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Genetic analysis of bacterial formaldehyde-fixing enzyme system and tis application of useful compound production

细菌甲醛固定酶系统的遗传分析及其在有用化合物生产中的应用

基本信息

批准号：
11460041
负责人：
KATO Nobuo
金额：
$ 9.02万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

项目摘要

1) Genetic analysis of bacterial formaldehyde-fixing enzyme system : The genes for formaldehydefixing enzyme system in the ribulose monophosphate pathway, involving 3-hexulose-6-phosphate synthase (EPS) and 6-phospho-3-hexuloisomerase (PHI), were cloned from the facultative methylotroph, Mycobacterium gastri MB 19, and the obligate methylotroph, Methylomonas aminofaciens 77a. The two strain were different in the gene organization and regulation mechanism for the gene expression. The homologous proteins to HPS and PHI are found in a variety of strains of Archaea and Bacteria.2) Purification of HPS and PHI from a methylotrophic and thermophilic bacterium, Bacillus brevis, and their gene analyses : HPS and PHI were purified from B. brevis and characterized A DNA fragment containing hpi and phi was cloned. From the genetic analysis for the fragment, gene cluster is an operon for hps/phi, whose expression occurred only the presence of methanol or formaldehyde.3) Regulation in the expression of formaldehyde-fixing (hps/phi) operon in Bacillus subtilist : The gene product of yckH, which locates upstream of hps/phi operon, was purified and characterized. Several nucleotide sequence on which yckH bound specifically were found. From these and another results, it is concluded that YckH is essential for the transcription of hps/phi operon.4) Expression and purification of HPS and PHI from a hyper thermophilic Archaean, Pyrococcus horikoshii : DNA fragments corresponding hps and phi were constructed from a DNA fragment (PH1938) of P. horikoshii. It has been confirmed that the two DNA fragments and PH1938 are enough to expression of the active enzymes for formaldehyde fixation. This is the first evidence that genes for formaldehyde fixation are active in an Archaeon.

1)细菌甲醛固定酶系统的遗传分析：从兼性甲基营养菌胃分枝杆菌MB 19和专性甲基营养菌产氨甲基单胞菌77 a中克隆了核酮糖单磷酸途径中甲醛固定酶系统的基因，包括3-己酮糖-6-磷酸合成酶（EPS）和6-磷酸-3-己酮糖异构酶（PHI）。这两个菌株在基因组织和基因表达调控机制上存在差异。2）短芽孢杆菌（Bacillusbrevis）中HPS和PHI的分离纯化及其基因分析：从B中分离纯化HPS和PHI。克隆了含有hpi和phi的DNA片段。3）枯草芽孢杆菌（Bacillus subtilist）中甲醛固定（formalde-fixing，hps/phi）操纵子表达的调控：纯化并鉴定了位于hps/phi操纵子上游的yckH基因产物。4）超嗜热细菌Pyrococcus horikoshii中HPS和PHI的表达和纯化：从Pyrococcus horikoshii的DNA片段（PH 1938）中构建了对应于HPS和PHI的DNA片段。证实这两个DNA片段和PH 1938足以表达甲醛固定活性酶。这是第一个证据表明，甲醛固定的基因是活跃的一个细胞。