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 Engineering,Nanjing Tech University,Nanjing 211816,China

Keywords:Dopa decarboxylase Dopamine Enzyme catalysis Fed-batch bioconversion

ABSTRACT 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 dihydroxyphenylalanine (dopa) were 0.02 mmol·ml-1·s-1,2.328 mmol·ml-1,10435.90 s-1 and 4482.77 ml·mmol-1·s-1,respectively.The highest DDC activity was observed at the condition of pH 7.5 and 45 °C.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 °C,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.

1.Introduction

Dopamine,a neurotransmitter identified by Arvid Carlsson in the 1950s,is an important therapeutic agent which can transmit pulsed chemicals in brain cells [1–3].In recent years,dopamine has been widely applied as a substrate to produce medicines and materials.In medicine,dopamine was adopted for Parkinson’s and Alzheimer’s disease,and its derivatives,such as norepinephrine and epinephrine,have been applied for emergency rescue of COVID-19 [4,5].In addition,dopamine could form a composite layer on the surface of solid materials under alkaline condition and impart hydrophilicity and biocompatibility to material without changing its functional groups,such as catechol and amino [6].However,the synthesis of dopamine is limited in complex and pollution-prone chemical process [7].As the great concerns on global environment and consumption of petroleum resources,an environment-friendly synthesis craft of dopamine is highly desired.

In living organisms,the dopamine is synthesized from the L-dopa by the catalysis of dopa decarboxylase(DDC;EC 4.1.1.28).DDC,one of the aromatic amino acid decarboxylase,is a pyridoxal-5′-phosphate (PLP) -dependent enzyme and possesses stereospecific α-decarboxylation,which was firstly discovered in mammalian tissue by Holtz et al.in 1938[8,9].To date,DDC has been characterized from human kidney[10],rat[11]and integument of Calliphora vicina Larve [12].However,the preparation of DDC was limited to the extraction from cells,which is quite deficient for industrial biological applications.Thus,the heterologous expression and production of DDC in the model organism,such as Escherichia coli,is highly desired for industrial production of dopamine.

Recently,based on the catalysis of DDC,several stains,such as Citrobacter freundii,Erwinia herbicola[13]and E.coli[14],have been engineered to produce dopamine via the direct fermentation strategy.However,the highest production is only 60 mg·L-1,which is far below the industrial demand [14].Enzymatic method takes the advantages of simple composition of reaction mixture,high conversion rate of substrate,and convenience of recovery and has been widely used for production of various value-added chemicals such as cyclic adenosine monophosphate of 18.45 g·L-1[15],5-aminovaleramide of 240.7 g·L-1[16] and phosphatidylserine of 32.5 g·L-1[17].Therefore,enzymatic production of dopamine from dopa by DDC is worth to investigate for industrialization of dopamine.

In this work,a DDC gene from Harmonia axyridis was cloned and expressed in model organism E.coli BL21(DE3).The conditions of heterologous expression of DDC including temperature of expression,concentration of IPTG and induction time were investigated.After purification,the enzymatic property was characterized and applied into production of dopamine.The reaction temperature,pH,and the concentration of PLP and L-dopa,metal ions and presence of oxygen were optimized to further enhance the titer and yield of dopamine.The fed-batch bio-production of dopamine from L-dopa under the optimal condition was carried out to produce dopamine.

2.Materials and Methods

2.1.Plasmids,strains and growth conditions

The amino acid sequence of DDC (NCBI GenBank:KU820948.1)was obtained from NCBI and coding gene was synthesized by Gen-Script (Nanjing,China).The gene encoding DDC was codonoptimized,synthesized and cloned in pET28a between Eco RI and Hind III by GenScript to form recombinant plasmid pET28a-DDC.For the DDC purification,the termination codon of TAA in DDC was mutated to TAT and a His-tag was ligated.The pET28a-DDC was transformed into E.coli BL21(DE3) to construct the recombinant strain of E.coli BL21(DE3)/pET28a-DDC (BL21-28a-DDC).The strains used in this study were all cultured in Luria-Bertani(LB) medium consisting of 10 g·L-1peptone,5 g·L-1yeast extract and 5 g·L-1sodium chloride with appropriate antibiotics.Solid LB medium was extra added 20 g·L-1agar in liquid LB medium.The concentration of kanamycin was 50 mg·L-1.The recombinant E.coli was cultured at 37 °C and 200 r·min-1.

