Applied Microbiology and Biotechnology

, Volume 97, Issue 1, pp 247–257 | Cite as

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

  • Thi Mai Hoa Bach
  • Ryotaro Hara
  • Kuniki Kino
  • Iwao Ohtsu
  • Nobuyuki Yoshida
  • Hiroshi Takagi
Applied genetics and molecular biotechnology

Abstract

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.

Keywords

l-Proline cis-4-Hydroxy-l-proline N-Acetyl cis-4-hydroxy-l-proline l-Proline cis-4-hydroxylase N-Acetyltransferase Mpr1 

Notes

Acknowledgments

We greatly appreciate Dr. Akira Nishimura and Ryo Nasuno (Nara Institute of Science and Technology, Japan) for their helpful assistance and discussion on this work. We thank Dr. Goh Matsuo (Institute for Advanced Biosciences, Keio University) and Dr. Hirotada Mori (Nara Institute of Science and Technology, Japan) providing N-acetyl CHOP, E. coli strains, and plasmids for this works. This work was supported by a Grant-in-Aid for Scientific Research (B) (22380061) from Japan Society for the Promotion of Science to H.T. and Global COE Program in NAIST from the Ministry of Education, Science, Culture, Sports and Technology of Japan.

Supplementary material

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ESM 1 (DOC 384 kb)

