) 831870, 2: 1209958–1211265, 3: 4169907–4171268); Arthrobacter induced by 0.1 mM IPTG. Lane 5: bacterium (harboring pGEX-6p-1) aurescens TC1 (GI: 119947346), 4: 1309108–1310400, 5: 4192248– non-induced by IPTG. Lane 6: bacterium (harboring pGEX-6p-1) 4193636, 6: 4374834–4376153. The regions in the boxes were areas induced by 0.1 mM IPTG. The protein molecular weight ladder is for degenerate primers design. (b) 12 % SDS-PAGE analysis of the Unstained Protein Molecular Weight Marker (Fermentas, Canada) purified AmSHMT, the bands in the ellipses show the GST 1692 Ann Microbiol (2015) 65:1689–1698 Table 2 conditions used for TAIL-PCR Construction of engineering bacteria Reaction Cycle no. Thermal condition In order to construct engineering bacteria for L-serine produc- Primary 1 94 °C, 2 min; 94 °C, 1 min tion, glyA genes from Arthrobacter sp. and E. coli were cloned 5 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min into pET-15b vectors, respectively, using the same restriction 1 94 °C, 30 s; 25 °C, 3 min; 72 °C, 2 min enzyme cutting sites (NdeI and BamHI). The recombinant 10 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min expression plasmids were transformed into E. coli BL21 (DE3). The engineering bacteria were induced as described 15 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min in the section of expression and purification of SHMT. The two engineering bacteria were designated as pET-15b-AmglyA 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min and pET-15b-EcglyA,respectively. 1 72 °C, 10 min 110°C,5min Secondary 1 94 °C, 5 min Enzymatic reactions for L-serine production 16 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min Under the same conditions, the two engineering bacteria were 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min inoculated, induced, cultivated, and collected. They were 1 72 °C, 10 min washed with phosphate buffer (0.2 M, pH 8.0) and centrifuged 110°C,5min (8,000 rpm, 2 min) twice, collected (each was 3,000 mg, wet Tertiary The cycle no. and thermal condition were the same as the weight) and stored in a refrigerator at −80 °C for about 4 h. secondary reaction. The bacteria were thawed at 37 °C. With phosphate buffer (0.2 M, pH 8.0), the bacteria were resuspended in the 15-ml reaction system consisting of glycine (0.133 M), formalde- hyde (13.3 mM), β-mercaptoethanol (0.2 M), PLP (0.4 mM), (1 ml, pH 7.8, sodium phosphate buffer), which contained and THFA (5 mM). 50 mM DL-3-phenylserine, 50 μM PLP, 1 mM Na EDTA Enzymatic reactions were processed at 30 °C, 150 rpm for (ethylene diamine tetraacetic acid), and 25 mM sodium sul- 24 h. A sample of 200 μlwas collectedevery2hfor 24 h; fate. When cells were used, 0.03 % (w/v) cetyltrimethyl meanwhile, formaldehyde was added to a final concentration ammonium bromide (CTAB) was added. The reaction of 13.3 mM and pH was adjusted to 7.0–7.5. L-Serine con- proceeded for 1 h at 30 °C, and the production of centration was measured by RP-HPLC with pre-column benzaldehyde was measured by its maximum absorption derivatization. value at 279 nm (Zuo et al. 2007). One unit of enzyme activity was defined as the quantity of enzyme capabil- ity of releasing 1.0 μmol benzaldehyde per hour (benz- Sample preparation and RP-HPLC analysis aldehyde as standard). Specific activity was expressed as units/mg protein. O-Phthalaldehyde (OPA) regent (4 mg/ml) and borate buffer (0.4 M, pH 10.4) were used for precolumn derivatization. Solvent A (pH 5.8) consisted of 25 mM sodium acetate buffer Enzyme characterization and tetrahydrofuran (95/5, v/v), and Solvent B of methanol (Zhao et al. 2012). The optimal pH of SHMTwas determined at 30 °C in different Twenty microliters of the enzymatic reaction solution buffers at pH 5.8–9.5, namely sodium phosphate buffer was diluted to an appropriate concentration (10–100 (pH 5.8–8.0) and sodium carbonate buffer (pH 8.7–9.5). The pmol/μl) with ultrapure water. Then 100 μl of diluted optimal temperature of SHMT was determined at 0 to 55 °C enzymatic reaction solution or standard amino acid dilution under optimal pH. The pH stability of SHMT was determined (10–100 pmol/μl) was injected into a 1.5-ml centrifuge by incubating the enzyme at the optimal temperature for 3 h at tube, followed by the addition of 600 μl of borate buffer different pH (pH 6.5–9.3), followed by the measurement of and 300 μl OPA derivatization reagent once the timing the enzyme activity under standard conditions. The thermo- began. After being mixed adequately, the solution was stability of SHMT was determined by incubating the enzyme filtered through a 0.22 μmorganic membrane. at 20 to 50 °C under optimal pH for 3 h and then measuring the Five minutes later, the sample was injected into the column enzyme activity under standard conditions. for RP-HPLC analysis. Effects of the metal ions and chemical compounds on L-Serine and glycine were assayed by RP-HPLC (1260 enzyme activity were determined in the standard reaction infinity quaternary LC system, Agilent Technologies) on a system for 1 h (Li et al. 2009). column of Agilent Eclipse XDB-C18 (250 mm×4.6 mm, Ann Microbiol (2015) 65:1689–1698 1693 5 μm) as described by Jiang et al. (Jiang et al. 2013), with moderate modifications. Results Strain isolation By comparing the SHMT activity of isolated strains, the strain (Arthrobacter sp., MCCC 1A05493) with the highest activity was isolated, whose SHMT was designated as AmSHMT. DNA matching and amino acid sequence analysis A sequence (1,574 bp) was obtained by matching the PglyA (936 bp) with the flanking fragments (335 and 613 bp) identified by TAIL-PCR (Fig. 2). Then the Fig. 2 PglyA gene fragment and its flanking gene fragments. (a) A part of whole glyA gene sequence (1,323 bp) was obtained the full-long AmglyA gene (PglyA, 936 bp) was amplified using using the ORF search tool from SoftBerry (http:// degenerate primers DP-F and DP-R. (b) The left flanking sequence (335 bp) was amplified through TAIL-PCR technology. (c) The right linux1.softberry.com/berry.phtml). The AmSHMT flanking sequence (613 bp) was amplified through TAIL-PCR exhibited 58 % amino acid identity with the known technology SHMT from E. coli and encoded a protein with 440 amino acids and a deduced molecular mass of 47. LTNKYAEGYPGRRYYGG (61–77) and GGHLTHG 3 kDa. A phylogenetic tree was constructed to verify (134–140) (Hong et al. 1999), were also found in further the evolutionary relationship among AmSHMT AmSHMT. and other known SHMTs (Fig. 3). The conserved active site T/ST/STTHKT/SL in all known SHMT proteins (Garrow et al. 1993)wasfound Expression, purification, and characterization of AmSHMT in AmSHMT (235–242) in the form of TSTTHKTL (Fig. 4). Another significant homologous sequence AmSHMT was expressed, purified, and its molecular mass GQQGGP (268–273), a glycine-rich region, had been was determined by SDS-PAGE analysis (Fig. 1b). SHMT proposed to be essential for PLP binding (Usha et al. showed optimal activity at pH 7.8, and retained over 75 % 1994). Two other well-known conserved sequences, of the maximal activity between pH 7.0 and 8.0. Furthermore, Fig. 3 Phylogenetic analysis of AmSHMT. The phylogenetic tree of AmSHMT was constructed using the neighbor-joining method (MEGA 5.05). Except for AmSHMT, the other SHMT sequences were obtained from GenBank and PDB (http://www. rcsb.org/pdb/home/home.do). The numbers at node indicate the bootstrap percentages of 1,000 resamples. The units at the bottom of the tree indicate the number of substitution events 1694 Ann Microbiol (2015) 65:1689–1698 Fig. 4 Multiple sequence alignment of SHMTs. The sequences in the boxes are conserved amino acid residues 2+ 2+ 2+ the enzyme was sensitive to low pH, displaying less than 20 % was weakly enhanced by Mg ,Ca ,Pb and EDTA, but 2+ 2+ 2+ 2+ 2+ of its maximal activity at pH 6.5 and nearly no activity below was strongly inhibited by Hg ,Co ,Cu ,Mn and Fe . pH 5.8 (Fig. 5a). Without any stabilizer, the purified SHMT In addition, SDS and CTAB also inhibited SHMTactivity, and + + + was apparently stable under weakly alkaline conditions K ,Na and NH showed no appreciable impact on the (pH 7–7.4), retaining over 85 % of the maximal activity after SHMT activity. 