2.2.Production of DDC

The strain was induced by isopropyl-beta-D-thiogalactopyrano side (IPTG) when OD600reached a predetermined value.Then the strain was cultured for 10 h to produce enzyme of DDC.To determine the optimal condition for DDC production,the related factors including the concentration of IPTG,temperature of expression and biomass of induction were investigated.The temperature of induction was set at 20°C,25°C,30°C and 37°C to investigate the effect of temperature on DDC expression.Meanwhile,the OD600and the concentration of IPTG was 0.4,and 0.5 mmol·L-1,respectively.Different concentrations (0.25,0.5,1,0 and 2.0 mmol·L-1) of IPTG were added into medium with induction temperature of 20 °C and OD600of 0.4.When OD600reached 0.4,0.8,1.0 and 1.4,0.5 mmol·L-1of IPTG was added with the induction temperature of 20°C.After cultivation for 10 h,the cells were harvested by centrifugation (6000 r·min-1,5 min,4 °C),and re-suspended in 0.2 mol·L-1PBS buffer (pH 7.0).The crude extract was obtained by sonicating the cells for 15 min(2 s worktime with a 2 s interval).

2.3.Purification of DDC

DDC was purified by exchange chromatography.All steps of purification were operated at 4 °C to keep enzyme activity.The crude extract was obtained by the abovementioned method.A fast protein liquid chromatography (FPLC) system (AKTA pure 150;GE healthcare Co.,Fairfield,USA) was adopted to purify the crude extract of DDC.The Ni-nitrilotriacetic acid affinity chromatography(Ni–NTA) column (His TrapTMFF5 ml) column equilibrating with 0.1 mol·L-1PBS at pH 7.0.To remove the imidazole and concentrate the enzyme solution,the eluted solution was centrifuged in a 10 kDa ultrafiltration tube (Millipore,USA).The purified DDC were analyzed with polyacrylamide gel electrophoresis (SDSPAGE) consisted of a 3% stacking gel and a 10% separating gel.

2.4.Characterization of DDC

L-dopa was employed as substrate to investigate enzymatic property.The optimal temperature for DDC was determined at 30–55 °C with interval of 5 °C.The purified DDC was incubated in PBS buffer with pH of 7.0 for 3 h at 30–55 °C to determine the thermostability.The optimal pH and pH stability were investigated from pH 6.0 to 8.5 by different kinds of buffer and the incubation time was 3 h at 37 °C.The metal ions including 20 mmol·L-1of Li+,Na+,K+,Mg2+,Ca2+,Ba2+,Co2+,Mn2+,Ni2+,Cu2+,Zn2+,Fe2+and Al3+were selected to investigate the metal ions preference of DDC in this study.One unit of DDC activity (U) was defined as the amount of enzyme required to synthesis 1 μg of dopamine from L-dopa per minute at 40°C.The bioconversion system consisted of 2 g·L-1of L-dopa,0.1 mmol·L-1of PLP and 3 g·L-1of supernatant of sonicating cells (soluble protein),lasted for 10 min at 40 °C with pH of 7.0.According to the method of Lineweaver,the kinetics parameters,comprising of maximum velocity (Vmax),the constant(Km)and catalytic number(Kcat)are estimated by linear regression from double-reciprocal plots [18].

