Applied Microbiology and Biotechnology

, Volume 69, Issue 2, pp 170–177

The solvent-tolerant Pseudomonas putida S12 as host for the production of cinnamic acid from glucose

  • Karin Nijkamp
  • Nicole van Luijk
  • Jan A. M. de Bont
  • Jan Wery
Applied Genetics and Molecular Biotechnology


A Pseudomonas putida S12 strain was constructed that efficiently produced the fine chemical cinnamic acid from glucose or glycerol via the central metabolite phenylalanine. The gene encoding phenylalanine ammonia lyase from the yeast Rhodosporidium toruloides was introduced. Phenylalanine availability was the main bottleneck in cinnamic acid production, which could not be overcome by the overexpressing enzymes of the phenylalanine biosynthesis pathway. A successful approach in abolishing this limitation was the generation of a bank of random mutants and selection on the toxic phenylalanine anti-metabolite m-fluoro-phenylalanine. Following high-throughput screening, a mutant strain was obtained that, under optimised culture conditions, accumulated over 5 mM of cinnamic acid with a yield (Cmol%) of 6.7%.


  1. Adelberg EA, Mandel M, Chen CC (1965) Optimal conditions for mutagenesis by N-methyl-N′-nitro-N-nitrosoguanidine in Escherichia coli. Biochem Biophys Res Commun 18:788–795CrossRefGoogle Scholar
  2. Anson JG, Gilbert HJ, Oram JD, Minton NP (1987) Complete nucleotide sequence of the Rhodosporidium toruloides gene coding for phenylalanine ammonia-lyase. Gene 58:189–199PubMedGoogle Scholar
  3. Arias-Barrau E, Olivera ER, Luengo JM, Fernandez C, Galan B, Garcia JL, Diaz E, Minambres B (2004) The homogentisate pathway: a central catabolic pathway involved in the degradation of l-phenylalanine, l-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida. J Bacteriol 186(15):5062–5077PubMedGoogle Scholar
  4. Backman K, O’Connor MJ, Maruya A, Rudd E, McKay D, Balakrishnan R, Radjai M, DiPasquantonio V, Shoda D, Hatch R et al (1990) Genetic engineering of metabolic pathways applied to the production of phenylalanine. Ann NY Acad Sci 589:16–24PubMedGoogle Scholar
  5. Benkrief R, Ranarivelo Y, Skaltsounis A, Tillequin F, Koch M, Pusset J, Sevenet T (1998) Monoterpene alkaloids, iridois and phenylpropanoid glycosides from Osmanthus austrocaledonica. Phytochemistry 47(5):825–832CrossRefGoogle Scholar
  6. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  7. Burt S (2004) Essential oils: their antibacterial properties and potential applications in foods—a review. Int J Food Microbiol 94(3):223–253PubMedGoogle Scholar
  8. Byng GS, Whitaker RJ, Jensen RA (1983) Evolution of l-phenylalanine biosynthesis in rRNA homology group I of Pseudomonas. Arch Microbiol 136(3):163–168PubMedGoogle Scholar
  9. Calabrese JC, Jordan DB, Boodhoo A, Sariaslani S, Vannelli T (2004) Crystal structure of phenylalanine ammonia lyase: multiple helix dipoles implicated in catalysis. Biochemistry 43(36):11403–11416PubMedGoogle Scholar
  10. De Bont JAM (1998) Solvent-tolerant bacteria in biocatalysis. Trends Biotechnol 16:493–499CrossRefGoogle Scholar
  11. Dopheide TA, Crewther P, Davidson BE (1972) Chorismate mutase-prephenate dehydratase from Escherichia coli K-12. II. Kinetic properties. J Biol Chem 247(14):4447–4452PubMedGoogle Scholar
  12. El-Batal AI (2002) Continuous production of l-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor. Acta Microbiol Pol 51(2):153–169PubMedGoogle Scholar