References

  1. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) The construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Methods Mol Biol 416:171–181CrossRefGoogle Scholar
  2. Bach TMH, Hibi T, Nasuno R, Matsuo G, Sasano Y, Takagi H (2012) Production of N-acetyl cis-4-hydroxy-L-proline by the yeast N-acetyltransferase Mpr1. J Biosci Bioeng. doi: 10.1016/j.jbiosc.2012.03.014
  3. Campos E, Montella C, Garces F, Baldoma L, Aguilar J, Badia J (2007) Aerobic l-ascorbate metabolism and associated oxidative stress in Escherichia coli. Microbiology 153:3399–3408CrossRefGoogle Scholar
  4. Cohen IK, Diegelmann RF (1978) Effect of N-acetyl-cis-4-hydroxyproline on collagen synthesis. Exp Mol Pathol 28:58–64CrossRefGoogle Scholar
  5. Du X, Takagi H (2005) N-Acetyltransferase Mpr1 confers freeze tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species. J Biochem 138:391–397CrossRefGoogle Scholar
  6. Du X, Takagi H (2007) N-Acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species. Appl Microbiol Biotechnol 75:1343–1351CrossRefGoogle Scholar
  7. Edwards N, Anderson CM, Gatfield KM, Jevons MP, Ganapathy V, Thwaites DT (2011) Amino acid derivatives are substrates or non-transported inhibitors of the amino acid transporter PAT2 (slc36a2). Biochim Biophys Acta 1808:260–270CrossRefGoogle Scholar
  8. Eldridge CF, Bunge RP, Bunge MB (1988) Effect of cis-4-hydroxy-l-proline, an inhibitor of Schwann cell differentiation, on the secretion of collagens and noncollagenous protein by Schwann cells. Exp Cell Res 174:491–501CrossRefGoogle Scholar
  9. Fowden L (1955) Azetidine-2-carboxylic acid: a new constituent of plant. Nature 176:347–348CrossRefGoogle Scholar
  10. Grothe S, Krogsrud RL, McClellan DJ, Milner JL, Wood JM (1986) Proline transport and osmotic stress response in Escherichia coli K-12. J Bacteriol 166:253–259Google Scholar
  11. Hara R, Kino K (2009) Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline. Biochem Biophys Res Commun 379:882–886CrossRefGoogle Scholar
  12. Joel R, Darwin JP (1971) Incorporation of cis-hydroxyproline into protocollagen and collagen. J Biol Chem 246:1549–1555Google Scholar
  13. Katz E, Kamal F, Mason K (1978) Biosynthesis of trans-4-hydroxy-l-proline by Streptomyces griseoviridus. J Biol Chem 254:6684–6690Google Scholar
  14. Kerr JS, Ruppert CL, Tozzi CA, Neubauer JA, Frankel HM, Yu SY, Riley DJ (1987) Reduction of chronic hypoxic pulmonary hypertension in the rat by an inhibitor of collagen production. Am Rev Respir Dis 135:300–306Google Scholar
  15. Lawrence CC, Sobey WJ, Field RA, Baldwin JE, Schofield CJ (1996) Purification and initial characterization of proline 4-hydroxylase from Streptomyces griseoviridus P8648: a 2-oxoacid, ferrous-dependent dioxygenase involved in etamycin biosynthesis. Biochem J 313:185–191Google Scholar
  16. Ling M, Allen SW, Wood JM (1994) Sequence analysis identifies the proline dehydrogenase and Δ1-pyrroline-5-carboxylate dehydrogenase domains of multifunctional Escherichia coli PutA protein. J Mol Biol 243:950–956CrossRefGoogle Scholar
  17. Miwa H, Hiyama C, Yamamoto M (1985) High performance liquid chromatography of short and long chain fatty acids as 2-nitrophenylhydrazides. J Chromatogr 321:165–174CrossRefGoogle Scholar
  18. Mori H, Shibasaki Y, Uozaki K, Ochiai K, Ozaki A (1996) Detection of novel proline 3-hydroxylase activities in Streptomyces and Bacillus spp. by regio- and stereospecific hydroxylation of l-proline. Appl Environ Microbiol 62:1903–1907Google Scholar
  19. Mori H, Shibasaki T, Yano K, Ozaki A (1997) Purification and cloning of a proline 3-hydroxylase, a novel enzyme which hydroxylate free l-proline to cis-3-hydroxy-l-proline. J Bacteriol 179:5677–5683Google Scholar
  20. Mueller C, Emmrich J, Jaster R, Braun D, Liebe S, Sparmann G (2006) Cis-hydroxyproline-induced inhibition of pancreatic cancer cell growth is mediated by endoplasmic reticulum stress. World J Gastroenterol 12:1569–1576Google Scholar
  21. Myllyla R, Kuutti-Savolainen ER, Kivirikko KI (1978) The role of ascorbate in the prolyl hydroxylase reaction. Biochem Biophys Res Commun 83:441–448CrossRefGoogle Scholar
  22. Nishimura A, Kotani T, Sasano Y, Takagi H (2010) An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced arginine synthesis and its physiological role. FEMS Yeast Res 10:687–698CrossRefGoogle Scholar
  23. Nomura M, Takagi H (2004) Role of the yeast acetyltransferase Mpr1 in oxidative stress: regulation of oxygen reactive species caused by a toxic proline catabolism intermediate. Proc Natl Acad Sci USA 101:12616–12621CrossRefGoogle Scholar
