Optimization of Expression Conditions Enhances Production Of

J. Microbiol. Biotechnol. (2015), 25(10), 1709–1713 http://dx.doi.org/10.4014/jmb.1506.06034 Research Article Review jmb

Optimization of Expression Conditions Enhances Production of Sepiapterin, a Precursor for Tetrahydrobiopterin Biosynthesis, in Recombinant Escherichia coli Eun-Hee Park1, Won-Heong Lee2, Mi-Hee Jang1, and Myoung-Dong Kim1*

1Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 200-701, Republic of Korea 2Department of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

Received: June 15, 2015

Revised: July 16, 2015 Sepiapterin is a precursor for the synthesis of tetrahydrobiopterin (BH4), which is a well- Accepted: July 18, 2015 known cofactor for aromatic amino acid hydroxylation and nitric oxide synthesis in higher mammals. In this study, a recombinant Escherichia coli BL21(DE3) strain harboring cyanobacterial guanosine 5’-triphosphate cyclohydrolase 1 (GCH1) and human 6-

First published online pyruvoyltetrahydropterin synthase (PTPS) genes was constructed to produce sepiapterin. The July 22, 2015 optimum conditions for T7 promoter–driven expression of GCH1 and PTPS were 30°C and *Corresponding author 0.1 mM isopropyl-β-D-thioglucopyranoside (IPTG). The maximum sepiapterin concentration Phone: +82-33-250-6458; of 88.1 ± 2.4 mg/l was obtained in a batch cultivation of the recombinant E. coli, corresponding Fax: +82-33-259-5565; to an 18-fold increase in sepiapterin production compared with the control condition (37°C E-mail: [email protected] and 1 mM IPTG). pISSN 1017-7825, eISSN 1738-8872

Tetrahydrobiopterin (BH4) is a well-known essential ovarian cancer cells. cofactor for phenylalanine hydroxylase [5], tyrosine The BH4 biosynthetic pathway begins from GTP as a hydroxylase [11], tryptophan hydroxylase [8], fatty acid starting material in the de novo pathway (Fig. 1). In the first glycerylether oxygenase [18], and nitric oxide synthase [6]. reaction, GTP is converted to dihydroneopterin triphosphate

Decreased levels of BH4 in the cerebrospinal fluid have (NH2TP) by GTP-cyclohydrolase 1 (GCH1) (E.C. 3.5.4.16) been documented in neurological diseases presenting [12], and then NH2TP is converted to 6-pyruvoyl- phenotypically without hyperphenylalaninemia, such as tetrahydropterin (6PPH4) by 6-pyruvoyltetrahydropterin

Parkinson’s disease [3, 19], autism [4], depression [20], and synthase (PTPS) (E.C. 4.2.3.12). Finally, BH4 is produced Alzheimer’s disease [1]. Sepiapterin can be used as a through a reaction catalyzed by sepiapterin reductase (SR) precursor of the salvage pathway for BH4 synthesis as well (E.C. 1.1.1.153) [19]. Alternatively, aldose reductase (E.C. as a substitute to BH4. When sepiapterin is added to 1.1.1.21) catalyzes the reduction of the C-2’ oxo group in mammalian tissue culture instead of BH4, it can show the PPH4, yielding 6-lactoyltetrahydropterin (LPH4) [10], same effect as BH4 [16]. Sepiapterin is more stable than BH4 which is further reduced to BH4 by SR or is converted and its synthesis is not affected by the cellular BH4 level, nonenzymatically to its dihydro form, sepiapterin. Escherichia which suggests that sepiapterin can replace BH4 when the coli is unable to produce either BH4 or sepiapterin because latter is absent [15]. Sepiapterin administration can prevent it lacks BH4 biosynthetic enzymes; however, in this renal damage in a rat model of acute kidney injury after study we demonstrate that simple introduction of two suprarenal aortic clamping [7] and reduces postischemic heterologous biosynthetic enzymes and optimization of the injury in the rat heart [17]. Cho et al. [2] reported that culture conditions can lead to the formation of considerable sepiapterin inhibits the proliferation and migration of amounts of sepiapterin in E. coli.

