Development of high-functional production processes using microbial cyclic-amide metabolism

利用微生物环酰胺代谢开发高功能生产工艺

• 批准号: 13660089
• 负责人: OGAWA Jun
• 金额: $ 2.3万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13660089/
• 关键词: cyclic-amide; cyclic-imide; pyruvate; fumarate; pyrimidine; purine; barbiturate; α-mercapto acid; チアゾリジンジオン; アラントイン; アラントイナーゼ; プリン塩基; ピリミジン塩基; バルビチュラーゼ; TCAサイクル; L-リンゴ酸; ブロモピルビン酸; Pseudomonas putida

Novel processes for the production of useful compounds using microbial cyclic-amide metabolism were developed. A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using the cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 286 mM pyruvate was produced from 500 mM fumarate in 27 h. Bromopyruvate-resistant mutants were derived from P. putida s52, and their pyruvate production was examined. One of the mutants, strain No. 15160 showed much higher pyruvate production than the parent strain. Using the mutant cells cultivated in the medium containing 2% (w/v) fumarate as the catalysts, 770 mM pyruvate was produced from 1000 mM fumrate in 72 h. Brevibacterium linens C-l was found to produce a novel cyclic amidohydrolase which stereoselectively hydrolyzes 5-substituted thiazolidinedione and wasuseful for the production of optically active α-mercapto acids. The gene encoding the enzyme was cloned from the chromosomal DNA of B. lines C-1 and sequenced. It contains an open reading frame consisting of 936 nucleotides corresponding to 312 amino acid residues. The deduced amino acid sequence exhibited no apparent homology with other protein sequences in the protein databases. The enzyme was expressed in recombinant E. coli cells. The E. coli transformant, E. coli BL21/pETBTH, was a good catalyst for stereo selective production of .S-3-phenyl-2-mercaptopropionic (S-PMPA) from RS-5-Benzyl-thiazolidinedione. The optical purity of S-PMPA produced was approximately 80% e.e.

开发了使用微生物环酰胺代谢生产有用化合物的新方法。琥珀酰亚胺同化细菌恶臭假单胞菌S52被发现是从富马酸生产丙酮酸的有效生产者。使用在含有2%（w/v）富马酸的培养基中培养的细胞作为催化剂，在27小时内从500 mM富马酸产生286 mM丙酮酸。从恶臭假单胞菌s52中获得溴代丁二酸抗性突变体，并检测其丙酮酸产量。其中一株突变株15160的丙酮酸产量比亲本菌株高得多。使用在含有2%（w/v）富马酸盐的培养基中培养的突变体细胞作为催化剂，在72小时内从1000 mM富马酸盐产生770 mM丙酮酸盐。亚麻短杆菌（Brevibacterium linens）C-1产生一种新型环状酰胺水解酶，它能立体选择性地水解5-取代噻唑烷二酮，并可用于光学活性α-巯基酸的合成。从B的染色体DNA中克隆了编码该酶的基因。线C-1并测序。它含有一个开放的阅读框架，由936个核苷酸组成，对应于312个氨基酸残基。推导的氨基酸序列与蛋白质数据库中的其他蛋白质序列没有明显的同源性。在重组E. coli细胞。急诊coli、大肠杆菌E. coli BL 21/pETBTH，对RS-5-苄基噻唑烷二酮立体选择性合成S-3-苯基-2-巯基丙酸（S-PMPA）具有良好的催化作用。制备的S-PMPA的光学纯度约为80% e.e.。

项目成果

小川 順: "微生物における環状アミド代謝の多様性とその応用"化学と生物. 39(5). 333-339 (2001)

小川淳：“微生物中环酰胺代谢的多样性及其应用”化学与生物学 39(5) (2001)。

Soong, C. L., J. Ogawa, E. Sakuradani and S. Shimizu.: "Barbiturase, a novel zinc-containing amidohydrolase involved in oxidative pyrimidine metabolism"J. Biol. Chem.. 277(9). 7051-7058 (2002)

Soong, C. L., J. Okawa, E. Sakuradani 和 S. Shimizu.：“巴比妥酶，一种参与氧化嘧啶代谢的新型含锌酰胺水解酶”J.

C.-L.Soong et al.: "Novel amidohydrolytic reactions in oxidative pyrimidine metabolism : Analysis of the barbiturase reaction and discovery of a novel enzyme, ureidomalonase"Biochem. Biophys. Res. Commun.. 286. 222-226 (2001)

C.-L.Soong 等人：“氧化嘧啶代谢中的新型酰胺水解反应：巴比妥酶反应的分析和新型酶脲醛丙二酸酶的发现”Biochem。

Ogawa, J., and S. Shimizu: "Industrial microbial enzymes: their discovery by screening and use in large-scale production of useful chemicals in Japan"Curr. Opinion Biotechnol.. 13. 367-375 (2002)

小川 J. 和 S. Shimizu：“工业微生物酶：通过筛选发现它们并在日本大规模生产有用化学品中使用”Curr。

J.Ogawa, S.Shimizu: "Industrial microbial enzymes : their discovery by screening and use in large-scale production of useful chemicals in Japan"Curr. Opin. Biotechnol.. 13. 365-375 (2002)

J.Okawa，S.Shimizu：“工业微生物酶：通过筛选发现它们并在日本大规模生产有用化学品中使用”Curr。