  24. Ogawa-Mitsuhashi K, Sagane K, Kuromitsu J, Takagi H, Tsukahara K (2009) MPR1 as a novel selection marker in Saccharomyces cerevisiae. Yeast 26:587–593CrossRefGoogle Scholar
  25. Penny JB, Karin MF (2000) Hydrolytic editing by class II aminoacyl-tRNA synthetase. Proc Natl Acad Sci USA 97:8916–8920CrossRefGoogle Scholar
  26. Peter R, Hellenbrand J, Mengerink Y, Van der Wal SJ (2004) On-line determination of carboxylic acids, aldehydes and ketones by high-performance liquid chromatography-diode array detection-atmospheric pressure chemical ionization mass spectrometry after derivatization with 2-nitrophenylhydrazine. J Chromatogr A 1031:35–50CrossRefGoogle Scholar
  27. Poiani GJ, Riley DJ, Fox JD, Kemnitzer JE, Gean KF, Kohn J (1994) Conjugates of cis-4-hydroxy-l-proline and poly(PEG-Lys), a water soluble poly(ether urethane): synthesis and evaluation of antifibrotic effects in vitro and in vivo. Bioconjugate Chem 5:621–630CrossRefGoogle Scholar
  28. Poiani GJ, Kemnitzer JE, Fox JD, Tozzi CA, Kohn J, Riley DJ (1997) Polymeric carrier of proline analogue with antifibrotic effect in pulmonary vascular remodeling. Am J Respir Crit Care Med 155:1384–1390Google Scholar
  29. Riley DJ, Berg RA, Edelman NH, Prockop DJ (1980) Prevention of collagen deposition following pulmonary oxygen toxicity in the rat by cis-4-hydroxy-l-proline. J Clin Invest 65:643–651CrossRefGoogle Scholar
  30. Rowe RC, Sheskey PJ, Weller PJ (2003) Polyethylene glycols. In: Rowe RC, Sheskey PJ, Weller PJ (eds) Handbook of pharmaceutical excipients, 6th edn. Pharmaceutical Press, London, pp 517–522Google Scholar
  31. Shibasaki T, Mori H, Chiba S, Ozaki (1999) Microbial proline 4-hydroxylase screening and gene cloning. Appl Environ Microbiol 65:4028–4031Google Scholar
  32. Shibasaki T, Hashimoto S, Mori H, Ozaki A (2000a) Construction of novel hydroxyproline-producing recombinant Escherichia coli by introducing a proline 4-hydroxylase gene. J Biosci Bioeng 90:522–525Google Scholar
  33. Shibasaki T, Mori H, Ozaki A (2000b) Enzymatic production of trans-4-hydroxy-l-proline by regio- and stereospecific hydroxylation of l-proline. Biosci Biotechnol Biochem 64:746–750CrossRefGoogle Scholar
  34. Shichiri M, Hoshikawa C, Nakamori S, Takagi H (2001) A novel acetyltransferase found in Saccharomyces cerevisiae Σ1278b that detoxifies a proline analogue, azetidine-2-carboxylic acid. J Biol Chem 276:41998–42002CrossRefGoogle Scholar
  35. Takagi H, Shichiri M, Takemura M, Mohri M, Nakamori S (2000) Saccharomyces cerevisiae Σ1278b has novel genes of the N-acetyltransferase gene superfamily required for l-proline analogue resistance. J Bacteriol 182:4249–4256CrossRefGoogle Scholar
  36. Tan EM, Ryhane LU (1971) Proline analogues inhibit human skin fibroblast growth and collagen production in culture. J Invest Dermatol 80:261–267CrossRefGoogle Scholar
  37. Tristram H, Neale S (1968) The activity and specificity of the proline permease in wild-type and analogue-resistant strains of Escherichia coli. J Gen Microbiol 50:121–137Google Scholar
  38. Tsai FY, Zhang XH, Ulanov A, Widholm JM (2010) The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue l-azetindine-2-carboxylic acid as a selectable marker system for plant transformation. J Exp Bot 61:2561–2573CrossRefGoogle Scholar
  39. Wada M, Okabe K, Kataoka M, Shimizu S, Yokota A, Takagi H (2008) Distribution of l-azetidine-2-carboxylate N-acetyltransferase in yeast. Biosci Biotechnol Biochem 72:582–586CrossRefGoogle Scholar
  40. Wood JM (1987) Membrane association of proline dehydrogenase in Escherichia coli is redox dependent. Proc Natl Acad Sci USA 84:373–377CrossRefGoogle Scholar
  41. Wright NP, Nolan S (2001) N-Acetyl-l-hydroxyproline: chromosome aberration test in CHL cells in vitro. SPL Project Number: 732/092. SafePharm Laboratories Ltd, DerbyGoogle Scholar
  42. Yomota C, Ohnishi (2007) Determination of biotin following derivatization with 2-nitrophenylhydrazine by high-performance liquid chromatography with on-line UV detection and electrospray-ionization mass spectrometry. J Chromatogr A 1142:231–235CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Thi Mai Hoa Bach
    • 1
  • Ryotaro Hara
    • 2
  • Kuniki Kino
    • 2
  • Iwao Ohtsu
    • 1
  • Nobuyuki Yoshida
    • 1
  • Hiroshi Takagi
    • 1
  1. 1.Graduate School of Biological SciencesNara Institute of Science and TechnologyNaraJapan
  2. 2.Department of Applied Chemistry, Faculty of Science and EngineeringWaseda UniversityTokyoJapan

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