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Characterization and application of a recombinant Dopa decarboxylase from Harmonia axyridis for the efficient biosynthesis of dopamine

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Chinese Journal of Chemical Engineering 2022年第1期35卷 449-456页

作者： Siyuan Gao Yuanke Guo Chen Ma Ding Ma Kequan Chen Pingkai Ouyang Xin Wang State Key Laboratory of Materials-Oriented Chemical Engineering College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjing 211816China

Here,a Dopa decarboxylase(DDC)from Harmonia axyridis was heterogeneously expressed in Escherichia coli for the efficient biosynthesis of dopamine.For the production of recombinant DDC,the cultivation conditions including IPTG concentration,temperature and induction time were optimized and obtained an optimal specific enzyme activity of 51.72 U·mg^(-1) crude extracts.After the purification of DDC with a recovery yield of 68.79%,its activity was further characterized.The Vmax,Km,Kcat,and Kcat/Km of DDC for d ihyd roxy pheny la la nine(dopa)were 0.02 mmol·ml^(-1)·s^(-1),2.328 mmol·ml^(-1),10435.90 s^(-1) and4482.77 ml,mmol respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45℃.With the purified DDC,the feasibility to produce dopamine from L-dopa was evaluated.The optimal yield was determined at the following bioconversion conditions:pH of 7,0,the reaction temperature of 40℃,0.4 mmol·L^(-1) of PLP and 4 g·L^(-1) of L-dopa,Subsequently,a fed-batch process for the production of dopamine was developed and the effect of oxygen was evaluated.The titer,yield and productivity of dopamine reached up to 21.99 g·L^(-1)80.88%and 14.66 g·L^(-1)·h^(-1) at 90 min under anaerobic condition.

关键词： Dopa decarboxylase dopamine Enzyme catalysis Fed-batch bioconversion

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Expression,purification of human Dopa decarboxylase and the establishment of high-throughput inhibitors screening model

Expression,purification of human DOPA decarboxylase and the ...

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全国第九届海洋生物技术与创新药物学术会议

作者： Qiu junqiang Zhang yuanyuan He peimin Zhou yueyang Zhang qinghua Wu fang Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources Ministry of EducationCollege of Fisheries and Life ScienceShanghai Ocean University Shanghai Center for System Biomedicine Shanghai Jiao tong University

Objective:Parkinson’s disease is one of common nervous system degenerative diseases.Dopa decarboxylase(DDC)has been thought as one of the drug target for Parkinson’s disease,but there is lacking of a high-throughput screening inhibitors model for Dopa decarboxylase.Therefore,our study aims to establish a high-throughput screening inhibitors model.Method:Cloning,expression and purification of DDC and PEPC which is to test the activity of DDC in the coupling reaction.CO content is detected,based on a series of enzyme-linked reactions.Result:The pure enzymes of DDC and PEPC were obtained.We set up a high-throughput screening model for inhibitors of DDC,and 2 inhibitors were found from 70 natural compound product.Conclusion:A suitable high-throughput screening model is set up and two inhibitors of Dopa decarboxylase have been found.

关键词： Dopa decarboxylase Parkinson’s disease Inhibitors High-throughput screening model

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A secreted phospholipase A_(2) (BmsPLA_(2)) regulates melanization of immunity through BmDDC in the silkworm Bombyx mori

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Insect Science 2023年第6期30卷 1579-1594页

作者： Tian Li Gemin Wang Wei He Guiqin Li Chunyang Wang Jiamei Zhao Pan Chen Meiwei Guo Ping Chen College of Sericulture Textile and Biomass SciencesSouthwest UniversityChongqingChina Drug Discovery Research Center Southwest Medical UniversityLuzhouChina State Key Laboratory of Silkworm Genome Biology Southwest UniversityChongqingChina

Insect immune-associated phospholipase A_(2) (PLA_(2)) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA_(2) on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA_(2) gene (BmsPLA_(2)), and its protein had characteristic features of secreted PLA_(2) (sPLA_(2)). After injection of bacteria, BmsPLA_(2) expression and sPLA_(2) activity in hemolymph significantly increased. BmsPLA_(2) fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA_(2) was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA_(2) by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA_(2) overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA_(2) inhibitor dosage. These results demonstrated that BmsPLA_(2) plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA_(2) after injection of bacteria, implying that BmsPLA_(2) was activated prior to BmDDC. Moreover, the alteration of BmsPLA_(2) by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA_(2) regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.