October 2015 ⎪ Vol. 25⎪ No. 10 1710 Park et al.

methods described by Sambrook and Russell [13]. To determine the optimal expression conditions for the GCH1 and PTPS genes, various sets of the induction temperature and concentration of isopropyl-β-D- thiogalactopyranoside (IPTG) were tested. E. coli BL21(DE3) cells harboring pME721 were grown in semi-synthetic medium (100 ml) at 37°C until the dry cell mass reached

0.3 g/l. IPTG was then added to induce T7 promoter– driven expression of both genes. To verify the expression of the GCH1 and PTPS genes, cells induced for 4 h were harvested by centrifugation at 10,000 ×g for 5 min at 4°C, resuspended in lysis buffer [13], and disrupted using an ultrasonic processor (Cole-Parmer, Vernon Hills, IL, USA). Crude extracts were fractionated into soluble and insoluble fractions by centrifugation at 16,000 ×g for 10 min at 4°C. Fig. 1. The sepiapterin biosynthetic pathway from glycerol. Soluble and insoluble protein fractions were analyzed by GAP, glyceraldehyde 3’-phosphate; IMP, inosine 5’-monophosphate; XMP, xanthosine 5’-monophospate; GMP, guanosine 5’-monophosphate; sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE; 10% polyacrylamide). Gels were stained with GDP, guanosine 5’-diphosphate; GTP, guanosine 5’-triphosphate; NH2TP, Coomassie Brilliant Blue R-250. The dry cell mass 7,8-dihydroneopterin; 6PPH4, 6-pyruvoyltetrahydropterin; LPH4, 6- lactoyltetrahydropterin; 1’-oxo-TP, 1’-oxo-2’-hydroxypropyltetrahydropterin. concentration was determined from the optical density of the culture broth measured at 600 nm and a predetermined standard curve. To determine sepiapterin concentrations, Escherichia coli TOP10 and BL21(DE3) strains (Invitrogen, filtered (0.2 µm) culture broth and crude cell extract were Carlsbad, CA, USA) were used as hosts for genetic injected into an Inertsil ODS-3 column (GL Sciences Inc., manipulation and sepiapterin production, respectively. Tokyo, Japan) installed on the HPLC system and Plasmid pETDuet-1 (pBR322 replicon, Ampr) was purchased equilibrated at 40°C with 10 mM sodium phosphate (pH from Merck Biosciences (Darmstadt, Germany) and used to 6.0). The column was eluted with the same buffer at a flow express GCH1 and PTPS. Luria-Bertani (LB) medium [9] rate of 1.2 ml/min. Detection was performed using a UV/ and a semi-synthetic medium [14] supplemented with Visible detector (Shimadzu, Japan). LC-MS/MS analyses glycerol (2% (w/v)) as a carbon source were used for were performed on a Thermo Scientific TSQ Quantum plasmid DNA preparation and sepiapterin production, Ultra triple quadrupole mass spectrometer (Thermo Electron respectively. Ampicillin (Sigma-Aldrich, St. Louis, MO, Corp., WI, USA) in the positive electrospray ionization USA) was added at a final concentration of 50 mg/l to mode with the following operation parameters: capillary select plasmid-harboring E. coli cells. Cyanobacterial GCH1 voltage, 3.6 kV; ion source, 5.5 kV; temperature, 320°C; (GenBank Accession No. AY120852.1; 0.7 kb) and human mobile phase, 10% methanol; and flow rate, 200 µl/min. PTPS (GenBank Accession No. NM_000317.2; 0.4 kb) genes Commercial human sepiapterin (Sigma-Aldrich) was used were synthesized by Bioneer (Daejeon, Korea). The primers as a standard for analysis. All experiments were performed used for PCR were GCH1-F (5’-AATT CCATGGTGT in triplicate. TGAGGTTTCTTGTGACGAT-3’), GCH1-R (5’-AATTCT To examine the effects of IPTG concentration and GCAGTTAGGCAAATGCGGCAGGTT-3’), PTPS-F (5’- induction temperature on the expression of GCH1 and AATTCATATGAGCACGGAAGGTGGTGG-3’), and PTPS- PTPS, recombinant E. coli BL21(DE3) cultures were induced R (5’-AATTCTCGAGCTATTCTCCTTTATAAACCA-3’); with various concentrations of IPTG and incubated at 25°C, plasmids pGEMT-GCH1 (pME752) and pGEMT-PTPS 30°C, or 37°C. As shown in Fig. 2, SDS-PAGE analysis of (pME935) was used as respective templates. The amplified recombinant E. coli extracts indicated that GCH1 was mostly products were digested with NcoI/PstI and NdeI/XhoI, expressed as insoluble inclusion bodies. The induction respectively, and cloned into pETDuet-1. The resulting temperature of 30°C was optimal based on the total plasmid, pME721 (6.6 kb), was sequenced using vector- amount of GCH1 produced. When IPTG was added at a and insert-specific primers. All DNA manipulations and final concentration of 0.1 mM, the fraction of soluble GCH1 bacterial transformations were performed according to the was higher than with other IPTG concentrations; the total

