Song Fuqiang, Chen Wujiu, Wu Fengli, Wang Xiaoshuang, Lu Fuping, Wang Qinhong
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin University of Science & Technology, Tianjin 300457, China.
CAS Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Science, Tianjin 300308, China.
Sheng Wu Gong Cheng Xue Bao. 2021 Dec 25;37(12):4266-4276. doi: 10.13345/j.cjb.210076.
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.
多巴胺是多种天然抗氧化化合物的前体。在体内，多巴胺作为一种神经递质，调节中枢神经系统的多种生理功能。因此，多巴胺被用于各种类型休克的临床治疗。多巴胺可以由工程微生物生产，但效率较低。在本研究中，将猪的多巴脱羧酶基因（Ssddc）克隆到不同拷贝数的质粒中，并转化到先前构建的能够生产L-多巴的大肠杆菌T004菌株中。所得菌株能够直接从葡萄糖生产多巴胺。为了进一步提高多巴胺的产量，应用基于序列的同源比对挖掘（SHAM）策略筛选更高效的多巴脱羧酶，从100个候选基因中选择了5个多巴脱羧酶基因。在摇瓶发酵中，人类的多巴脱羧酶基因（Hsddc）显示出最高的多巴胺产量（3.33 g/L），而果蝇的多巴脱羧酶基因（Dmddc）显示出最低的L-多巴残留浓度（0.02 g/L）。在5 L补料分批发酵中，两种工程菌株生产的多巴胺分别达到13.3 g/L和16.2 g/L。L-多巴的残留浓度分别为0.45 g/L和0.23 g/L。最后，将Ssddc和Dmddc基因整合到大肠杆菌T004的基因组中，获得遗传稳定的多巴胺生产菌株。在5 L补料分批发酵中，生产出17.7 g/L的多巴胺，这是迄今为止报道的最高产量。
