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Gluconate Dehydratase

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Gluconate Dehydratase Europäisches Patentamt *EP001496113A1* (19) European Patent Office Office européen des brevets (11) EP 1 496 113 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.7: C12N 9/88, C12N 15/55, 12.01.2005 Bulletin 2005/02 C12N 15/63, C12P 7/58 (21) Application number: 04254114.4 (22) Date of filing: 08.07.2004 (84) Designated Contracting States: • Ishibashi, Hiroki, c/o Mitsui Chemicals Inc. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Omuta-shi, Fukuoka 836-8610 (JP) HU IE IT LI LU MC NL PL PT RO SE SI SK TR • Fukuiri, Yasushi, c/o Mitsui Chemicals Inc. Designated Extension States: Omuta-shi, Fukuoka 836-8610 (JP) AL HR LT LV MK • Sakuma, Atsushi, c/o Mitsui Chemicals Inc. Omuta-shi, Fukuoka 836-8610 (JP) (30) Priority: 10.07.2003 JP 2003194680 • Komatsu, Hironori, c/o Mitsui Chemicals Inc. Sodegaura-shi, Chiba 299-0265 (JP) (71) Applicant: MITSUI CHEMICALS, INC. • Ando, Tomoyuki, c/o Mitsui Chemicals Inc. Tokyo (JP) Sodegaura-shi, Chiba 299-0265 (JP) • Togashi, Kazuhiko, c/o Mitsui Chemicals Inc. (72) Inventors: Sodegaura-shi, Chiba 299-0265 (JP) • Miyake, Hitoki, c/o Mitsui Chemicals Inc. • Umetani, Hideki, c/o Mitsui Chemicals Inc. Mobara-shi, Chiba, 297-0017 (JP) Sodegaura-shi, Chiba 299-0265 (JP) • Yamaki, Toshifumi, c/o Mitsui Chemicals Inc. Mobara-shi, Chiba, 297-0017 (JP) (74) Representative: Paget, Hugh Charles Edward et al • Oikawa, Toshihiro, c/o Mitsui Chemicals Inc. Mewburn Ellis LLP Mobara-shi, Chiba, 297-0017 (JP) York House • Nakamura, Takeshi, New Energy and Industrial 23 Kingsway Kawasaki-shi, Kanagawa 212-8554 (JP) London WC2B 6HP (GB) (54) Gluconate dehydratase (57) A novel gluconate dehydratase derived from gluconate dehydratase or a transformed cell containing Achromobacter xylosoxidans and a gene encoding the the gene with an aldonic acid, the corresponding 2-keto- gluconate dehydratase are provided. By reacting the 3-deoxyaldonic acid can be efficiently produced. EP 1 496 113 A1 Printed by Jouve, 75001 PARIS (FR) EP 1 496 113 A1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates to a novel gluconate dehydratase capable of efficiently producing a 2-keto- 3-deoxyaldonic acid from aldonic acid; a base sequence encoding the gluconate dehydratase; a plasmid comprising the base sequence; and a cell transformed by the plasmid. The present invention also relates to a process using the gluconate dehydratase or the transformed cell for producing a 2-keto-3-deoxyaldonic acid and a 2-deoxyaldonic acid or 2-deoxyaldose whose carbon number is reduced by 1. 10 BACKGROUND [0002] 2-keto-3-deoxyaldonic acids are useful as pharmaceutical material. For example, 2-keto-3-deoxyaldonic acids are decarboxylated to 2-deoxyaldonic acids or 2-deoxyaldoses whose carbon number is reduced by 1. These sub- 15 stances are thought of for raw materials of antibiotics, antiviral agents, antisense drugs, and other drugs and medicines. [0003] On the other hand, enzymes have been known which catalyze a reaction for producing 2-keto-3-deoxyaldonic acids by dehydration of aldonic acids. For example, a gluconate dehydratase (EC4. 2. 1. 39) for dehydrating D-gluconic acid to synthesize 2-keto-3-deoxy-D-gluconic acid is derived from Clostridium pasteurianum (Analytical Biochemistry 61, 275 (1974)), Alcaligenes sp. strain M250 (Methods in Enzymology 41, 99 (1975)), or Sulfolobus solfataricus (Bio- 20 technol. Lett. 8,497 (1986)). Unfortunately, it is reported that the gluconate dehydratase derived from Clostridium pas- teurianum is liable to oxidize with air and is thus rapidly deactivated in the presence of air. The gluconate dehydratase derived from Alcaligenes sp. strain M250 is not thermally stable. A report has taught that the dehydration activity of the gluconate dehydratase is 50% degraded in storage at 0°C for 7 days in a