3 h at 45 °C. However, it was unstable under acidic conditions In terms of activity, the AmSHMT (287.9 units/mg) was or in strongly alkaline environment, especially when the pH 2.75-fold higher than the EcSHMT (104.7 units/mg) under was over 9.0 (Fig. 5c). standard assay conditions. The maximal activity of SHMT was observed at 45 °C (Fig. 5b). However, SHMT retained over 50 % of its maximal Production of L-serine by resting cells reaction activity after 3 h incubation under pH 7.8 (Fig. 5c), and decreased rapidly in activity at temperatures over 40 °C. After 48 h incubation at 28 °C as described in section of the The effects of metal ions and chemical reagents on SHMT resting cells reaction system, 1.8±0.3 mg/ml L-serine was are shown in Table 3, indicating that the activity of the enzyme obtained by RP-HPLC analysis. Ann Microbiol (2015) 65:1689–1698 1695 Fig. 5 Effects of temperature and pH on the activity. (a)Effect of pH on 45 °C, the purified enzyme was pre-treated at a different pH for 3 h. Then the activity of AmSHMT. Assays were conducted in buffers over a pH assays were conducted under standard conditions and the enzyme activity range from 5.8 to 9.5 at 30 °C for 1 h, under standard conditions. The without pre-treatment was taken as 100 %. (d) Effect of temperature on maximal activity was taken as 100 %. (b) Effect of temperature on the the stability of AmSHMT. At the optimal pH 7.8, the purified enzyme was activity of AmSHMT. Assays were conducted at the optimal pH 7.8, pre-treated at a different temperature for 3 h. Then assays were conducted under standard conditions. The maximal activity was taken as 100 %. under standard conditions and the enzyme activity without pre-treatment (c) Effect of pH on the stability of AmSHMT. At the optimal temperature was taken as 100 % Production of L-serine by enzymatic reaction SHMT, a member of the α-family of PLP-dependent en- zymes (Mehta and Christen 2000), exists as a dimer in Using the enzymatic reaction system described in the Psychromonas ingrahamii (Siglioccolo et al. 2010) and is methods, L-serine was detected by RP-HPLC analysis 24 h ubiquitous for generating one-carbon fragments for the syn- later (Figs. 6 and 7a). The L-serine concentration at the 12th thesis of nucleotides, methionine, thymidylate, and choline hour was calculated to be 93.6 mM in the pET-15b-AmglyA (Appaji Rao et al. 2003). This enzyme is also useful in the system, but 71.1 mM in the pET-15b-EcglyA system, indicat- synthesis of serine using glycine and formaldehyde. There- ing that the former was 70.3 % in the molecular conversion fore, studying the enzymatic properties of SHMT can provide rate, which was 1.32-fold higher than the latter (53.4 %). guidance for the industrial production of L-serine. Using the resting cells reaction system, 1.8±0.3 mg/ml L- serine was produced. The reasons for the low yield might be that the metabolic pathways in vivo are so complex that L- serine can be quickly degraded and converted to other sub- Discussion stances. For instance, even the Methylobacterium sp. strain MN43, which has the highest glycine conversion rate ever In this study, we obtained Arthrobacter sp., a methanol-using strain with high SHMT activity, which was first reported in reported, can degrade 32 g/l L-serine in 2 days (Hagishita et al. 1996). 1972 as a new species excreting L-glutamic acid (Nand and Rao 1972). The glyA gene was cloned by TAIL-PCR, an From Fig. 5a, it can be seen that the two engineering bacteria produced little L-serine during the first 6 h, but efficient PCR strategy, using AD1-10 primers separately. However, only AD8 and AD10 primers were better suited both of them could produce L-serine rapidly 6 h later, especially engineering bacterium pET-15b-AmglyA.This for PCR amplification, and all the other AD primers often led to non-targeted, dispersive or small fractional products, prob- was probably because the coenzyme (PLP and THFA) and substrate were binding to SHMT during this time, but 6 h ably due to codon usage and the cycling conditions. In any later when the binding process was completed, the conver- case, choosing more AD primers is helpful for TAIL-PCR sion reaction would proceed rapidly and more L-serine amplification. 1696 Ann Microbiol (2015) 65:1689–1698 Table 3 Effects of metal ions and chemical reagents on the activity of purified SHMT* Metal ion and chemical Concentration Relative activity reagent (mM) (%) None 0 100 % 2+ a Hg 1 − 2+ b Co 1 65.0±0.2 K 1 99.4±0.1 Na 1 98.7±0.4 NH 1 99.5±0.7 2+ Mg 1101.4±0.6 2+ Ca 1103.7±0.4 2+ Cu 1 48.9±0.8 2+ Zn 1 83.1±0.1 2+ Mn 1 63.1±0.4 2+ Fe 1 68.3±0.1 2+ Pb 1101.9±0.3 EDTA 1 % 102.1±0.3 CTAB 0.03 % 71.5±0.5 SDS 0.1 % – *The data are the average of three replicates Unmeasured data Relative activity ± the standard deviation Fig. 6 HPLC detection of the L-serine in the enzymatic reaction system. All assays were performed in the standard conditions and the activity (a) Determination of glycine and L-serine standards; (b) Determination of measured without additional reagents and ions was taken as 100 % L-serine synthesis by pET-15b-AmglyA in the enzymatic reaction system at the 12th hour; (c) Determination of L-serine synthesis by pET-15b- EcglyA in the enzymatic reaction system at the 12th hour could be produced during 6–12 h. At the 14th hour, the concentration of L-serine significantly decreased in the generate glycine and formaldehyde. At the 24th hour, the L- serine concentration showed a significant decrease in the pET-15b-AmglyA reaction system, while that of the pET- 15b-EcglyA reaction system showed a steady increase, pET-15b-AmglyA reaction system, but little variation in the resulting in a significant descrease in L-serine conversion pET-15b-EcglyA system compared with that at the 14th in both systems, which could be attributed to the possibility hour. The possible reason was that 14 h later when the that the coenzymes were almost entirely consumed in the THFA and PLP had been almost completely consumed, former reaction system; thus, the enzymatic reaction would AmSHMT still retained a little activity, and thus the enzy- be favourably performed in the reverse reaction direction to matic conversion sequentially proceeded in the reverse Fig. 7 HPLC and SDS-PAGE analysis of the induced AmSHMT and and pET-15b-EcglyAwere treated in the same conditions. Lanes 1, 2, and EcSHMT. (a) HPLC analysis of the L-serine concentration in the 3 indicate the three replicates of induced EcSHMT from engineering enzymatic reaction system during 24 h. (b) SDS-PAGE analysis of the bacteria pET-15b-EcglyA; lanes 4, 5, and 6 indicate the three replicates induced AmSHMT and EcSHMT. Engineering bacteria pET-15b-AmglyA of induced AmSHMT from engineering bacteria pET-15b-AmglyA Ann Microbiol (2015) 65:1689–1698 1697 Acknowledgments This work was supported by grants from China reaction direction. In the pET-15b-EcglyA reaction system, National Natural Sciences Foundation (No. 31270162) and a project the EcSHMT almost completely lost activity and the re- funded by the Priority Academic Program Development of Jiangsu verse reaction was blocked, so that at the 24th hour, the L- Higher Education Institutions. serine concentration almost remained constant. Conflict of interests The authors claim that they have no competing The above analyses indicated that the pET-15b-AmglyA interests. And there are not any non-financial competing interests. engineering bacterium has more potential for industrial appli- cations, especially at the 12th hour, when the L-serine con- centration reached the maximum value, because its faster conversion rate was conducive to time saving and cost reduc- References ing. However, the present work cannot meet the requirements of industrial applications and much work needs to done to Amedeo P, Habu Y, Afsar K, Scheid OM, Paszkowski J (2000) further improve the L-serine yield. Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 405:203–206 The two engineering bacteria were treated under the Appaji Rao N, Ambili M, Jala V, Subramanya H, Savithri H (2003) same conditions, and interestingly, AmSHMT was found Structure-function relationship in serine hydroxymethyltransferase. to be lower than EcSHMT in the expression level Biochim Biophys Acta 1647:24–29 (Fig. 5b), which might be caused by codon usage bias or Barra D, Martini F, Angelaccio S, Bossa F, Gavilanes F, Peterson D, Bullis B, Schirch L (1983) Sequence homology between prokary- other reasons. However, even under such conditions, pET- otic and eukaryotic forms of serine hydroxymethyltransferase. 15b-AmglyA was still higher than pET-15b-EcglyA in the Biochem Biophys Res Commun 116:1007–1012 conversion rate during 12 h. So in the next study, the Blakley RL (1955) The interconversion of serine and glycine: participa- codons of AmSHMT should be optimized to improve its tion of pyridoxal phosphate. Biochem J 61:315–323 Byrne PC, Sanders P, Snell K (1992) Nucleotide sequence and expression expression in E. coli (Carbone et al. 2003). Many published of a cDNA encoding rabbit liver cytosolic serine reports provide helpful information on how to improve the hydroxymethyltransferase. Biochem J 286:117–123 yield. With the triparental mating method, a new recombi- Carbone A, Zinovyev A, Kepes F (2003) Codon adaptation index as a nant strain MB202 was screened, whose SHMT activity measure of dominating codon bias. Bioinformatics 19:2005–2015 Das S, Lyla P, Khan SA (2006) Marine microbial diversity and ecology: was approximately 3.5-fold higher than that of the parent importance and future perspectives. Curr Sci 90:1325–1335 strain, and L-serine output was 4.4-fold higher (Shen et al. Emelyanov A, Gao Y, Naqvi NI, Parinov S (2006) Trans-kingdom trans- 2010). SdaA-encoded L-serine dehydratase has been dem- position of the maize dissociation element. Genetics 174:1095–1104 onstrated to be involved in L-serine degradation (Netzer Garrow T, Brenner A, Whitehead V, Chen XN, Duncan R, Korenberg J, Shane B (1993) Cloning of human cDNAs encoding mitochondrial et al. 2004). With the presence of L-serine dehydratase in and cytosolic serine hydroxymethyltransferases and chromosomal Arthrobacter globiformis SK-200, 80 % of the L-serine in localization. J Biol Chem 268:11910–11916 the medium was consumed in 2 days (Tani et al. 1978). Hagishita T, Yoshida T, Izumi Y, Mitsunaga T (1996) Efficient L-serine When glyA expression was reduced and L-serine production from methanol and glycine by resting cells of dehydratase activity was missing, L-serine production in- Methylobacterium sp. strain MN43. Biosci Biotechnol Biochem 60:1604–1607 creased up to 9.04 mg/ml (Peters-Wendisch et al. 2005). By Hamilton B, Hsiao H, Swann W, Anderson D, Delente J (1985) Manufacture using error-prone PCR, DNA shuffling and high through- of L-amino acids with bioreactors. Trends Biotechnol 3:64–68 put screening, a mutant with approximately eightfold Hong MC, Wu ML, Chang MC (1999) Cloning and complete nucleotide higher enzyme activity and 41-fold higher enzyme produc- sequence of Acinetobacter radioresistens CMC-1 AglyA gene encoding serine hydroxymethyltransferase. FEMS Microbiol Lett tivity than the wild-type parent was obtained (Zuo et al. 170:413–418 2007). Hsiao H, Walter J, Anderson D, Hamilton B (1988) Enzymatic produc- tion of amino acids. Biotechnol Genet Eng Rev 6:179–219 Izumi Y, Yoshida T, Miyazaki SS, Mitsunaga T, Ohshiro T, Shimao M, Miyata A, Tanabe T (1993) L-Serine production by a methylotroph and its related enzymes. Appl Microbiol Biotechnol 39:427–432 Conclusions Jiang W, Xia B, Liu Z (2013) A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, charac- The SHMT in the present study is a novel one from terization and l-serine production. Microbiol Res 168:477–484 Jones C, Priest D (1976) Metabolism of glutamine and ammonia in rat Arthrobacter sp., which was stable in weakly alkali condi- liver: the effects of N-acetylglutamate and phosphate. Arch tions, showing optimal activity at pH 7.8 and 45 °C, and a Biochem Biophys 174:305–311 2.75-fold higher activity than the corresponding enzyme from Li C, Hong Y, Shao Z, Lin L, Huang X, Liu P, Wu G, Meng X, Liu Z (2009) Novel alkali-stable, cellulase-free xylanase from deep-sea E. coli. The molecular conversion rate of L-serine in the pET- Kocuria sp. Mn22. J Microbiol Biotechnol 19:873–880 15b-AmglyA system was 70.3 % and 1.32-fold higher than Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C, Dong J, Xiao L, that in the pET-15b-EcglyA system (53.4 %). Based on these Liu Z (2009) Improved catalytic efficiency of endo-β-1,4-glucanase characteristics, this strain and its enzyme have the potential for from Bacillus subtilis BME-15 by directed evolution. Appl further research and industrial use. Microbiol Biotechnol 82:671–679 1698 Ann Microbiol (2015) 65:1689–1698 Liu YG, Huang N (1998) Efficient amplification of insert end sequences Methylobacterium sp MB200. Appl Biochem Biotechnol 160:740– from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol Biol Report 16:175–175 Siglioccolo A, Bossa F, Pascarella S (2010) Structural adaptation of serine Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: auto- hydroxymethyltransferase to low temperatures. Int J Biol Macromol matable amplification and sequencing of insert end fragments from 46:37–46 P1 and YAC clones for chromosome walking. Genomics 25:674– Sun J (2000) Study on synthesis of L-serine catalyzed by SHMT recom- 681 binant bacteria. Dissertation, China Pharmaceutical University Mehta PK, Christen P (2000) The molecular evolution of pyridoxal-5′- Sun J, Wu W, Chen Z (2000) Synthesis of coenzyme THFA and deter- phosphate-dependent enzymes. Adv Enzymol Relat Areas Mol Biol mination of its relative activity. Pharm Biotechnol 7:38–41 74:129–184 Tani Y, Kanagawa T, Hanpongkittikun A, Ogata K, Yamada H (1978) Miyata A, Yoshida T, Yamaguchi K, Yokoyama C, Tanabe T, Toh H, Production of L-Serine by a methanol-utilizing bacterium, Mitsunaga T, Izumi Y (1993) Molecular cloning and expression of Arthrobacter globiformis SK-200. Agric Biol Chem 42:2275–2279 the gene for serine hydroxymethyltransferase from an obligate Ulevitch RJ, Kallen RG (1977) Studies of the reactions of lamb liver methylotroph Hyphomicrobium methylovorum GM2. Eur J serine hydroxymethylase with L-phenylalanine: kinetic isotope ef- Biochem 212:745–750 fects upon quinonoid intermediate formation. Biochemistry 16: Nand K, Rao D (1972) Arthrobacter mysorens-a new species excreting L- 5350–5354 glutamic acid. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Usha R, Savithri HS, Appaji Rao N (1994) The primary structure of sheep 127:324–331 liver cytosolic serine hydroxymethyltransferase and an analysis Netzer R, Peters-Wendisch P, Eggeling L, Sahm H (2004) Cometabolism of the evolutionary relationships among serine of a nongrowth substrate: L-serine utilization by Corynebacterium hydroxymethyltransferases. Biochim et Biophys Acta (BBA)- glutamicum. Appl Environ Microbiol 70:7148–7155 Protein Struct Mol Enzymol 1204:75–83 Peters-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H, Vidal L, Calveras J, Clapes P, Ferrer P, Caminal G (2005) Recombinant Eggeling L (2005) Metabolic engineering of Corynebacterium production of serine hydroxymethyl transferase from Streptococcus glutamicum for L-serine production. Appl Environ Microbiol 71: thermophilus and its preliminary evaluation as a biocatalyst. Appl 7139–7144 Microbiol Biotechnol 68:489–497 Rao DN, Appaji Rao N (1982) Purification and regulatory properties of Zhao M, Ma Y, Dai L, Zhang D, Li J, Yuan W, Li Y, Zhou H (2012) A mung bean (Vigna radiata L.) serine hydroxymethyltransferase. high-performance liquid chromatographic method for simultaneous Plant Physiol 69:11–18 determination of 21 free amino acids in Tea. Food Anal Methods 6: Schirch V, Hopkins S, Villar E, Angelaccio S (1985) Serine 69–75 hydroxymethyltransferase from Escherichia coli: purification and Zuo Z-Y, Zheng Z-L, Liu Z-G, Yi Q-M, Zou G-L (2007) Cloning, DNA properties. J Bacteriol 163:1–7 shuffling and expression of serine hydroxymethyltransferase gene Shen P, Chao H, Jiang C, Long Z, Wang C, Wu B (2010) Enhancing from Escherichia coli strain AB90054. Enzym Microb Technol 40: production of L-serine by increasing the glyA Gene expression in 569–577 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Characterization of a novel serine hydroxymethyltransferase isolated from marine bacterium Arthrobacter sp. and its application on L-serine production

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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10.1007/s13213-014-1008-7
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Ann Microbiol (2015) 65:1689–1698 DOI 10.1007/s13213-014-1008-7 ORIGINAL ARTICLE Characterization of a novel serine hydroxymethyltransferase isolated from marine bacterium Arthrobacter sp. and its application on L-serine production Junjie Huang & Lin Chen & Nan Hu & Wei Jiang & Gaobing Wu & Ziduo Liu Received: 27 June 2014 /Accepted: 21 November 2014 /Published online: 7 January 2015 Springer-Verlag Berlin Heidelberg and the University of Milan 2015 Abstract The current L-serine production relies mainly on Introduction cellular or enzymatic conversion from the precursor glycine plus a C1 compound. To date, only several reports have been Only several reports have been published on L-serine produc- published on L-serine production from glycine and methanol tion from glycine and methanol by methylotrophic bacteria by methylotrophic bacteria with the serine pathway. This work with the serine pathway (Hagishita et al. 1996;Shen et al. aimed to isolate a novel serine hydroxymethyltransferase 2010), and all the microorganisms used in the studies reported (SHMT) from the methanol-using Arthrobacter sp. and use were from the terrestrial environment. However, many other it for L-serine production with the enzymatic conversion bacteria also probably have the same function. The marine method. Here, A novel glyA gene was isolated from the realm covers 70 % of the earth’s surface, and provides the methanol-using Arthrobacter sp. by thermal asymmetric in- largest inhabitable space for living organisms, particularly terlaced PCR (TAIL-PCR), encoding a serine microbes (Das et al. 2006); thus isolating some novel and hydroxymethyltransferase (SHMT) with 440 amino acids, useful marine bacteria is feasible and important. belonging to the α-family of fold type I, and pyridoxal-5- Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is a phosphate (PLP) dependent enzymes. The enzyme was stable ubiquitous, highly conserved pyridoxal-5-phosphate (PLP)- in weakly alkali conditions, showing the optimal activity at dependent enzyme with tetrahydrofolate (THFA) as the C pH 7.8 and 45 °C, and a 2.75-fold increase in activity over the acceptor (Blakley 1955), and has been purified and extensive- corresponding enzyme of Escherichia coli. Two methods ly studied in animals (Jones and Priest 1976; Ulevitch and (resting cells reaction and enzymatic conversion) were Kallen 1977), plants (Rao and Appaji Rao 1982), and bacteria employed to produce serine. Using glycine (133 mM) and (Barra et al. 1983). SHMT plays an important role in the formaldehyde (13.3 mM) as substrates to produce serine by assimilation of C compounds, yielding the main L-serine enzymatic reaction, 93.6 mM L-serine was obtained with a intermediate. The enzymes involved in L-serine synthesis 70.4 % molar conversion rate from glycine to L-serine. Thus, are methanol dehydrogenase (EC 1.1.1.244) and SHMT, the the characteristics of this novel strain and its enzyme suggest latter of which is coded by the glyA gene. The former catalyzes that it has the potential for further research and industrial use. the oxidation of methanol to formaldehyde and the latter converts formaldehyde and glycine to L-serine by reaction. It is well-known that two molecules of glycine are needed for . . Keywords Arthrobacter sp. SHMT L-Serine enzymatic the synthesis of one molecule of L-serine, and the theoretical . . production Thermal asymmetric interlaced PCR RP-HPLC production yield is 50 % (mol/mol) (Izumi et al. 1993). If half of the C units are displaced by low-cost formaldehyde, the : : : : J. Huang L. Chen W. Jiang G. Wu Z. Liu (*) theoretical ratio of glycine conversion may be 100 % (Izumi State Key Laboratory of Agricultural Microbiology, College of Life et al. 1993). Therefore, isolating a strain with high methanol Science and Technology, Huazhong Agricultural University, dehydrogenase and SHMT activity is the key, and in this Wuhan 430070, China e-mail: lzd@mail.hazu.edu.cn study, one methylotrophic strain Arthrobacter sp. with high SHMT activity was isolated. N. Hu The study of enzymatic properties can contribute to a better College of Biotechnology and Pharmaceutical Engineering, Nanjing use of SHMT. In previous reports, many glyA genes were Tech University, Nanjing 211800, People’s Republic of China 1690 Ann Microbiol (2015) 65:1689–1698 Table 1 Primersused in thisstudy obtained by shotgun technology (Hamilton et al. 1985), direct PCR amplification based on the known genome information Primer 5′ to 3′ Reference (Schirch et al. 1985; Vidal et al. 2005), cDNA library (Byrne et al. 1992;Garrowetal. 1993), southern-blot hybridization DP-F CTSACCAAYAARTACGC This work CGAGGGYT (Miyata et al. 1993), and a genomic library constructed with DP-R ACCATSGGCGGSCKSGG This work the application of a probe (Shen et al. 2010). PCR amplifica- RTCGAAGG tion would be convenient only with known genome informa- SP1-F GGTCGAAGATCTTGGCT This work tion. Otherwise the aforementioned methods would be time- GAGCGTTTG SP2-F GAAGACGTTGTTGCCAA This work consuming, and thus libraries such as EMBL and GenBank GGGCATC with information about various genomes and nucleic acids SP3-F CGCAGGTGGAGATCACT This work would facilitate the search for information about relevant GTGAATCG SP1-R AACAATCATCTTCGGCT This work genes. In this work, the partial glyA (PglyA)sequence was GGTGCTCG amplified with degenerate primers designed based on the SP2-R TGGTAGGTTTCCTCATC This work result of multiple sequence alignment before the flanking CACGCCGTA sequences of PglyA were obtained by thermal asymmetric SP3-R AGCTTCATGCCGTGGGT This work CAAATGG interlaced PCR (TAIL-PCR). AD1 TGWGNAGSANCASAGA (Liu and Whittier 1995) The current L-serine production relies mainly on cellular or AD2 AGWGNAGWANCAWAGG (Liu and Whittier 1995) enzymatic conversion from the precursor glycine plus a C AD3 CAWCGICNGAIASGAA (Liu and Whittier 1995) compound (Peters-Wendisch et al. 2005). Because there is no AD4 TCSTIGNCITWGGA (Liu and Whittier 1995) need to add any coenzymes PLP and tetrahydrofolate (THFA) AD5 NGTCGASWGANAWGAA (Liu and Huang 1998) into