2.5.Development of bioconversion by purified DDC

The bioconversion was operated in a 10 ml tube with 5 ml of purified enzyme at a final concentration of 2.5 g·L-1and a 5 ml mixture with L-dopa,PLP and metal ions solved in buffers of different pH to investigate the optimal bio-catalysis condition.The effects of reaction pH and temperature,metal ions,concentration of PLP and substrate on catalysis were studied.The temperature ranged from 20 to 60 °C with interval of 5 °C.The pH controlled from 4.0 to 10.6 by different kinds of buffer.The concentration of PLP was from 0 to 1.0 mmol·L-1with the interval of 0.1 mmol·L-1.20 mmol·L-1of metal ions including Na+,Fe2+,Ca2+,Cu2+,Co2+,Zn2+,Mg2+,Mn2+,Fe3+,and Al3+was determined to study the effects of metal ions on bioconversion.

Fed-batch bioconversion was used to further improve the production of dopamine.The bioconversion system consisted of initial 5 g·L-1of L-dopa,0.4 mmol·L-1of PLP and 0.5 g·L-1of purified DDC.The fed-batch bioconversion was set in 500 ml flask at 40 °C and pH 7.0 under aerobic and anaerobic condition respectively.Concentration of L-dopa and dopamine was measured at 15,30,45,60,90 and 120 min under aerobic condition and at 10,20,30,40,55,70,90 and 120 min under anaerobic condition.5 g·L-1of L-dopa powder was supplemented into reaction system when it was almost exhausted.The anaerobic bioconversion was operated in anaerobic box.As the dopa was almost not consumed at the last batch both in the aerobic and anaerobic condition,the titer,yield and productivity were calculated at the bioconversion of 90 min.The yield was calculated by dividing the concentration of dopamine by the concentration of dopa added in reaction.

2.6.Analysis methods

The situation of expression was presented by SDS-PAGE analysis and enzyme specific activity.L-dopa and dopamine were treated with trifluoroacetic acid (TFA).A high-performance liquid chromatography(HPLC)system(Agilent 1260,USA)equipped with a TC-C18 column (150 mm × 4.6 mm,5 μm,USA) was used to detect L-dopa and dopamine.The temperature of detecting was at 25°C.The compounds detected at a wavelength of 280 nm with a UV detector.The mobile phase(supplied at 1 ml·min-1)consisted of a solution of 0.1% TFA solution-acetonitrile (96:4).The appearance times of dopamine and dopa were 8.6 minute and 10.1 minute,respectively.

3.Results and Discussion

3.1.Production of DDC from Harmonia axyridis in E.coli

The application of DDC was limited by its low content in most tissues.For high production of DDC,an efficient expression system is required.E.coli host is an outstanding cell factory for production of recombinant proteins for the reasons of its relative simplicity,inexpensive and fast high-density cultivation,the well-known genetic background and the large number of genetic tools[19,20].E.coli could be an ideal host for rapid and economical production of recombinant DDC.Meanwhile,Wang et al.reported the presence of DDC in Harmonia axyridis[21].Here,DDC from Harmonia axyridis was determined to expressed in E.coli.

The results of expression of DDC in E.coli was represented in Fig.1(a).According to the SDS-PAGE analysis,the molecular weight of DDC was about 50 kDa.The activity of DDC was further identified.As shown in Fig.1(b),there was no dopamine detected in the control strain of E.coli BL21(DE3),while 0.72 g·L-1of dopamine was synthesized from 2 g·L-1of L-dopa with the recombinant strain of E.coli (BL21-28a-DDC.The results confirmed that DDC from Harmonia axyridis could functionally expressed in E.coli BL21(DE3).

