Genetics of Prokaryotes, Faculty of Biology & CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
Organic Chemistry III, Faculty of Chemistry & CeBiTec, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
J Biotechnol. 2019 Feb 10;291:7-16. doi: 10.1016/j.jbiotec.2018.12.008. Epub 2018 Dec 20.
Halogenated compounds, like 7-chloro-l-tryptophan, are important intermediates or components of bioactive substances relevant for the pharmaceutical, chemical and agrochemical industries. About 20% of all pharmaceutical small molecule drugs and around 30% of all active compounds in agrochemistry are halogenated. Chemical halogenation procedures usually are characterized by the use of hazardous or even highly toxic chemicals. Recently, a biocatalytic process for l-tryptophan halogenation at the gram-scale using FAD-dependent halogenase and NADH-dependent flavin reductase enzymes has been described. Many proteinogenic amino acids are produced by fermentation using Corynebacterium glutamicum. The fermentative production of l-glutamate and l-lysine, for example, is operated at the million-ton scale. However, fermentative production of halogenated amino acids has not yet been described. In this study, fermentative production of the halogenated amino acid 7-chloro-l-tryptophan from sugars, ammonium and chloride salts was achieved. This required metabolic engineering of an l-tryptophan producing C. glutamicum strain for expression of the genes coding for FAD-dependent halogenase RebH and NADH-dependent flavin reductase RebF from Lechevalieria aerocolonigenes. Chlorination of l-tryptophan to 7-chloro-l-tryptophan by recombinant C. glutamicum was improved by optimizing the RBS of rebH. Metabolic engineering enabled production of 7-chloro-l-tryptophan and l-tryptophan from the alternative carbon sources arabinose, glucosamine and xylose.
卤代化合物，如 7-氯-l-色氨酸，是医药、化工和农化行业中生物活性物质的重要中间体或组成部分。大约 20%的医药小分子药物和约 30%的农化活性化合物都是卤代的。化学卤化过程通常使用危险甚至剧毒化学品。最近，已经描述了一种使用依赖 FAD 的卤化酶和依赖 NADH 的黄素还原酶的克规模 l-色氨酸卤化的生物催化过程。许多蛋白质氨基酸是通过使用谷氨酸棒杆菌进行发酵生产的。例如，l-谷氨酸和 l-赖氨酸的发酵生产已经达到百万吨级。然而，卤代氨基酸的发酵生产尚未得到描述。在这项研究中，从糖、铵盐和氯化物盐中通过发酵生产了卤代氨基酸 7-氯-l-色氨酸。这需要对生产 l-色氨酸的谷氨酸棒杆菌菌株进行代谢工程改造，以表达来自 Lechevalieria aerocolonigenes 的依赖 FAD 的卤化酶 RebH 和依赖 NADH 的黄素还原酶 RebF 的基因。通过优化 RebH 的 RBS，提高了重组谷氨酸棒杆菌对 l-色氨酸的氯化作用，生成 7-氯-l-色氨酸。代谢工程使 7-氯-l-色氨酸和 l-色氨酸能够从替代碳源阿拉伯糖、葡糖胺和木糖中生产。