the reaction system, a resting cells system is a useful AD6 NTCGASTWTSGWGTT (Liu and Huang 1998) method to produce L-serine from glycine and methanol by AD7 WGTGNAGWANCANAGA (Liu and Huang 1998) methylotrophic bacteria with the serine pathway (Izumi et al. AD8 WCAGNTGWTNGTNCTG (Emelyanov et al. 2006) 1993; Hagishita et al. 1996). Another useful method is the enzymatic reaction, manufacturing L-serine by constructing AD9 WGCNAGTNAGWANAAG (Amedeo et al. 2000) engineering bacteria and adding coenzymes (Hsiao et al. AD10 AWGCANGNCWGANATA (Amedeo et al. 2000) 1988), which has been rarely reported. In China, the enzymat- glyA-F CGGGATCCATGAGCAACC This work ic reaction method is mainly used for L-serine production with AGACTTTTGAA glyA-R CCGCTCGAGCTACTCGGA This work the enzyme primarily from Escherichia coli SHMT (Sun AACCTTTGGCA 2000; Zuo et al. 2007) In this paper, the two methods (resting cells reaction and IUPAC ambiguity codes, M=A/C, R=A/G, W=A/T, S=G/C, Y=C/T, enzymatic conversion) have been tried for L-serine produc- K=G/T, and N=A/G/C/T. I indicate inosine. F, forward; R, reverse. Restriction sites BamH1 and Xho1in primers glyA-F/R are underlined, tion, and the latter method was used to evaluate the difference respectively; Start and stop codons are in bold, respectively between the Arthrobacter sp. SHMT engineering bacterium and the Escherichia coli SHMT engineering bacterium in L- serine production. Media and growth conditions Materials and methods Escherichia coli strains were incubated in Luria-Bertani (LB) Materials medium, supplemented with ampicillin (100 μg/ml) if neces- sary, and other bacteria were cultured in agar 2216 medium. Restriction enzymes, pMD18-T vectors, T4 DNA ligases, Taq Screening media I and II were used for the screening of strains DNA polymerases, and DNA markers were purchased from with SHMT activity. Screening medium I consisted of meth- TaKaRa Company (Dalian, China). Kits for plasmid extrac- anol 1.2 % (v/v), (NH ) SO 37.84 mM, KH PO 7.34 mM, 4 2 4 2 4 tion and DNA purification were obtained from Qiagen Com- K HPO ·3H O 13.15 mM, MgSO ·7H O0.81 mM, MnSO · 2 4 2 4 2 4 −3 pany (Germany). All oligonucleotide primers (Table 1)were H O 0.012 mM, FeSO ·7H O7.19×10 mM, biotin 2.05× 2 4 2 −4 −4 synthesized and all DNA fragments were sequenced by 10 mM, thiamine hydrochloride 3.32×10 mM, NaNO GenScript Company (Nanjing, China). All the chemicals were 35.30 mM, and NaCl 34.22 mM, with the pH of the mixture of HPLC grade or biotechnology grade and purchased from being adjusted to 7.5. The components listed above were also Sigma unless specially noted. The THFA used in the enzy- used in the mixture of screening medium II except that the matic reaction to produce L-serine was independently synthe- concentration of methanol was increased to 5 % (w/v), and sized by our laboratory (Sun et al. 2000). 1.5 % (w/v) agar and 0.27 M glycine were added. Ann Microbiol (2015) 65:1689–1698 1691 Microorganism isolation at 94 °C for 30 s, 58 °C for 30 s and 72 °C for 1 min; and one final additional cycle at 72 °C for 10 min. The PCR product The bacteria were cultured by enrichment culture in screening was gel-purified, digested with BamH I and Xho I, and then culture medium I at 28 °C, and 1.2 % (v/v) methanol was ligated into the pGEX-6p-1 vector (Amersham Biosciences) added once every 2 days. After 7–10 days of growth, bacterial (Lin et al. 2009). The recombinant plasmids were verified by suspension was diluted to 10,000 times, 100,000 times, and DNA sequencing and designated as pGEX-6p-AmglyA. one million times with sterile water. Then these samples were Using the same method described above, the glyA gene spread onto plates filled with medium II, followed by 4–5days (Gene ID: 947022) from E. coli was also ligated into the of incubation at 28 °C. Finally, Arthrobacter sp. was isolated pGEX-6p-1 vector and designated as pGEX-6p-EcglyA. and preserved for further use for its high SHMT activity. Expression and purification of SHMT Gene cloning To optimize the expression of SHMT, the pGEX-6p-AmglyA To amplify the PglyA gene, degenerate primers DP-F and DP- plasmids were transformed into E. coli BL21 (DE3) compe- R(Table 1) were designed based on the result of multiple tent cells (Stratagene, USA). The transformants were cultured sequence alignment (Fig. 1a)of glyA gene sequences which in Luria–Bertani broth at 37 °C until the cells reached an belong to the genus of Arthrobacter (Arthrobacter sp. FB24 optical density of 0.6–0.8 at 600 nm. Then, protein expression and Arthrobacter aurescens TC1) and were obtained from the was induced by adding IPTG to a final concentration of 1 mM. NCBI database. After 6–8 h, the cells were harvested and disrupted with High Flanking fragments of PglyAwere amplified by TAIL-PCR Pressure Homogenizer (NS100IL 2 K, Niro Soavi, Germany). (Liu and Huang 1998) using ten arbitrary degenerate primers, SHMT of Arthrobacter sp. (AmSHMT) was purified using AD1-10 (Table 1), and six nested specific primers, SP1-3 F glutathione-S-transferase (GST)-free affinity purification and SP1-3 R (Table 1), designed according to the sequencing method (Lin et al. 2009). SHMT of E. coli (EcSHMT) was also purified with the same method. result of PglyA. The cycling conditions for TAIL-PCR were listed in Table 2. The full ORF of glyA was obtained from the chromosome Enzyme assay DNA of A. mysorens by PCR with the forward and reverse primers (glyA-F and glyA-R, Table 1). The PCR reaction was Standard enzyme activity assay was conducted as described performed as follows: one cycle at 94 °C for 4 min; 30 cycles below. Moderate enzyme was added into the reaction system Fig. 1 Design of degenerate primers and SDS-PAGE analysis of the (glutathione-S-transferase) and fusion protein (AmSHMT and GST). purified AmSHMT. (a)The glyA gene sequences belonged to the genus of Lane1: protein marker. Lane 2: purified AmSHMT without GST. Lane Arthrobacter and were obtained from NCBI database (http://www.ncbi. 3: recombinant bacterium (harboring pGEX-6P-glyA) non-induced by nlm.nih.gov/). Arthrobacter sp. FB24 (GI: 116668568), 1: 830533– IPTG. Lane 4: recombinant bacterium (harboring pGEX-6P-glyA) 831870, 2: 1209958–1211265, 3: 4169907–4171268); Arthrobacter induced by 0.1 mM IPTG. Lane 5: bacterium (harboring pGEX-6p-1) aurescens TC1 (GI: 119947346), 4: 1309108–1310400, 5: 4192248– non-induced by IPTG. Lane 6: bacterium (harboring pGEX-6p-1) 4193636, 6: 4374834–4376153. The regions in the boxes were areas induced by 0.1 mM IPTG. The protein molecular weight ladder is for degenerate primers design. (b) 12 % SDS-PAGE analysis of the Unstained Protein Molecular Weight Marker (Fermentas, Canada) purified AmSHMT, the bands in the ellipses show the GST 1692 Ann Microbiol (2015) 65:1689–1698 Table 2 conditions used for TAIL-PCR Construction of engineering bacteria Reaction Cycle no. Thermal condition In order to construct engineering bacteria for L-serine produc- Primary 1 94 °C, 2 min; 94 °C, 1 min tion, glyA genes from Arthrobacter sp. and E. coli were cloned 5 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min into pET-15b vectors, respectively, using the same restriction 1 94 °C, 30 s; 25 °C, 3 min; 72 °C, 2 min enzyme cutting sites (NdeI and BamHI). The recombinant 10 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min expression plasmids were transformed into E. coli BL21 (DE3). The engineering bacteria were induced as described 15 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min in the section of expression and purification of SHMT. The two engineering bacteria were designated as pET-15b-AmglyA 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min and pET-15b-EcglyA,respectively. 