关键词： Dopa decarboxylase immunity melanization phospholipase A_(2) silkworm

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Melanin synthesis genes BgTH and BgDdc affect body color and cuticle permeability in Blattella germanica

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Insect Science 2022年第6期29卷 1552-1568页

作者： Tian-Tian Bai Xiao-Jin Pei Tong-Xian Liu Yong-Liang Fan Shi-Ze Zhang State Key Laboratory of Crop Stress Biology for Arid Areas and Key Laboratory of lIntegrated Pest Management on Crops in Northwestern Loess Plateau Ministry of AgricultureNorthwest A&F UniversityYanglingShaanxiChina Present address:Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology and Institute of Insect Science and Technology School of Life SciencesSouth China Normal UniversityGuangzhou510631China

Melanin is involved in cuticle pigmentation and sclerotization of insects,which is critical for maintaining structural integrity and functional completeness of insect cuti-cle.The 2 key enzymes of tyrosine hydroxylase(TH)and Dopa decarboxylase(DDC)predicted in melanin biosynthesis are usually conserved in insects.However,it is unclear whether their function is related to epidermal permeability.In this study,we identified and cloned the gene sequences of BgTH and BgDdc from Blattella germanica,and revealed that they both showed a high expression at the molting,and BgTH was abundant in the head and integument while BgDdc was expressed highest in the fat body.Using RNA in-terference(RNAi),we found that knockdown of BgTH caused molting obstacles in some cockroaches,with the survivors showing pale color and softer integuments,while knock-down of BgDdc was viable and generated an abnormal light brown body color.Desiccation assay showed that the dsBgTH-injected adults died earlier than control groups under a dry atmosphere,but dsBgDdc-injected cockroaches did not.In contrast,when dsRNA-treated cockroaches were reared under a high humidity condition,almost no cockroaches died in all treatments.Furthermore,with eosin Y staining assay,we found that BgTH-RNAi resulted in a higher cuticular permeability,and BgDdc-RNAi also caused slight dye pen-etration.These results demonstrate that BgTH and BgDdc function in body pigmentation and affect the waterproofing ability of the cuticle,and the reduction of cuticular perme-ability may be achieved through cuticle melanization.

关键词： cuticular permeability desiccation tolerance Dopa decarboxylase German cockroach pigmentation tyrosine hydroxylase

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The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana

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Insect Science 2016年第4期23卷 513-519页

作者： Thomas R. Lemonds Jin Liu Aleksandar Popadic Biological Sciences Department Wayne State University Detroit Michigan USA

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes （TH and DDC）, in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not dopa melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.

关键词： Dopa decarboxylase insect pigmentation melanin pathway Periplanetaamericana tyrosine hydroxylase

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小地老虎黑色素信号通路关键基因研究

小地老虎黑色素信号通路关键基因研究

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作者：杨洋华东师范大学

学位级别：硕士

小地老虎(Agrotis ipsilon)是一种危害性极其严重的农业害虫。针对小地老虎的防治主要是以化学农药为主,但随着农药的频繁使用,小地老虎对化学农药的抗药性呈逐年增强的趋势,农药安全问题日益严重,为了达到稳定控制害虫的目的,亟需找到... 详细信息

