Key Laboratory of Biomass Chemical Engineering (Education Ministry), College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China; Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Institute of Biological Engineering, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310027, China.
Metab Eng. 2018 Jul;48:279-287. doi: 10.1016/j.ymben.2018.06.007. Epub 2018 Jun 19.
Cellular metabolic networks should be carefully balanced using metabolic engineering to produce the desired products at the industrial scale. As the precursor for the biosynthesis of the neurotransmitter serotonin, 5-hydroxytryptophan (5-HTP) is effective in treating a variety of diseases, such as depression, fibromyalgia, obesity, and cerebellar ataxia. Due to the lack of an efficient synthetic method, commercial production of 5-HTP is only achieved by extracting from the seeds of Griffonia Smplicifolia. This study reports efficient microbial production of 5-HTP via metabolically engineered Escherichia coli. Firstly, human tryptophan hydroxylase I (TPH1) gene was functionally expressed. For endogenous supply of the cofactor tetrahydrobiopterin (BH4), human BH4 biosynthesis and regeneration pathway was reconstituted. Whole-cell bioconversion resulted in high-level production of 5-HTP (~1.2 g/L) from 2 g/L L-tryptophan in shake flasks. Further metabolic engineering efforts were employed to achieve 5-HTP biosynthesis from simple carbon sources. The whole biosynthetic pathway was divided into three functional modules, L-tryptophan module, the hydroxylation module, and the BH4 module. By reducing the copy number of L-tryptophan module, replacing TPH1 with a more stable mutant form, and promoter regulation of the BH4 module, 5-HTP was produced at a final titer of 1.3 g/L in the shake flask and 5.1 g/L in a fed-batch fermenter with glycerol as the carbon source, both of which were the highest ever reported for microbial production of 5-HTP.
细胞代谢网络应该通过代谢工程进行仔细的平衡，以在工业规模上生产所需的产品。5-羟色氨酸（5-HTP）作为神经递质血清素生物合成的前体，在治疗抑郁症、纤维肌痛、肥胖症和小脑共济失调等多种疾病方面非常有效。由于缺乏有效的合成方法，5-HTP 的商业生产只能通过从格里菲尼亚种子中提取来实现。本研究通过代谢工程大肠杆菌报告了 5-HTP 的高效微生物生产。首先，功能性表达了人色氨酸羟化酶 I（TPH1）基因。为了内源供应辅酶四氢生物蝶呤（BH4），重建了人 BH4 生物合成和再生途径。全细胞生物转化导致从 2 g/L L-色氨酸在摇瓶中产生高水平的 5-HTP（~1.2 g/L）。进一步的代谢工程努力用于实现从简单碳源合成 5-HTP。整个生物合成途径分为三个功能模块，L-色氨酸模块、羟化模块和 BH4 模块。通过降低 L-色氨酸模块的拷贝数，用更稳定的突变体形式替代 TPH1，并对 BH4 模块进行启动子调控，5-HTP 在摇瓶中的终浓度达到 1.3 g/L，在以甘油为碳源的分批补料发酵罐中达到 5.1 g/L，这是微生物生产 5-HTP 的最高记录。