1 72 °C, 10 min 110°C,5min Secondary 1 94 °C, 5 min Enzymatic reactions for L-serine production 16 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min 94 °C, 30 s; 65 °C, 1 min; 72 °C, 2 min Under the same conditions, the two engineering bacteria were 94 °C, 30 s; 44 °C, 1 min; 72 °C, 2 min inoculated, induced, cultivated, and collected. They were 1 72 °C, 10 min washed with phosphate buffer (0.2 M, pH 8.0) and centrifuged 110°C,5min (8,000 rpm, 2 min) twice, collected (each was 3,000 mg, wet Tertiary The cycle no. and thermal condition were the same as the weight) and stored in a refrigerator at −80 °C for about 4 h. secondary reaction. The bacteria were thawed at 37 °C. With phosphate buffer (0.2 M, pH 8.0), the bacteria were resuspended in the 15-ml reaction system consisting of glycine (0.133 M), formalde- hyde (13.3 mM), β-mercaptoethanol (0.2 M), PLP (0.4 mM), (1 ml, pH 7.8, sodium phosphate buffer), which contained and THFA (5 mM). 50 mM DL-3-phenylserine, 50 μM PLP, 1 mM Na EDTA Enzymatic reactions were processed at 30 °C, 150 rpm for (ethylene diamine tetraacetic acid), and 25 mM sodium sul- 24 h. A sample of 200 μlwas collectedevery2hfor 24 h; fate. When cells were used, 0.03 % (w/v) cetyltrimethyl meanwhile, formaldehyde was added to a final concentration ammonium bromide (CTAB) was added. The reaction of 13.3 mM and pH was adjusted to 7.0–7.5. L-Serine con- proceeded for 1 h at 30 °C, and the production of centration was measured by RP-HPLC with pre-column benzaldehyde was measured by its maximum absorption derivatization. value at 279 nm (Zuo et al. 2007). One unit of enzyme activity was defined as the quantity of enzyme capabil- ity of releasing 1.0 μmol benzaldehyde per hour (benz- Sample preparation and RP-HPLC analysis aldehyde as standard). Specific activity was expressed as units/mg protein. O-Phthalaldehyde (OPA) regent (4 mg/ml) and borate buffer (0.4 M, pH 10.4) were used for precolumn derivatization. Solvent A (pH 5.8) consisted of 25 mM sodium acetate buffer Enzyme characterization and tetrahydrofuran (95/5, v/v), and Solvent B of methanol (Zhao et al. 2012). The optimal pH of SHMTwas determined at 30 °C in different Twenty microliters of the enzymatic reaction solution buffers at pH 5.8–9.5, namely sodium phosphate buffer was diluted to an appropriate concentration (10–100 (pH 5.8–8.0) and sodium carbonate buffer (pH 8.7–9.5). The pmol/μl) with ultrapure water. Then 100 μl of diluted optimal temperature of SHMT was determined at 0 to 55 °C enzymatic reaction solution or standard amino acid dilution under optimal pH. The pH stability of SHMT was determined (10–100 pmol/μl) was injected into a 1.5-ml centrifuge by incubating the enzyme at the optimal temperature for 3 h at tube, followed by the addition of 600 μl of borate buffer different pH (pH 6.5–9.3), followed by the measurement of and 300 μl OPA derivatization reagent once the timing the enzyme activity under standard conditions. The thermo- began. After being mixed adequately, the solution was stability of SHMT was determined by incubating the enzyme filtered through a 0.22 μmorganic membrane. at 20 to 50 °C under optimal pH for 3 h and then measuring the Five minutes later, the sample was injected into the column enzyme activity under standard conditions. for RP-HPLC analysis. Effects of the metal ions and chemical compounds on L-Serine and glycine were assayed by RP-HPLC (1260 enzyme activity were determined in the standard reaction infinity quaternary LC system, Agilent Technologies) on a system for 1 h (Li et al. 2009). column of Agilent Eclipse XDB-C18 (250 mm×4.6 mm, Ann Microbiol (2015) 65:1689–1698 1693 5 μm) as described by Jiang et al. (Jiang et al. 2013), with moderate modifications. Results Strain isolation By comparing the SHMT activity of isolated strains, the strain (Arthrobacter sp., MCCC 1A05493) with the highest activity was isolated, whose SHMT was designated as AmSHMT. DNA matching and amino acid sequence analysis A sequence (1,574 bp) was obtained by matching the PglyA (936 bp) with the flanking fragments (335 and 613 bp) identified by TAIL-PCR (Fig. 2). Then the Fig. 2 PglyA gene fragment and its flanking gene fragments. (a) A part of whole glyA gene sequence (1,323 bp) was obtained the full-long AmglyA gene (PglyA, 936 bp) was amplified using using the ORF search tool from SoftBerry (http:// degenerate primers DP-F and DP-R. (b) The left flanking sequence (335 bp) was amplified through TAIL-PCR technology. (c) The right linux1.softberry.com/berry.phtml). The AmSHMT flanking sequence (613 bp) was amplified through TAIL-PCR exhibited 58 % amino acid identity with the known technology SHMT from E. coli and encoded a protein with 440 amino acids and a deduced molecular mass of 47. LTNKYAEGYPGRRYYGG (61–77) and GGHLTHG 3 kDa. A phylogenetic tree was constructed to verify (134–140) (Hong et al. 1999), were also found in further the evolutionary relationship among AmSHMT AmSHMT. and other known SHMTs (Fig. 3). The conserved active site T/ST/STTHKT/SL in all known SHMT proteins (Garrow et al. 1993)wasfound Expression, purification, and characterization of AmSHMT in AmSHMT (235–242) in the form of TSTTHKTL (Fig. 4). Another significant homologous sequence AmSHMT was expressed, purified, and its molecular mass GQQGGP (268–273), a glycine-rich region, had been was determined by SDS-PAGE analysis (Fig. 1b). SHMT proposed to be essential for PLP binding (Usha et al. showed optimal activity at pH 7.8, and retained over 75 % 1994). Two other well-known conserved sequences, of the maximal activity between pH 7.0 and 8.0. Furthermore, Fig. 3 Phylogenetic analysis of AmSHMT. The phylogenetic tree of AmSHMT was constructed using the neighbor-joining method (MEGA 5.05). Except for AmSHMT, the other SHMT sequences were obtained from GenBank and PDB (http://www. rcsb.org/pdb/home/home.do). The numbers at node indicate the bootstrap percentages of 1,000 resamples. The units at the bottom of the tree indicate the number of substitution events 1694 Ann Microbiol (2015) 65:1689–1698 Fig. 4 Multiple sequence alignment of SHMTs. The sequences in the boxes are conserved amino acid residues 2+ 2+ 2+ the enzyme was sensitive to low pH, displaying less than 20 % was weakly enhanced by Mg ,Ca ,Pb and EDTA, but 2+ 2+ 2+ 2+ 2+ of its maximal activity at pH 6.5 and nearly no activity below was strongly inhibited by Hg ,Co ,Cu ,Mn and Fe . pH 5.8 (Fig. 5a). Without any stabilizer, the purified SHMT In addition, SDS and CTAB also inhibited SHMTactivity, and + + + was apparently stable under weakly alkaline conditions K ,Na and NH showed no appreciable impact on the (pH 7–7.4), retaining over 85 % of the maximal activity after SHMT activity. 