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

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.

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

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.

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

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.

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

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.