The expression of DDC in E.coli suffered from the inclusion bodies in our study.In order to improve the amount of soluble protein,the induction conditions were further studied.The expression of exogenous gene was severely affected by induction temperature[19,22],so the effect of induction temperature was firstly investigated.As shown in Fig.2(a),the soluble expression of DDC obviously increased with the decrease of temperature and the insoluble body almost disappeared under the induction temperature of 20 °C.The highest specific enzyme activity was up to 51.72 U·mg-1at induction temperature of 20 °C,which is 301.6%higher than that of 37 °C (Fig.2(b)).The induction OD600and the concentration of IPTG were another important factor that affected protein expression during the cultivation phase [16].In order to enhance soluble expression of DDC,the optimization of induction OD600and the concentration of IPTG was performed.The optimal concentration of IPTG and induction OD600for DDC expression was 0.5 mmol·L-1and around 0.4,respectively,which is 33.37%,21.61% more than those of 2 mmol·L-1and 1.6,respectively(Fig.2(c) and 2(d)).

3.2.Purification and characterization of DDC

To identify the specific properties of the recombinant DDC,the enzyme was purified by exchange chromatography and the results are shown in Table 1.The specific enzyme activity after purification was up to 665 U·mg-1.Additionally,the recovery yield reached 68.79% eluted with 300 mmol·L-1imidazole from a Ni-NTA resin.

The activity of purified DDC in different pH and temperature are shown in Fig.3.The highest enzyme activity was found at pH of 7.5,which was 60.0%,23.35%,3.18%,24.45% and 26.95% higher than that at pH of 6.0,6.5,7.0,8.0 and 8.5,respectively (Fig.3(a)).From Fig.3(b),at pH of 7.5 and 8.0,the activity of DDC exhibited relative stability,where the retained relative activity of DDC was 85.69%and 84.52%of its activity after incubation for 8 h.However,the stability of DDC was poor at pH of 6.0 and 6.5 with a relative activity of 61% and 61.83%,respectively.Meanwhile,higher activity of DDC was observed at the temperature between 35 °C and 50 °C,and showed an optimal activity at temperature of 45 °C,which was 33.57%,9.25%,6.19%,7.39% and 44.52% higher than that of 30 °C,35 °C,40 °C,50 °C and 55 °C,respectively.According to Fig.3d,DDC was stable between 30 and 45°C during the whole 8 h.A significant decrease in DDC activity could be observed at 50 °C after incubation for 6 h and 55 °C after incubation for 2 h,respectively.

As a PLP-dependent enzyme,the activity of DDC was affected by concentration of PLP (Fig.2(e)).The relative activity of DDC was low to 4.67%without the addition of PLP.When the PLP concentration was more than 15 mmol·L-1,the DDC activity was almost not detected.The relative activity of DDC reached maximum when the concentration of PLP was 0.2 mmol·L-1,which outstripped 7.49%,8.31%,60.75% and 86.73% than that in the PLP concentration of 1,2,5 and 10 mmol·L-1.This phenomenon was attributed to the competitive inhibition between excessive PLP and DDC [23].The effects of metal ions on DDC were also investigated.As shown in Fig.3(f) and Table 2,the activity of DDC was inhibited by Ni2+,Cu2+,Zn2+,Fe2+,Al3+.The addition of Co2+and Mn2+did not affect DDC activity.In addition,the kinetic parameters for DDC(Table 3)was researched in this study.According to the[s]-velocity plots of DDC (not shown),the Vmax,Km,Kcatand Kcat/Kmof DDC for L-dopa were 0.02 mmol·ml-1·s-1,2.328 mmol·ml-1,10435.90 s-1and 4482.77 ml·mmol-1·s-1,respectively.

Table 2 Effects of metal ions on the activity of purified DDC.

Table 3 Kinetic parameters of DDC.

Fig.1.The identification of expression and activity of DDC in recombinant E.coli.(a)SDS-PAGE analysis of DDC produced under 20°C(Line M is marker and Line 1 is DDC expressed at 20 °C) (b) The production of dopamine by the crude extracts of E.coli BL21(DE3) and E.col BL21-28a-DDC.

Fig.2.Optimization of cultivation conditions for expression of DDC in recombinant E.coli BL21-28a-DDC.(a)SDS-PAGE analysis of DDC expressed at 20 and 37°C(Line M is marker.Line 1,2,3,and 4 are the sediment and supernatant of DDC producing at 37°C and 20°C,respectively).(b)The effect of temperature on expression of DDC;(c)The effect of IPTG concentration on expression of DDC;(d) The effect of induction time on expression of DDC.

