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

研究了以甲醇和甘氨酸为原料，采用丝氨酸甲基化途径生产l -丝氨酸的工艺。我们筛选了一种高产菌——专性甲基化菌——methyovorum。抗甘氨酸突变体GM2的丝氨酸产量提高（34 mg/ml）。此外，还研究了其他甲基营养细菌在静息细胞反应中从甲醇和甘氨酸产生l -丝氨酸的能力。在具有l -丝氨酸生产力的菌株中，菌株MN43的生产力最高。在优化条件下，该菌的l -丝氨酸产率为71 mg/ml。加入1 mM CdCl_2可完全抑制该菌对l -丝氨酸的高降解活性，使甘氨酸转化为l -丝氨酸的摩尔转化率提高（约100%）。发现GM2菌株甲醇脱氢酶和丝氨酸羟甲基转移酶（SHMT）活性升高。由于目前对甲基化营养菌丝氨酸途径的系统表征研究甚少，本文不仅对上述两种酶进行了纯化，还对丝氨酸酰氧基氨基转移酶、羟丙酮酸还原酶、甘油激酶等三种酶进行了纯化。经克隆得到SHMT基因，其氨基酸序列与大肠杆菌（55%）和兔肝（44%）具有同源性，而其酶在免疫化学上与菌丝菌菌株和哺乳动物肝脏以外的微生物具有明显的区别。

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.