J. Microbiol. Biotechnol. Sepiapterin Production in Recombinant Escherichia coli 1711

Fig. 2. SDS-PAGE analysis of GCH1 and PTPS expression in Electrospray ionization mass spectra of the sepiapterin recombinant E. coli BL21(DE3). Fig. 3. standard (A) and cell-free culture broth (B), and HPLC Cells were harvested after 4-h induction with IPTG at 25°C (A), 30°C (B), or 37°C (C). T, S, and I denote total, soluble, and insoluble protein analysis of cell-free culture broth (C). fractions, respectively. Arrows indicate protein bands that match the Cell-free culture broth of recombinant E. coli BL21(DE3) was obtained estimated molecular mass of GCH1 and PTPS. Lane M, molecular after 4-h induction by 0.1 mM IPTG at 30°C. weight marker. The gel was stained with Coomassie Brilliant Blue R-250. the recombinant E. coli BL21(DE3) strain harboring plasmid pME721. Electrospray ionization-MS spectra of both the GCH1 level was similar at all tested IPTG concentrations. sepiapterin standard (Fig. 3A) and culture broth of However, no noticeable amount of soluble PTPS was recombinant E. coli (Fig. 3B) showed a compound with m/z detected under any induction conditions, indicating that 234.02. HPLC analysis revealed that a compound with a further studies are needed to increase the expression level retention time identical to that of the sepiapterin standard of cyanobacterial PTPS or to clone other PTPS genes was produced in recombinant E. coli BL21(DE3)/pME721 that may encode more soluble proteins. The maximum (Fig. 3C). Sepiapterin was not produced in the control sepiapterin concentration of 88.1 ± 2.4 mg/l was obtained strain harboring the empty vector (pETDuet-1). from recombinant E. coli BL21(DE3) cultures induced with Previously, sepiapterin production in E. coli was attempted 0.1 mM IPTG at 30°C, which is consistent with the SDS- by expression of cyanobacterial GCH1 and human PTPS PAGE analysis of protein expression patterns. However, [21]. That study focused only on the confirmation of sepiapterin was not detected in the crude cell extract under sepiapterin production in recombinant E. coli and suggested the same experimental conditions. Chromatographic analysis that one or more E. coli aldo-keto reductases could convert was carried out to confirm the production of sepiapterin in 6PPH4 to LPH4, a non-enzymatic precursor of sepiapterin,

October 2015 ⎪ Vol. 25⎪ No. 10 1712 Park et al.

Table 1. Summary of sepiapterin production in recombinant E. coli BL21(DE3) under different induction conditions.a Induction IPTG Dry cell Sepiapterin Specific sepiapterin temperature (°C) concentration (mM) concentration (g/l) concentration (mg/l) productivity (mg/g·h) 25 0 3.5 ± 0.2 7.5 ± 0.3 0.09 ± 0.03 0.1 3.2 ± 0.3 53.7 ± 0.8 0.70 ± 0.05 0.5 2.5 ± 0.7 37.1 ± 0.2 0.62 ± 0.09 1.0 2.1 ± 0.1 25.4 ± 0.9 0.50 ± 0.04 30 0 4.9 ± 0.3 9.0 ± 0.5 0.08 ± 0.00 0.1 3.7 ± 0.2 88.1 ± 2.4 1.01 ± 0.10 0.5 2.9 ± 0.1 58.7 ± 3.9 0.84 ± 0.08 1.0 2.8 ± 0.2 46.3 ± 3.2 0.69 ± 0.09 37 0 4.3 ± 0.2 5.2 ± 0.3 0.05 ± 0.02 0.1 4.4 ± 0.2 44.2 ± 1.9 0.42 ± 0.04 0.5 4.7 ± 0.3 5.4 ± 0.3 0.05 ± 0.01 1.0 4.7 ± 0.1 7.2 ± 0.2 0.06 ± 0.00 aValues are means ± standard error from three independent experiments. In all experiments, shake-flask cultivation was performed for 24 h. which indicated that sepiapterin can be synthesized and Technology (NRF-2009-0066557). M.D. Kim was supported without introduction of additional enzymes catalyzing the by “Cooperative Research Program for Agriculture Science conversion of PPH4 to sepiapterin. However, the maximum & Technology Development (Project No. PJ009477),” Rural yield of sepiapterin was estimated to be approximately Development Administration, Republic of Korea. 33 mg/l even though its extracellular secretion was successful, which might be explained by the leaky expression References of GCH1 and PTPS allowing sepiapterin accumulation even in the absence of IPTG [21]. 1. Barford PA, Blair JA, Eggar C, Hamon C, Morar C, In this study, culture conditions for sepiapterin production Whitburn SB. 1984. Tetrahydrobiopterin metabolism in the in E. coli were optimized through a series of batch temporal lobe of patients dying with senile dementia of fermentations. As shown in Table 1, sepiapterin production Alzheimer type. J. Neurol. Neurosurg. Psychiatry 47: 736-738. was maximized by adjusting the incubation temperature 2. Cho YR, Kim SH, Ko HY, Kim MD, Choi SW, Seo DW. 2011. Sepiapterin inhibits cell proliferation and migration of and IPTG concentration. Undesirable sepiapterin formation ovarian cancer cells via down-regulation of p70S6K- without IPTG induction was minimal under all culture dependent VEGFR-2 expression. Oncol. Rep. 26: 861-867. conditions, possibly because the cells were grown in a 3. Curtius HC, Müldner H, Niederwieser A. 1982. semi-synthetic medium. It should be easy to perform Tetrahydrobiopterin: efficacy in endogenous depression and fed-batch fermentation to produce higher amounts of Parkinson’s disease. J. Neural Transm. 55: 301-308. sepiapterin on the basis of our current results. 4. Frye RE, Huffman LC, Elliott GR. 2010. Tetrahydrobiopterin In conclusion, this study describes a straightforward as a novel therapeutic intervention for autism. Neurotherapeutics method to improve the heterologous expression of two 7: 241-249. genes essential for sepiapterin biosynthesis in recombinant 5. Kaufman S. 1958. A new cofactor required for the enzymatic E. coli. Simple optimization of the expression conditions conversion of phenylalanine to tyrosine. J. Biol. Chem. 230: resulted in an 18-fold increase in sepiapterin production in 931-939. comparison with the control conditions. 6. Kwon NS, Nathan CF, Stuehr DJ. 1989. Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J. Biol. Chem. 264: 20496-20501. Acknowledgments 7. Legrand M, Kandil A, Payen D, Ince C. 2011. Effects of sepiapterin infusion on renal oxygenation and early acute This study was supported by the Basic Science Research renal injury after suprarenal aortic clamping in rats. J. Program through the National Research Foundation of Cardiovasc. Pharmacol. 58: 192-198. Korea (NRF) funded by the Ministry of Education, Science 8. Lovenberg W, Jequier E, Sjoerdsma A. 1967. Tryptophan