tris(hydroxymethyl)aminomethane (here- inafter referred to as Tris) buffer solution (pH 8.0 to 8.8) containing 1 mM of sodium D-gluconate and 1 mM of magnesium 25 chloride. Hence, these microorganisms or enzymes derived from these microorganisms are not suitable for industrial production because of the difficulty in handling, such as degradation of the reactivity in a process. The gluconate dehydratase derived from Sulfolobus solfataricus has not yet been purified, and accordingly, its scientific characteristics have not been known. For synthesis of 2-keto-3-deoxy-D-gluconic acid with use of the Sulfolobus solfataricus as it is, this microorganism has activity of decomposing the product 2-keto-3-deoxy-D-gluconic acid, thus causing the yield to 30 decrease. [0004] As described above, no gluconate dehydratase that is industrially applicable in practice has been discovered, and no industrial process for efficiently producing a 2-keto-3-deoxyaldonic acid has been established. SUMMARY OF THE INVENTION 35 [0005] Accordingly, an object of the present invention is to provide a novel gluconate dehydratase having a ther- mostability and storage stability so as to be industrially useful in practice and capable of efficiently producing a 2-keto- 3-deoxyaldonic acid, which is useful for pharmaceutical material, from the corresponding aldonic acid, and to a process for producing a 2-keto-3-deoxyaldonic acid using the gluconate dehydratase. 40 [0006] Another object of the present invention is to provide a base sequence encoding the gluconate dehydratase, a plasmid comprising the base sequence, and a cell transformed with the plasmid. The base sequence is advanta- geously used in the production of the gluconate dehydratase and of 2-keto-3-deoxyaldonic acids using the gluconate dehydratase. [0007] The inventors of the present invention have conducted intensive research to find that Achromobacter xylos- 45 oxidans strain ATCC 9220 exhibits a high activity of dehydrating gluconic acid. [0008] A paper on the Alcaligenes sp. strain M250 said that the strain M250 is the same type as the ATCC 9220 strain, and the gluconate dehydratase derived from Alcaligenes sp. strain M250 is reported to be unstable, as described above. However, the inventors found that D-gluconic acid acted upon by Achromobacter xylosoxidans strain ATCC 9220 maintains its activity stably even at a reaction temperature of 50°C and efficiently produces 2-keto-3-deoxy-D- 50 gluconic acid. [0009] Then, the inventors isolated a gluconate dehydratase from bacterial cells of this strain by various purification techniques, and allowed the gluconate dehydratase to stand at 55°C for 2 hours. As a result, it was found that the gluconate dehydratase is so heat-resistant as to maintain at least 95% of the activity. [0010] In spite of the report that the dehydration activity of the gluconate dehydratase derived from Alcaligenes sp. 55 strain M250 is 50% degraded in storage at 0°C for 7 days in a Tris buffer (pH 8.0 to 8.8) containing 1 mM of sodium D-gluconate and 1 mM of magnesium chloride, the inventors found that the gluconate dehydratase of the present invention is so stable as to maintain the activity stably for one month under the same conditions. [0011] In addition, while it has been reported that the molecular weight of the gluconate dehydratase purified from 2 EP 1 496 113 A1 Alcaligenes sp. strain M250 determined by gel permeation chromatography is 270,000 ± 2,500, the molecular weight of the gluconate dehydratase of the present invention is different and is 188,000 ± 2,500. [0012] Moreover, the inventors successfully determined the amino acid sequence of the gluconate dehydratase, as shown in sequence ID No: 1 of the sequence listing. 