  13. Faulkner JDB, Anson JG, Tuite MF, Minton NP (1994) High-level expression of the phenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloides in Saccharomyces cerevisiae and Escherichia coli using a bifunctional expression system. Gene 143:13–20PubMedGoogle Scholar
  14. Fiske MJ, Whitaker RJ, Jensen RA (1983) Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa. J Bacteriol 154(2):623–631PubMedGoogle Scholar
  15. Fritz RR, Hodgins DS, Abell CW (1976) Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals. J Biol Chem 251:4646–4650PubMedGoogle Scholar
  16. Frost JW, Draths KM (1995) Biocatalytic syntheses of aromatics from d-glucose: renewable microbial sources of aromatic compounds. Annu Rev Microbiol 49:557–579PubMedGoogle Scholar
  17. Gibson F, Pittard J (1968) Pathways of biosynthesis of aromatic amino acids and vitamins and their control in microorganisms. Bacteriol Rev 32(4 Pt 2):465–492PubMedGoogle Scholar
  18. Hanson KR, Havir EA (1972) The enzymatic elimination of ammonia. In: Boyer P (ed) The enzymes. Academic, New York, pp 75–167Google Scholar
  19. Hanson KR, Havir EA (1981) Phenylalanine ammonia-lyase. In: Stumpf PK, Conn EE (eds) The biochemistry of plants. A comprehensive treatise. Academic, New York, pp 577–625Google Scholar
  20. Hartmans S, Smits J, van der Werf M, Volkering F, de Bont JAM (1989) Metabolism of styrene oxide and 2-phenylethanol in the styrene-degrading Xanthobacter strain 124X. Appl Environ Microbiol 55:2850–2855PubMedGoogle Scholar
  21. Hartmans S, van der Werf MJ, de Bont JA (1990) Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase. Appl Environ Microbiol 56(5):1347–1351PubMedGoogle Scholar
  22. Hodgins DS (1971) Yeast phenylalanine ammonia-lyase; purification, properties, and the identification of catalytically essential dehydroalanine. J Biol Chem 246(9):2977–2985PubMedGoogle Scholar
  23. Hoskins JA (1984) The occurrence, metabolism and toxicity of cinnamic acid and related compounds. J Appl Toxicol 4(6):283–292PubMedGoogle Scholar
  24. Hüsken LE, Beeftink R, de Bont JA, Wery J (2001) High-rate 3-methylcatechol production in Pseudomonas putida strains by means of a novel expression system. Appl Microbiol Biotechnol 55(5):557–571Google Scholar
  25. Isken S, de Bont JA (1996) Active efflux of toluene in a solvent-resistant bacterium. J Bacteriol 178(20):6056–6058PubMedGoogle Scholar
  26. Jimenez JI, Minambres B, Garcia JL, Diaz E (2002) Genomic analysis of the aromatic catabolic pathways from Pseudomonas putida KT2440. Environ Microbiol 4(12):824–841PubMedGoogle Scholar
  27. Kieboom J, Dennis JJ, de Bont JA, Zylstra GJ (1998) Identification and molecular characterization of an efflux pump involved in Pseudomonas putida S12 solvent tolerance. J Biol Chem 273(1):85–91PubMedGoogle Scholar
  28. Lee J, Lee SY, Park S, Middelberg AP (1999) Control of fed-batch fermentations. Biotechnol Adv 17(1):29–48PubMedGoogle Scholar
  29. Lessie TG (1984) Alternative pathways of carbohydrate utilization in Pseudomonads. Annu Rev Microbiol 38:359–387PubMedGoogle Scholar
  30. Miyamoto K, Sasaki M, Minamisawa Y, Kurahashi Y, Kano H, Ishikawa S (2004) Evaluation of in vivo biocompatibility and biodegradation of photocrosslinked hyaluronate hydrogels (HADgels). J Biomed Mater Res 70A(4):550–559CrossRefGoogle Scholar
  31. Ogino T, Garner C, Markley JL, Herrmann KM (1982) Biosynthesis of aromatic compounds: 13C NMR spectroscopy of whole Escherichia coli cells. Proc Natl Acad Sci U S A 79(19):5828–5832PubMedGoogle Scholar