J. Microbiol. Biotechnol. Sepiapterin Production in Recombinant Escherichia coli 1713

hydroxylation: measurement in pineal gland, brainstem, and 16. Tiefenbacher CP. 2001. Tetrahydrobiopterin: a critical carcinoid tumor. Science 155: 217-219. cofactor for eNOS and a strategy in the treatment of 9. Miller H. 1987. Practical aspects of preparing phage and endothelial dysfunction? Am. J. Physiol. Heart Circ. Physiol. plasmid DNA: growth, maintenance, and storage of bacteria 280: 2484-2488. and bacteriophage. Methods Enzymol. 152: 145-170. 17. Tiefenbacher CP, Lee CH, Kapitza J, Dietz V, Niroomand F. 10. Milstien S, Kaufman S. 1989. Immunological studies on the 2003. Sepiapterin reduces postischemic injury in the rat participation of 6-pyruvoyl tetrahydropterin (2’-oxo) reductase, heart. Pflugers Arch. 447: 1-7. an aldose reductase, in tetrahydrobiopterin biosynthesis. 18. Tietz A, Lindberg M, Kennedy EP. 1964. A new pteridine- Biochem. Biophys. Res. Commun. 165: 845-850. requiring enzyme system for the oxidation of glyceryl 11. Nagatsu T, Levitt M, Udenfriend S. 1964. Tyrosine ethers. J. Biol. Chem. 239: 4081-4090. hydroxylase: the initial step in norepinephrine biosynthesis. 19. Tobin JE, Cui J, Wilk JB, Latourelle JC, Laramie JM, McKee J. Biol. Chem. 239: 2910-2917. AC, et al. 2007. Sepiapterin reductase expression is increased 12. Nasser A, Møller LB. 2014. GCH1 variants, tetrahydrobiopterin in Parkinson's disease brain tissue. Brain Res. 1139: 42-47. and their effects on pain sensitivity. Scand. J. Pain 5: 121-128. 20. Van Amsterdam JG, Opperhuizen A. 1999. Nitric oxide and 13. Sambrook J, Russell DW. 2001. Molecular Cloning. Cold biopterin in depression and stress. Psychiatry Res. 85: 33-38. Spring Harbor Laboratory Press, New York. 21. Woo HJ, Kang JY, Choi YK, Park YS. 2002. Production of 14. Sherman F. 2002. Getting started with yeast. Methods sepiapterin in Escherichia coli by coexpression of cyanobacterial Enzymol. 350: 3-41. GTP cyclohydrolase I and human 6-pyruvoyltetrahydropterin 15. Thöny B, Auerbach G, Blau N. 2000. Tetrahydrobiopterin synthase. Appl. Environ. Microbiol. 68: 3138-3140. biosynthesis, regeneration and functions. Biochem. J. 347: 1-16.

October 2015 ⎪ Vol. 25⎪ No. 10