Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.

中文翻译：

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用于生产四氢生物蝶呤的大肠杆菌基因工程。


四氢生物蝶呤 (BH4) 是哺乳动物中多种酶的必需辅助因子。在体内，它是由 GTP 通过 GTP 环化水解酶 I (GCHI)、6-丙酮酰四氢蝶呤合酶 (PTPS) 和墨蝶呤还原酶 (SPR) 的三步途径合成的。 BH4是一种用于治疗非典型高苯丙氨酸血症的药物。目前主要采用化学方法合成，步骤较多，原料昂贵，工序复杂。为了探索生产 BH4 的替代微生物方法，我们利用重组 DNA 技术构建了携带表达 GCHI、PTPS 和 SPR 酶基因的重组大肠杆菌 (E. coli) 菌株。这些菌株成功产生了BH4，并检测到BH4的氧化产物为二氢生物蝶呤和生物蝶呤。为了提高 BH4 的生产率，我们做了进一步的改进。首先，为了增加从头 GTP 供应，分离了 8-氮鸟嘌呤抗性突变体，并引入了额外的 guaBA 操纵子。其次，为了增强 GCHI 的活性，来自大肠杆菌的 folE 基因被来自枯草芽孢杆菌的 mtrA 基因取代。这些修饰为我们提供了表现出显着更高生产力的菌株，每升培养液中生物蝶呤含量高达 4.0 g。结果表明我们的方法有可能实现 BH4 的商业化生产。