Lyase Activities of Heterologous CpcS and CpcT for Phycocyanin Holo-β-subunit from Arthrospiraplatensis in Escherichia coli

YI Junjie,XU Di,,ZANG Xiaonan,YUAN Dingyang,ZHAO Bingran,TANG Li,TAN Yanning,and ZHANG Xuecheng

1)Key Laboratory of Marine Genetics and Breeding(Ocean University of China), Ministry of Education,Qingdao 266003, P.R.China

2) State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha 410125,P.R.China

? Ocean University of China,Science Press and Spring-Verlag Berlin Heidelberg 2014

1 Introduction

Phycobiliproteins(PBPs)are light-harvesting antenna proteins found in cyanobacteria,red algae and cryptomonads,which are assemblied into light-harvesting complexes called phycobilisomes(PBS)(Fairchildet al.,1992).Cyanobacterial PBS is normally composed of two PBPs,phycocyanin(PC)and allophycocyanin(APC)while phycoerythrin(PE)appeared occasionally(Glazer,1985).Instead of PE,heterocyst forming cyanobacteria contain phycoerythrocyanin(PEC)which serves the function of PE.The PBPs are usually polymers of a heterodimer composed of α and β subunit,each carries several bilins which link to cysteinyl residues with thioether bonds(Tooleyet al.,2001).In PC,three phycocyanobilin(PCB)attach to the heterodimer at α-84,β-84 and β-155(or consensus numbering).Such a structure greatly widens the light absorption spectrum and excitation lifetime,rendering it an excellent photoreceptor(Scheer and Zhao,2008).

The last step of PBP biosynthesis in cyanobacteria is the covalent attachment of chromophore to the apo-protein,in which three separate mechanisms,spontaneous attachment,autocatalytic attachment and PBP lyase catalysis may involve(Scheer and Zhao,2008).The earliest intensive study was on the highly specific α-PC PCB lyase,a heterodimer of CpcE and CpcF inSynechococcussp.PCC 7002(Fairchildet al.,1992; Zhouet al.,1992).Shortly,catalyzing mechanism of enzyme CpcE/CpcF to the formation of the α-subunit of PBPs became clear(Junget al.,1995; Zhaoet al.,2000; Storfet al.,2001; Zhaoet al.,2002; Li and Sherman,2002).In contrast,the identification and characterization of lyases functioning in the formation of the β-subunit of PBPs achieved less because the two bilin attachment sites have different stereochemical properties(Shenet al.,2006).Initially,Cobleyet al.(2002)deduced thatcpeTin cpeCDESTR operon encodes the lyase catalyzing the assembly of PE β-subunits inFremyella diplosiphon.Lately,four genes,cpcS,cpcT,cpcUandcpcV,in another operon were found functioning in such an assembly inSynechococcussp.PCC7002 and some PE-free cyanobacteria(Shenet al.,2004).ThecpeTparalog inSynechococcussp.PCC 7002,cpcT,was found to encode CpcT,a lyase responsible for attaching PCB to cysteine-153 of CpcB(Shenet al.,2006).In addition,CpcS1 and CpcT1 were evidenced to be the lyases attaching PCB to cysteine-84 and cysteine-155 of CpcB respectively inNostocsp.PCC 7120(Zhaoet al.,2006,2007b); whereas CpcS was found to function similarly but must in association with CpcU to form a heterodimeric bilin lyase(Shenet al.,2008; Saunéeet al.,2008).

PCB is synthesized from heme by ring opening at C-5 of the tetrapyrrole firstly and then reducing and isomerising.Heterologousin vivoPCB synthesis inE.colihas been achieved by co-expressing heme oxygenase 1(hox1)and PCB-ferredoxin oxidoreductase(pcyA)gene(Landgrafaet al.,2001).Further,holo-α-PC was synthesized inE.coliby co-expressingcpcAandcpcE/Fandhox1andpcyA(Tooleyet al.,2001).Recently,a lyase set were expressed and analyzed inE.coli(Tooley and Glazer,2002; Shenet al.,2006; Zhaoet al.,2007b; Guanet al.,2007; Geet al.,2009; Biswaset al.,2010).Accordingly,coexpression of various components inE.coliis an approach to understanding the assembly of PBS.

Arthrospira platensis,previously known asSpirulina platensis, is an economically important cyanobacterium for food and pharmaceutical industry.The PC ofArthrospiranot only has biological activities(e.g.,anti-oxidation,anti-tumor,and anti-inflammatory)but also has fluorescence properties,thus can be used for fluorescent tagging and possibly for photodynamic therapy of cancers(Nyman and Hynninen,2004; Benedettiet al.,2010; Rodríguez-Sánchezet al.,2012).Compared to natural PE recovery,the recombinant PE production inE.colican substantially reduce the cost; it avoids large-scale algal cultivation and complicated purification process using Expanded Bed Adsorption Chromtography(Niuet al.,2007).Recently,we have investigated the lyase activity of CpcE/F for α-PC fromA.platensis(Yiet al.,2011).In this study,we continued to investigate the activity of CpcS/U and CpcT expressed inE.coliin β-PC assembly.