3 h at 45 °C. However, it was unstable under acidic conditions In terms of activity, the AmSHMT (287.9 units/mg) was or in strongly alkaline environment, especially when the pH 2.75-fold higher than the EcSHMT (104.7 units/mg) under was over 9.0 (Fig. 5c). standard assay conditions. The maximal activity of SHMT was observed at 45 °C (Fig. 5b). However, SHMT retained over 50 % of its maximal Production of L-serine by resting cells reaction activity after 3 h incubation under pH 7.8 (Fig. 5c), and decreased rapidly in activity at temperatures over 40 °C. After 48 h incubation at 28 °C as described in section of the The effects of metal ions and chemical reagents on SHMT resting cells reaction system, 1.8±0.3 mg/ml L-serine was are shown in Table 3, indicating that the activity of the enzyme obtained by RP-HPLC analysis. Ann Microbiol (2015) 65:1689–1698 1695 Fig. 5 Effects of temperature and pH on the activity. (a)Effect of pH on 45 °C, the purified enzyme was pre-treated at a different pH for 3 h. Then the activity of AmSHMT. Assays were conducted in buffers over a pH assays were conducted under standard conditions and the enzyme activity range from 5.8 to 9.5 at 30 °C for 1 h, under standard conditions. The without pre-treatment was taken as 100 %. (d) Effect of temperature on maximal activity was taken as 100 %. (b) Effect of temperature on the the stability of AmSHMT. At the optimal pH 7.8, the purified enzyme was activity of AmSHMT. Assays were conducted at the optimal pH 7.8, pre-treated at a different temperature for 3 h. Then assays were conducted under standard conditions. The maximal activity was taken as 100 %. under standard conditions and the enzyme activity without pre-treatment (c) Effect of pH on the stability of AmSHMT. At the optimal temperature was taken as 100 % Production of L-serine by enzymatic reaction SHMT, a member of the α-family of PLP-dependent en- zymes (Mehta and Christen 2000), exists as a dimer in Using the enzymatic reaction system described in the Psychromonas ingrahamii (Siglioccolo et al. 2010) and is methods, L-serine was detected by RP-HPLC analysis 24 h ubiquitous for generating one-carbon fragments for the syn- later (Figs. 6 and 7a). The L-serine concentration at the 12th thesis of nucleotides, methionine, thymidylate, and choline hour was calculated to be 93.6 mM in the pET-15b-AmglyA (Appaji Rao et al. 2003). This enzyme is also useful in the system, but 71.1 mM in the pET-15b-EcglyA system, indicat- synthesis of serine using glycine and formaldehyde. There- ing that the former was 70.3 % in the molecular conversion fore, studying the enzymatic properties of SHMT can provide rate, which was 1.32-fold higher than the latter (53.4 %). guidance for the industrial production of L-serine. Using the resting cells reaction system, 1.8±0.3 mg/ml L- serine was produced. The reasons for the low yield might be that the metabolic pathways in vivo are so complex that L- serine can be quickly degraded and converted to other sub- Discussion stances. For instance, even the Methylobacterium sp. strain MN43, which has the highest glycine conversion rate ever In this study, we obtained Arthrobacter sp., a methanol-using strain with high SHMT activity, which was first reported in reported, can degrade 32 g/l L-serine in 2 days (Hagishita et al. 1996). 1972 as a new species excreting L-glutamic acid (Nand and Rao 1972). The glyA gene was cloned by TAIL-PCR, an From Fig. 5a, it can be seen that the two engineering bacteria produced little L-serine during the first 6 h, but efficient PCR strategy, using AD1-10 primers separately. However, only AD8 and AD10 primers were better suited both of them could produce L-serine rapidly 6 h later, especially engineering bacterium pET-15b-AmglyA.This for PCR amplification, and all the other AD primers often led to non-targeted, dispersive or small fractional products, prob- was probably because the coenzyme (PLP and THFA) and substrate were binding to SHMT during this time, but 6 h ably due to codon usage and the cycling conditions. In any later when the binding process was completed, the conver- case, choosing more AD primers is helpful for TAIL-PCR sion reaction would proceed rapidly and more L-serine amplification. 1696 Ann Microbiol (2015) 65:1689–1698 Table 3 Effects of metal ions and chemical reagents on the activity of purified SHMT* Metal ion and chemical Concentration Relative activity reagent (mM) (%) None 0 100 % 2+ a Hg 1 − 2+ b Co 1 65.0±0.2 K 1 99.4±0.1 Na 1 98.7±0.4 NH 1 99.5±0.7 2+ Mg 1101.4±0.6 2+ Ca 1103.7±0.4 2+ Cu 1 48.9±0.8 2+ Zn 1 83.1±0.1 2+ Mn 1 63.1±0.4 2+ Fe 1 68.3±0.1 2+ Pb 1101.9±0.3 EDTA 1 % 102.1±0.3 CTAB 0.03 % 71.5±0.5 SDS 0.1 % – *The data are the average of three replicates Unmeasured data Relative activity ± the standard deviation Fig. 6 HPLC detection of the L-serine in the enzymatic reaction system. All assays were performed in the standard conditions and the activity (a) Determination of glycine and L-serine standards; (b) Determination of measured without additional reagents and ions was taken as 100 % L-serine synthesis by pET-15b-AmglyA in the enzymatic reaction system at the 12th hour; (c) Determination of L-serine synthesis by pET-15b- EcglyA in the enzymatic reaction system at the 12th hour could be produced during 6–12 h. At the 14th hour, the concentration of L-serine significantly decreased in the generate glycine and formaldehyde. At the 24th hour, the L- serine concentration showed a significant decrease in the pET-15b-AmglyA reaction system, while that of the pET- 15b-EcglyA reaction system showed a steady increase, pET-15b-AmglyA reaction system, but little variation in the resulting in a significant descrease in L-serine conversion pET-15b-EcglyA system compared with that at the 14th in both systems, which could be attributed to the possibility hour. The possible reason was that 14 h later when the that the coenzymes were almost entirely consumed in the THFA and PLP had been almost completely consumed, former reaction system; thus, the enzymatic reaction would AmSHMT still retained a little activity, and thus the enzy- be favourably performed in the reverse reaction direction to matic conversion sequentially proceeded in the reverse Fig. 7 HPLC and SDS-PAGE analysis of the induced AmSHMT and and pET-15b-EcglyAwere treated in the same conditions. Lanes 1, 2, and EcSHMT. (a) HPLC analysis of the L-serine concentration in the 3 indicate the three replicates of induced EcSHMT from engineering enzymatic reaction system during 24 h. (b) SDS-PAGE analysis of the bacteria pET-15b-EcglyA; lanes 4, 5, and 6 indicate the three replicates induced AmSHMT and EcSHMT. Engineering bacteria pET-15b-AmglyA of induced AmSHMT from engineering bacteria pET-15b-AmglyA Ann Microbiol (2015) 65:1689–1698 1697 Acknowledgments This work was supported by grants from China reaction direction. In the pET-15b-EcglyA reaction system, National Natural Sciences Foundation (No. 31270162) and a project the EcSHMT almost completely lost activity and the re- funded by the Priority Academic Program Development of Jiangsu verse reaction was blocked, so that at the 24th hour, the L- Higher Education Institutions. serine concentration almost remained constant. Conflict of interests The authors claim that they have no competing The above analyses indicated that the pET-15b-AmglyA interests. And there are not any non-financial competing interests. engineering bacterium has more potential for industrial appli- cations, especially at the 12th hour, when the L-serine con- centration reached the maximum value, because its faster conversion rate was conducive to time saving and cost reduc- References ing. However, the present work cannot meet the requirements of industrial applications and much work needs to done to Amedeo P, Habu Y, Afsar K, Scheid OM, Paszkowski J (2000) further improve the L-serine yield. Disruption of the plant gene MOM releases transcriptional silencing of methylated genes. Nature 405:203–206 The two engineering bacteria were treated under the Appaji Rao N, Ambili M, Jala V, Subramanya H, Savithri H (2003) same conditions, and interestingly, AmSHMT was found Structure-function relationship in serine hydroxymethyltransferase. to be lower than EcSHMT in the expression level Biochim Biophys Acta 1647:24–29 (Fig. 5b), which might be caused by codon usage bias or Barra D, Martini F, Angelaccio S, Bossa F, Gavilanes F, Peterson D, Bullis B, Schirch L (1983) Sequence homology between prokary- other reasons. However, even under such conditions, pET- otic and eukaryotic forms of serine hydroxymethyltransferase. 