小地老虎(Agrotis ipsilon)是一种危害性极其严重的农业害虫。针对小地老虎的防治主要是以化学农药为主,但随着农药的频繁使用,小地老虎对化学农药的抗药性呈逐年增强的趋势,农药安全问题日益严重,为了达到稳定控制害虫的目的,亟需找到对环境友好型并能有效防治小地老虎的新方法。酪氨酸羟化酶(Tyrosine hydroxylase,TH)催化酪氨酸合成多巴(dopa),是昆虫黑色素合成通路中的第一步反应,dopa经多巴脱羧酶(Dopa decarboxylase,DDC)的催化形成多巴胺。多巴胺既是合成黑色素的前体物质,也是一种重要的神经递质,能够调控昆虫表皮黑色素的合成及多种代谢活动。本研究以小地老虎为实验对象,克隆获得小地老虎TH基因全长c DNA序列,并对TH和DDC的蛋白序列进行生物信息学分析;通过q RT-PCR技术对小地老虎不同发育时期及不同的组织中TH和DDC的时空表达水平进行相对定量研究;运用CRISPR/Cas9基因组编辑技术敲除Ai TH和Ai DDC,并检测是否在靶向基因的靶点位置发生了基因突变,筛选突变体并观察与野生型之间的表型差异,分析Ai TH和Ai DDC对小地老虎生长发育的调控作用,研究结果如下:1.构建TH基因和DDC基因的系统进化树发现,Ai TH基因与粘虫(Mythimna separate)和甜菜夜蛾(Spodoptera exigua)的亲缘关系更近,Ai DDC基因与粘虫(Mythimna separate)和甘蓝夜蛾(Mamestra brassicae)的亲缘关系更近些,都可与其他鳞翅目昆虫汇成一支。2.表达模式分析显示:(1)组织表达:对小地老虎各组织中两个基因的相对表达量进行检测后发现,TH和DDC基因在头部和表皮中表达水平较高,这与黑色素产生并发挥功能的场所相一致。(2)龄期表达:TH基因在生长发育的各个阶段都表达,DDC基因在幼虫蜕皮前后发生显著变化,与蜕皮激素和保幼激素的调控相关。3.将Cas9 m RNA及特定基因的sg RNA混合物注入小地老虎胚胎,基因组检测突变发现,Ai TH突变体的TH基因和Ai DDC突变体的DDC基因在靶点处碱基发生了不同类型的变异。4.与野生型相比,Ai TH突变体具体表型为:小地老虎在胚胎时期体型明显变小,发育受阻,不能正常孵化,部分胚胎在孵化成幼虫的第一天严重脱水死亡。敲除小地老虎中DDC基因后,突变个体中黑色素的合成作用明显减弱,低龄幼虫体表毛发毛囊处及头部几乎没有黑色素沉积现象,后期生存免疫力低下,在幼虫期死亡。部分出现嵌合体性状,头部有一半有黑色素的沉积。嵌合体突变体可以存活到成虫期,突变体成虫的翅膀、触角及足颜色偏浅黄,黑色素在身体的分布较少。5.通过实时定量PCR(q RT-PCR)检测分析了CRISPR/Cas9介导的Ai TH突变体中黑色素信号通路TH基因及其他关键基因在m RNA水平上的表达差异,分析显示TH基因在突变体中的表达发生显著性下调,PO基因发生显著性上调,aa NAT、tan、ebony基因发生显著性下调。这表明,Ai TH在小地老虎的生长和发育中起着重要作用,TH基因的突变影响黑色素通路对机体的调控。本研究主要利用基因组编辑CRISPR/Cas9技术对小地老虎的黑色素通路的基因进行了研究和功能验证,表明TH与DDC基因在小地老虎黑色素合成及生长发育中具有十分重要的作用。DDC基因可以作为研究小地老虎遗传转化的Marker基因。利用TH基因的致死性,为昆虫种群遗传调控防治害虫提供了潜在的靶标基因,为有效防控小地老虎种群暴发与危害提供了新思路。

关键词：小地老虎 CRISPR/Cas9 Tyrosine hydroxylase Dopa decarboxylase 遗传调控

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