Table 1 Purification of recombinant DDC by Ni-NTA resin.

3.3.Application of DDC to produce dopamine

To develop an efficient bioconversion process for dopamine production from L-dopa,the purified enzyme system was constructed and investigated.The bioconversion conditions,including reaction temperature,pH,the concentration of PLP,and metal ion additives,were optimized.As shown in Fig.4(a),the reaction pH ranged from 4.0 to 10.6.However,the dopamine production was only detected between pH of 6.0 and 9.0.Notably,the highest titer of dopamine(1.55 g·L-1) was found at pH of 7.0,which was 10.83,2.08 and 11.74 times higher compared to that at pH of 6.0,8.0 and 9.0,respectively.Thus,the pH 7.0 of the dopamine production environment is more suitable for the catalytic action of DDC.The reaction temperature was further investigated for the improvement of dopamine production.From Fig.4(b),with the increase of reaction temperature ranging from 20°C to 45°C,the dopamine production was increased and reached the highest titer of 1.55 g·L-1at 45 °C.When the reaction temperature was over than 45 °C,the yield of dopamine was gradually decreased with the increasing temperature.The results were in coincident with the abovementioned conclusion that the catalytic activity of DDC was highest at 45 °C.

According to Fig.4(c),the absence of PLP resulted in the significant decrease in dopamine yield.When the concentration of PLP was ranged from 0.2 mmol·L-1to 2 mmol·L-1,the optimum concentration of PLP was determined at 0.4 mmol·L-1with the dopamine titer of 1.48 g·L-1,while a decrease in dopamine production was observed in other concentrations of PLP.Actually,PLP is an expensive cofactor and would greatly increase cost of industrial production.The dopamine yield under the condition of 0.2 mmol·L-1of PLP was almost equal with that of 0.4 mmol·L-1.Thus,0.2 mmol·L-1of PLP was desired for dopamine production.The effect of metal ions on dopamine production was also investigated.As shown in Fig.4(d),the production of dopamine was inhibited to varying degrees in the presence of most metal ions(Ni2+,Cu2+,Zn2+,Fe2+,Al3+,Co2+and Mn2+).Compared with the characterization of DDC,the conditions for high DDC activity were similar with that for high titer of dopamine.However,the conditions,such as temperature and concentration of PLP,could be controlled at sub-optimal level to reduce the cost in industrial production.

Fig.3.Enzymatic property of DDC.(a) Effect of pH on DDC activity.(b) Effect of pH on stability of purified DDC,(c) Effect of temperature on DDC activity,(d) Effect of temperature on stability of purified DDC.(e) Effect of PLP concentration on DDC activity.(f) Effect of metal ions on DDC activity.

3.4.Fed-batch bioconversion of dopamine from dopa

The effect of L-dopa concentration on the dopamine production was further investigated and shown in Fig.5.It was found that dopamine production was increased with the increasing concentration of dopa.However,the dopamine yield reached the highest level of 85% when the concentration of L-dopa was 4 g·L-1,which suggested a substrate inhibition on DDC.In addition,the upper limit of L-dopa solubility was about 5 g·L-1at room temperature.Therefore,a fed-batch strategy to maintain L-dopa concentration was performed in a 500 ml shake flask.

Fig 4.Optimization of bioconversion conditions for dopamine production from dopa.(a) The effect of reaction pH on dopamine production.(b) The effect of reaction temperature on dopamine production.(c) The effect of PLP concentration on dopamine production.(d) The effect of metal ions on dopamine production.

