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Enzymzyic znd Molecular Genetic Studies of Serine Production by C_1-Microorganisms

C_1-微生物产生丝氨酸的酶znd分子遗传学研究

基本信息

批准号：
04660119
负责人：
IZUMI Yoshikazu
金额：
$ 1.22万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

The production process of L-serine from methanol and glycine has been developed using a methylotroph with serine pathway. We screened a high producer, Hypgomicrobium methyovorum, which is an obligate methylotroph. A glycine-resistant mutant GM2 showed improved serine production (34 mg/ml). Furthermore other methylotrophic bacteria were examined for their ability to produce L-serine from methanol and glycine in a resting cell reacton. Among strains exhibiting L-serine productivity, the strain MN43 WAS found to exhibit the highest productiveity. Under optimized conditions usng this bacterium 71 mg/ml L-serine was produced. The high L-serine degrading activity of this bacterium was entirely suppressed by adding 1 mM CdCl_2, resulting in an enhanced converison ratio of glycine to L-serine (ca. 100% molar conversion). The GM2 strain was found to have elevated activities of methanol dehvdrogenase and serine hydroxymethyltransferase (SHMT). Since thers has so for been little information on the systematic characterization of enzymes of serine pathway in methylotroph, not only the adove two enzymes but also other three enzymes, serineglyoxylate aminotransferase, hydroxypyruvate reductase, glycerate kinase, were purified to hmogeneity. The gene of SHMT was cloned and its deduced amino acid sequence had homology to those of Escherichia coli (55%) and rabbit liver (44%), whereas the enzyme of the bacterinum was immunochemically distinguishadble from those of microorganisms other than Hyphomicrobium strains and mammalian livers.

利用甲基营养菌的丝氨酸代谢途径，研究了由甲醇和甘氨酸生产L-丝氨酸的工艺。我们筛选了一个高生产商，Hypgomicrobium methyovorum，这是一个专性甲基营养菌。甘氨酸抗性突变体GM 2显示出提高的丝氨酸产量（34 mg/ml）。此外，还检测了其他甲基营养菌在静息细胞反应中从甲醇和甘氨酸生产L-丝氨酸的能力。在表现出L-丝氨酸生产力的菌株中，发现菌株MN 43表现出最高的生产力。在优化的条件下，该细菌可生产71 mg/ml L-丝氨酸。该菌高的L-丝氨酸降解活性被1 mM CdCl_2完全抑制，导致甘氨酸转化为L-丝氨酸的转化率提高（约10%）。100%摩尔转化率）。发现GM 2菌株的甲醇脱氢酶和丝氨酸羟甲基转移酶（SHMT）活性升高。由于目前对甲基营养菌丝氨酸途径酶的系统研究还不多，因此，我们不仅对上述两种酶进行了分离纯化，而且对丝氨酸乙醛酸转氨酶、羟基丙酮酸还原酶、甘油酸激酶等三种酶进行了分离纯化。克隆了SHMT基因，其推导的氨基酸序列与大肠杆菌（55%）和兔肝（44%）的同源性较高，而丝微菌属（Hyphomicrobium）以外的微生物和哺乳动物肝的SHMT酶与其他微生物和哺乳动物肝的SHMT酶的同源性较低。