异源表达多巴脱羧酶促进大肠杆菌从头合成多巴胺

Heterogeneous expression of DOPA decarboxylase to improve the production of dopamine in Escherichia coli

摘要:

多巴胺是多种天然抗氧化药物生物合成的前体物质，在人体内作为神经递质调控中枢神经系统的多种生理功能，常用于多种类型休克的临床治疗。目前，通过微生物合成技术已经实现了多巴胺的从头合成，但是合成效率很低。针对该问题，在左旋多巴 (l-DOPA) 大肠杆菌工程菌基础上，利用不同拷贝数质粒表达野猪Sus scrofa来源的多巴脱羧酶基因Ssddc，实现了葡萄糖到多巴胺的生产。为了进一步提高多巴胺合成效率，从100个候选基因中筛选出5个多巴脱羧酶基因进行测试，其中来源于人Homo sapiens多巴脱羧酶基因Hsddc的工程菌摇瓶发酵的多巴胺产量最高，达到3.33 g/L；而来源于果蝇Drosophila melanogaster多巴脱羧酶基因Dmddc的工程菌摇瓶发酵的左旋多巴残余量最低，仅有0.02 g/L；这两株工程菌分批补料发酵表明，多巴胺的产量可以分别达到13.3 g/L和16.2 g/L，左旋多巴残余量分别是0.45 g/L和0.23 g/L。将多巴脱羧酶基因Dmddc和Ssddc分别整合到基因组上，获得遗传稳定的工程菌，在分批补料发酵条件下，多巴胺产量最高达到17.7 g/L，是目前国内外报道的最高产量。

关键词:大肠杆菌，代谢工程，l-左旋多巴，多巴胺，多巴脱羧酶

Abstract:

Dopamine is the precursor of a variety of natural antioxidant compounds. In the body, dopamine acts as a neurotransmitter that regulates a variety of physiological functions of the central nervous system. Thus, dopamine is used for the clinical treatment of various types of shock. Dopamine could be produced by engineered microbes, but with low efficiency. In this study, DOPA decarboxylase gene from Sus scrofa (Ssddc) was cloned into plasmids with different copy numbers, and transformed into a previously developed l-DOPA producing strain Escherichia coli T004. The resulted strain was capable of producing dopamine from glucose directly. To further improve the production of dopamine, a sequence-based homology alignment mining (SHAM) strategy was applied to screen more efficient DOPA decarboxylases, and five DOPA decarboxylase genes were selected from 100 candidates. In shake-flask fermentation, the DOPA decarboxylase gene from Homo sapiens (Hsddc) showed the highest dopamine production (3.33 g/L), while the DOPA decarboxylase gene from Drosophila Melanogaster (Dmddc) showed the least residual l-DOPA concentration (0.02 g/L). In 5 L fed-batch fermentations, production of dopamine by the two engineered strains reached 13.3 g/L and 16.2 g/L, respectively. The residual concentrations of l-DOPA were 0.45 g/L and 0.23 g/L, respectively. Finally, the Ssddc and Dmddc genes were integrated into the genome of E. coli T004 to obtain genetically stable dopamine-producing strains. In 5 L fed-batch fermentation, 17.7 g/L of dopamine was produced, which records the highest titer reported to date.

Key words:Escherichia coli, metabolic engineering, l-DOPA, dopamine, DOPA decarboxylase