15b-AmglyA was still higher than pET-15b-EcglyA in the Biochem Biophys Res Commun 116:1007–1012 conversion rate during 12 h. So in the next study, the Blakley RL (1955) The interconversion of serine and glycine: participa- codons of AmSHMT should be optimized to improve its tion of pyridoxal phosphate. Biochem J 61:315–323 Byrne PC, Sanders P, Snell K (1992) Nucleotide sequence and expression expression in E. coli (Carbone et al. 2003). Many published of a cDNA encoding rabbit liver cytosolic serine reports provide helpful information on how to improve the hydroxymethyltransferase. Biochem J 286:117–123 yield. With the triparental mating method, a new recombi- Carbone A, Zinovyev A, Kepes F (2003) Codon adaptation index as a nant strain MB202 was screened, whose SHMT activity measure of dominating codon bias. Bioinformatics 19:2005–2015 Das S, Lyla P, Khan SA (2006) Marine microbial diversity and ecology: was approximately 3.5-fold higher than that of the parent importance and future perspectives. Curr Sci 90:1325–1335 strain, and L-serine output was 4.4-fold higher (Shen et al. Emelyanov A, Gao Y, Naqvi NI, Parinov S (2006) Trans-kingdom trans- 2010). SdaA-encoded L-serine dehydratase has been dem- position of the maize dissociation element. Genetics 174:1095–1104 onstrated to be involved in L-serine degradation (Netzer Garrow T, Brenner A, Whitehead V, Chen XN, Duncan R, Korenberg J, Shane B (1993) Cloning of human cDNAs encoding mitochondrial et al. 2004). With the presence of L-serine dehydratase in and cytosolic serine hydroxymethyltransferases and chromosomal Arthrobacter globiformis SK-200, 80 % of the L-serine in localization. J Biol Chem 268:11910–11916 the medium was consumed in 2 days (Tani et al. 1978). Hagishita T, Yoshida T, Izumi Y, Mitsunaga T (1996) Efficient L-serine When glyA expression was reduced and L-serine production from methanol and glycine by resting cells of dehydratase activity was missing, L-serine production in- Methylobacterium sp. strain MN43. Biosci Biotechnol Biochem 60:1604–1607 creased up to 9.04 mg/ml (Peters-Wendisch et al. 2005). By Hamilton B, Hsiao H, Swann W, Anderson D, Delente J (1985) Manufacture using error-prone PCR, DNA shuffling and high through- of L-amino acids with bioreactors. Trends Biotechnol 3:64–68 put screening, a mutant with approximately eightfold Hong MC, Wu ML, Chang MC (1999) Cloning and complete nucleotide higher enzyme activity and 41-fold higher enzyme produc- sequence of Acinetobacter radioresistens CMC-1 AglyA gene encoding serine hydroxymethyltransferase. FEMS Microbiol Lett tivity than the wild-type parent was obtained (Zuo et al. 170:413–418 2007). Hsiao H, Walter J, Anderson D, Hamilton B (1988) Enzymatic produc- tion of amino acids. Biotechnol Genet Eng Rev 6:179–219 Izumi Y, Yoshida T, Miyazaki SS, Mitsunaga T, Ohshiro T, Shimao M, Miyata A, Tanabe T (1993) L-Serine production by a methylotroph and its related enzymes. Appl Microbiol Biotechnol 39:427–432 Conclusions Jiang W, Xia B, Liu Z (2013) A serine hydroxymethyltransferase from marine bacterium Shewanella algae: Isolation, purification, charac- The SHMT in the present study is a novel one from terization and l-serine production. Microbiol Res 168:477–484 Jones C, Priest D (1976) Metabolism of glutamine and ammonia in rat Arthrobacter sp., which was stable in weakly alkali condi- liver: the effects of N-acetylglutamate and phosphate. Arch tions, showing optimal activity at pH 7.8 and 45 °C, and a Biochem Biophys 174:305–311 2.75-fold higher activity than the corresponding enzyme from Li C, Hong Y, Shao Z, Lin L, Huang X, Liu P, Wu G, Meng X, Liu Z (2009) Novel alkali-stable, cellulase-free xylanase from deep-sea E. coli. The molecular conversion rate of L-serine in the pET- Kocuria sp. Mn22. J Microbiol Biotechnol 19:873–880 15b-AmglyA system was 70.3 % and 1.32-fold higher than Lin L, Meng X, Liu P, Hong Y, Wu G, Huang X, Li C, Dong J, Xiao L, that in the pET-15b-EcglyA system (53.4 %). Based on these Liu Z (2009) Improved catalytic efficiency of endo-β-1,4-glucanase characteristics, this strain and its enzyme have the potential for from Bacillus subtilis BME-15 by directed evolution. Appl further research and industrial use. Microbiol Biotechnol 82:671–679 1698 Ann Microbiol (2015) 65:1689–1698 Liu YG, Huang N (1998) Efficient amplification of insert end sequences Methylobacterium sp MB200. Appl Biochem Biotechnol 160:740– from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR. Plant Mol Biol Report 16:175–175 Siglioccolo A, Bossa F, Pascarella S (2010) Structural adaptation of serine Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: auto- hydroxymethyltransferase to low temperatures. Int J Biol Macromol matable amplification and sequencing of insert end fragments from 46:37–46 P1 and YAC clones for chromosome walking. Genomics 25:674– Sun J (2000) Study on synthesis of L-serine catalyzed by SHMT recom- 681 binant bacteria. Dissertation, China Pharmaceutical University Mehta PK, Christen P (2000) The molecular evolution of pyridoxal-5′- Sun J, Wu W, Chen Z (2000) Synthesis of coenzyme THFA and deter- phosphate-dependent enzymes. Adv Enzymol Relat Areas Mol Biol mination of its relative activity. Pharm Biotechnol 7:38–41 74:129–184 Tani Y, Kanagawa T, Hanpongkittikun A, Ogata K, Yamada H (1978) Miyata A, Yoshida T, Yamaguchi K, Yokoyama C, Tanabe T, Toh H, Production of L-Serine by a methanol-utilizing bacterium, Mitsunaga T, Izumi Y (1993) Molecular cloning and expression of Arthrobacter globiformis SK-200. Agric Biol Chem 42:2275–2279 the gene for serine hydroxymethyltransferase from an obligate Ulevitch RJ, Kallen RG (1977) Studies of the reactions of lamb liver methylotroph Hyphomicrobium methylovorum GM2. Eur J serine hydroxymethylase with L-phenylalanine: kinetic isotope ef- Biochem 212:745–750 fects upon quinonoid intermediate formation. Biochemistry 16: Nand K, Rao D (1972) Arthrobacter mysorens-a new species excreting L- 5350–5354 glutamic acid. Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Usha R, Savithri HS, Appaji Rao N (1994) The primary structure of sheep 127:324–331 liver cytosolic serine hydroxymethyltransferase and an analysis Netzer R, Peters-Wendisch P, Eggeling L, Sahm H (2004) Cometabolism of the evolutionary relationships among serine of a nongrowth substrate: L-serine utilization by Corynebacterium hydroxymethyltransferases. Biochim et Biophys Acta (BBA)- glutamicum. Appl Environ Microbiol 70:7148–7155 Protein Struct Mol Enzymol 1204:75–83 Peters-Wendisch P, Stolz M, Etterich H, Kennerknecht N, Sahm H, Vidal L, Calveras J, Clapes P, Ferrer P, Caminal G (2005) Recombinant Eggeling L (2005) Metabolic engineering of Corynebacterium production of serine hydroxymethyl transferase from Streptococcus glutamicum for L-serine production. Appl Environ Microbiol 71: thermophilus and its preliminary evaluation as a biocatalyst. Appl 7139–7144 Microbiol Biotechnol 68:489–497 Rao DN, Appaji Rao N (1982) Purification and regulatory properties of Zhao M, Ma Y, Dai L, Zhang D, Li J, Yuan W, Li Y, Zhou H (2012) A mung bean (Vigna radiata L.) serine hydroxymethyltransferase. high-performance liquid chromatographic method for simultaneous Plant Physiol 69:11–18 determination of 21 free amino acids in Tea. Food Anal Methods 6: Schirch V, Hopkins S, Villar E, Angelaccio S (1985) Serine 69–75 hydroxymethyltransferase from Escherichia coli: purification and Zuo Z-Y, Zheng Z-L, Liu Z-G, Yi Q-M, Zou G-L (2007) Cloning, DNA properties. J Bacteriol 163:1–7 shuffling and expression of serine hydroxymethyltransferase gene Shen P, Chao H, Jiang C, Long Z, Wang C, Wu B (2010) Enhancing from Escherichia coli strain AB90054. Enzym Microb Technol 40: production of L-serine by increasing the glyA Gene expression in 569–577

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Annals of Microbiology – Springer Journals

Published: Jan 7, 2015

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