  32. Orndorff SA, Costantino N, Stewart D, Durham DR (1988) Strain improvement of Rhodotorula graminis for production of a novel l-phenylalanine ammonia-lyase. Appl Environ Microbiol 54(4):996–1002PubMedGoogle Scholar
  33. Ørum H, Rasmussen OF (1992) Expression in E. coli of the gene encoding phenylalanine ammonia-lyase from Rhodosporidium toruloides. Appl Microbiol Biotechnol 36:745–748PubMedGoogle Scholar
  34. Parales RE, Bruce NC, Schmid A, Wackett LP (2002) Minireview: biodegradation, biotransformation, and biocatalysis (B3). Appl Environ Microbiol 4699–4709Google Scholar
  35. Patel N, Pierson DL, Jensen RA (1977) Dual enzymatic routes to l-tyrosine and l-phenylalanine via pretyrosine in Pseudomonas aeruginosa. J Biol Chem 252(16):5839–5846PubMedGoogle Scholar
  36. Pine MJ (1978) Comparative physiological effects of incorporated amino acid analogs in Escherichia coli. Antimicrob Agents Chemother 13(4):676–685PubMedGoogle Scholar
  37. Polen T, Kramer M, Bongaerts J, Wubbolts M, Wendisch VF (2005) The global gene expression response of Escherichia coli to l-phenylalanine. J Biotechnol 115(3):221–237PubMedGoogle Scholar
  38. Rojas A, Duque E, Mosqueda G, Golden G, Hurtado A, Ramos JL, Segura A (2001) Three efflux pumps are required to provide efficient tolerance to toluene in Pseudomonas putida DOT-T1E. J Bacteriol 183(13):3967–3973PubMedGoogle Scholar
  39. Sambrook J, Maniatis T, Fritsch EF (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NYGoogle Scholar
  40. Sarkissian CN, Shao Z, Blain F, Peevers R, Su H, Heft R, Chang TMS, Scriver CR (1999) A different approach to treatment of phenylketonuria: phenylalanine degradation with recombinant phenylalanine ammonia lyase. Proc Natl Acad Sci U S A 96:2339–2344PubMedGoogle Scholar
  41. Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409(6817):258–268PubMedGoogle Scholar
  42. Wery J, de Bont JAM (2004) Solvent-tolerance of Pseudomonads: a new degree of freedom in biocatalysis, Chap 20. In: Ramos JL (ed) The pseudomonads. III. Kluwer Academic/Plenum Publishers, New YorkGoogle Scholar
  43. Wery J, Hidayat B, Kieboom J, de Bont JA (2001) An insertion sequence prepares Pseudomonas putida S12 for severe solvent stress. J Biol Chem 276(8):5700–5706PubMedGoogle Scholar
  44. Whitaker RJ, Fiske MJ, Jensen RA (1982) Pseudomonas aeruginosa possesses two novel regulatory isozymes of 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase. J Biol Chem 257(21):12789–12794PubMedGoogle Scholar
  45. Xiang L, Moore BS (2002) Inactivation, complementation and heterologous expression of encP, a novel bacterial phenylalanine ammonia-lyase gene. J Biol Chem 277(36):32505–32509PubMedGoogle Scholar
  46. Yamada S, Nabe K, Izuo N, Nakamichi K, Chibata I (1981) Production of l-phenylalanine from trans-cinnamic acid with Rhodotorula glutinis containing l-phenylalanine ammonia-lyase activity. Appl Environ Microbiol 42 (5): 773–778PubMedGoogle Scholar
  47. Zhang S, Pohnert G, Kongsaeree P, Wilson DB, Clardy J, Ganem B (1998) Chorismate mutase-prephenate dehydratase from Escherichia coli. Study of catalytic and regulatory domains using genetically engineered proteins. J Biol Chem 273(11):6248–6253PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Karin Nijkamp
    • 1
    • 3
  • Nicole van Luijk
    • 2
  • Jan A. M. de Bont
    • 1
  • Jan Wery
    • 1
  1. 1.TNO Quality of LifeBusiness Unit Bioconversion and Processes for Food IndustryApeldoornThe Netherlands
  2. 2.TNO Quality of LifeBusiness Unit MicrobiologyZeistThe Netherlands
  3. 3.ApeldoornThe Netherlands

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