Due to the fact that dopamine would self-polymerize in the presence of oxygen to form polydopamine [24],the fed-batch bioconversion was operated under the protection of cysteine hydrochloride,a reducing agent used to prevent oxidization of dopamine.As shown in Fig.6(a),after bioconversion of 90 minutesin aerobic condition,the dopamine production has almost reached the highest level,and the further supplementation of dopa was not consumed by the DDC.Therefore,the dopamine production at 90 min was employed to calculate the yield and productivity of dopamine.The dopamine titer,yield and productivity reached up to 15.15 g·L-1,78.01% and 10.1 g·L-1·h-1,respectively.Although cysteine hydrochloride was added into reaction system,the reac-tion system had gradually become black.Das et al.reported that dopamine would be degraded via oxidation within the observable time of experiments [14].To further avoid the oxidative polymerization of dopamine,an anaerobic reaction system was constructed with the entire reaction process completing in an anaerobic operation box.As is shown in Fig.6(b),the titer,yield and productivity of dopamine at 90 min were up to 21.99 g·L-1,80.88% and 14.66 g·L-1·h-1,respectively,which were 45.15%,3.68% and 45.15%higher than those in the aerobic system,respectively.Compared with the aerobic bio-catalysis process,the reaction mixture was stable and did not turn black during the whole bioconversion process.Thus,the presence of oxygen would affect the titer and yield of the reaction.The reason may be the coating formed by polydopamine covered the surface of the enzyme,which hindered the progress of the reaction[25].In order to enhance the titer and yield of dopamine,an anaerobic condition would be better in the conversion of L-dopa to dopamine.

Fig 5.Effect of the dopa concentration on dopamine production.Different concentration of dopa ranging from 1 g·L-1 to 5 g·L-1 was added into the reaction mixture.

Fig 6.Fed-batch bio-production of dopamine from dopa.(a) The bioconversion was conducted under the aerobic condition.(b) The bioconversion was conducted under the anaerobic condition.(The blue arrow represents the addition of 5 g·L-1-dopa powder.)

In fact,the production of dopamine has been reported that the 60 mg·L-1dopamine was obtained by Das et al.[14].To our knowledge,the dopamine production obtained here was the highest level that have been reported to date.Our result exhibited a promising approach for industrial scale production of dopamine from Ldopa by enzymatic bioconversion.Considering the existence of substrate inhibition,the engineering of dopa decarboxylase to decrease inhibition of product or optimization of bio-process to separate dopamine from reaction system would be desired in the following work.

4.Conclusions

In this study,a DDC from Harmonia axyridis was produced,purified and characterized for the bio-catalysis production of dopamine.The optimum activity of DDC was at pH 7.5 and 45 °C and it possessed great pH stability at 7.5 and 8.0 and thermal tolerance when temperature was less than 45°C.The values of Vmax,Km,Kcat,and Kcat/Kmfor L-dopa were 0.02 mmol·ml-1·s-1,2.328 mmol·ml-1,10435.90 s-1and 4482.77 ml·mmol-1·s-1,respectively.The purified DDC was applied into developing a bioconversion from L-dopa to dopamine.To realize the maximum of bio-catalysis,the reaction pH,reaction temperature and the concentration of PLP were optimized to 7.0,45 °C and 0.4 mmol·L-1,with a titer of 1.55 g·L-1and a molar yield of 99.9%of dopamine.Finally,aiming at industrial scale production of dopamine,a fed-batch bioprocess was employed to enhance the titer,yield and productivity of dopamine up to 21.99 g·L-1,80.88%and 14.66 g·L-1·h-1,respectively,which was highest since reported.

CRediT authorship contribution statement

Xin Wang and Siyuan Gao conceived the experiments,supervised the work,and confirmed the manuscript.Siyuan Gao and Yuanke Guo conducted the experiment,analyzed the data and drafted the manuscript.Yuanke Guo and Chen Ma conducted the experiment of pure enzyme and data analysis.Siyuan Gao.and Ding Ma conducted the application of DDC and fed-batch conversion.Kequan Chen analyzed and checked the data.Pingkai Ouyang was responsible for the project administration.All of the authors read and approved the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (21576134 and 21706126) and the National Key Research and Development Program (2016YFA0204300)