5 [0013] Furthermore, the inventors successfully produced a 2-keto-3-deoxyaldonic acid efficiently through preparing DNA containing the base sequence shown in SEQ ID No: 1 and a cell transformed with a plasmid containing a DNA fragment having the base sequence, preparing the gluconate dehydratase so as to be active, and reacting the corre- sponding aldonic acid with the transformed cell or processed product from the transformed cell. Thus, the inventors have accomplished the present invention. 10 [0014] According to an aspect of the present invention, a gluconate dehydratase capable of dehydrating D-gluconic acid to produce 2-keto-3-deoxy-D-gluconic acid is provided. The gluconate dehydratase maintains at least 95% of its enzyme activity after being allowed to stand in 30 mM tris(hydroxymethyl)aminomethane buffer solution with a pH of about 8.5 containing 1 mM of sodium D-gluconate and 1 mM of magnesium chloride at 55°C for 2 hours. [0015] Preferably, the gluconate dehydratase is derived from Achromobacter xylosoxidans. 15 [0016] The gluconate dehydratase may comprise (e.g., consist of) the amino acid sequence shown in SEQ No: 1. [0017] The gluconate dehydratase may comprise (e.g., consist of) an amino acid sequence having a homology of at least 70% with the amino acid sequence shown in SEQ ID No: 1. [0018] According to another aspect of the present invention, a polynucleotide (e.g., a DNA) or a gene encoding the gluconate dehydratase is provided. 20 [0019] The polynucleotide (e.g., DNA) or gene may comprise (e.g., consist of) the base sequence shown in SEQ ID No: 1. [0020] According to another aspect of the present invention, a polynucleotide (e.g., a DNA) or gene encoding the gluconate dehydratase is provided which comprises (e.g., consists of) a base sequence capable of hybridizing with the foregoing polynucleotide (e.g., DNA) or gene under stringent conditions. 25 [0021] According to another aspect of the present invention, a plasmid comprising (e.g., containing) any one of the polynucleotides (e.g., DNAs) or genes above is provided. [0022] The present invention is also directed to a transformed cell prepared by transforming a host cell with the plasmid.
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  • S41598-018-34741-9.Pdf
    www.nature.com/scientificreports OPEN The development of a new parameter for tracking post- transcriptional regulation Received: 26 June 2018 Accepted: 25 October 2018 allows the detailed map of the Published: xx xx xxxx Pseudomonas aeruginosa Crc regulon Fernando Corona1, Jose Antonio Reales-Calderón 2,3, Concha Gil2 & José Luis Martínez 1 Bacterial physiology is regulated at diferent levels, from mRNA synthesis to translational regulation and protein modifcation. Herein, we propose a parameter, dubbed post-transcriptional variation (PTV), that allows extracting information on post-transcriptional regulation from the combined analysis of transcriptomic and proteomic data. We have applied this parameter for getting a deeper insight in the regulon of the Pseudomonas aeruginosa post-transcriptional regulator Crc. P. aeruginosa is a free-living microorganism, and part of its ecological success relies on its capability of using a large number of carbon sources. The hierarchical assimilation of these sources when present in combination is regulated by Crc that, together with Hfq (the RNA-binding chaperon in the complex), impedes their translation when catabolite repression is triggered. Most studies on Crc regulation are based either in transcriptomics or in proteomics data, which cannot provide information on post-transcriptional regulation when analysed independently. Using the PTV parameter, we present a comprehensive map of the Crc post-transcriptional regulon. In addition of controlling the use of primary and secondary carbon sources, Crc regulates as well cell respiration, c-di-GMP mediated signalling, and iron utilization. Thus, besides controlling the hyerarchical assimilation of carbon sources, Crc is an important element for keeping bacterial homeostasis and, consequently, metabolic robustness.
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