2 Materials and Methods

2.1 Gene Cloningb

ThecpcB(DQ406671.1)was amplified fromA.platensisFACHB314 with primer 314cpcB-1(5’-CGCGGA TCCGAT GTT TGA TGC CTT CAC C-3’)and 314cpcB-2(5’-ACG AGC TCT TAT TAG GAA ACT GCT GCA GCA GC-3’).ThecpcS(EU145732.1),cpcU(EU145731.1),andcpcT(EU364770.1)were amplified fromSynechococcussp.PCC 7002 with primer 7002cpcS-1(5’-ACG AAG ATC T AA GGA GAT ATA CCA TGC AAA GCT TTG CGG ATG CC-3’)and 7002cpcS-2(5’-CCG CTC GAG CTA TCA CTA CCA ACC GCT AAT AGC GTA AAG-3’),7002cpcU-1(5’-ACG GAA TTC CAT ATG GAT ATC AAT GCC TTT ATC CATC-3’)and 7002cpcU-2(5’-CGG AAG ATC TTC ACT ATT AGT TAC TGG CTT CAG CGG-3’),and 7002cpcT-1(5’-ACG GAA TTC CAT ATG ATG TCC CAC TCT ACC GAT GCC C-3’)and 7002cpcT-2(5’-CGG AAG ATC T TC ACT ATT AAT GGG GTT GAA CTT CCC CAG-3’),respectively.Thehox1andpcyA(BA000022.2)were amplified fromSynechocystissp.PCC6803 with primer 6803hox1-1(5’-CGC GGA TCC ATG AGT GTC AAC TTA GCT TCC C-3’)and 6803hox1-2(5’-AGG AGC TCT CAC TAG CCT TCG GAG GTG G-3’)and 6803pcyA-1(5’-TGG AGC TC A TAA GGA GAT ATA CCA TGG CCG TCA CTG ATT TAA G-3’)and 6803pcyA-2(5’-AGG CGT CGA CTC ATT ATT GGA TAA CAT CAA ATA AGA CTT GG-3’),respectively.Underlines indicate the restriction sites.

2.2 Construction of Recombinant Plasmids

The amplifiedcpcBwas double-digested withBamHI andSacIand inserted into pACYCDuet-1(Novagen),yielding pACYCDuet-cpcB).ThecpcS,cpcU,andcpcTwere double-digested withBglII andXhoI,NdeI andBglII,andNdeI andBglII,and inserted into pACYCDuetcpcB,yielding pACYCDuet-cpcB-cpcS,pACYCDuetcpcB-cpcU,and pACYCDuet-cpcB-cpcT,respectively.ThecpcSwas double-digested withNdeI andBglII and inserted into pACYCDuet-cpcB-cpcU,yielding pACYCDuet-cpcB-cpcU-cpcS.Thehox1andpcyAwere doubledigested withBamHI andSacI andSacI andSalI,respectively,and inserted into pET-24a(+)(Invitrogen),yielding pET-hox1-pcyA.

2.3 Expression of Recombinant Proteins

Five pACYCDuet based recombinant plasmids each(Table 1)was co-transferred with pET-hox1-pcyAintoE.coliBL21(DE3)(Invitrogen).Recombinants were selected on agar plates containing 34 μg mL?1chloramphenicol and 100 μg mL?1kanamycin,and confirmed by re-amplification of inserts.

Table 1 Recombinants and plasmids they contained

The recombinants,100 mL each,were incubated at 37℃ till OD600reached 0.6.After induction of T7 RNA polymerase with 1.0 mmol L?1IPTG,they were incubated at 28℃ for 6 h,and harvested by centrifugation at 1700

r min?1for 25 min.The pellets were rinsed twice with double-distilled water and stored at ?20℃.

2.4 Emission Spectrum of β-PC

The cell pellet was thawed and resuspended in 5 mL of 0.1 mol L?1phosphate buffer solution(pH 7.2),ultrasonically lysed on ice for 6 min,and centrifuged at 10000 r min?1for 15 min.Then the fluorescence emission spectrum(exciting peak)of supernatant was read in a HITA-CHI F-4500 fluorescence spectrophotometer.The exciting peak was 590 nm.The excitation and emission slit width was 5 nm.The scan speed was 240 nm min?1.

3 Results

3.1 Expression of Recombinant Proteins

Depending on the genes expressed,five recombinants showed different intensities of blue-green color(Fig.1).In comparison with the control(BL21)which was greywhite,all the recombinants showed PCB ligation activity.Of these recombinants,BUS was the most intensive in blue-green color,indicating that CpcS/U had better effects than CpcS and CpcU separately.The second most intensive color was found in B,suggesting that β-PC has autocatalytic activity for the attachment to PCB.

Fig.1 Cell pellets of recombinants with E.coli BL21 as the control.

3.2 Fluorescence Emission Spectrum

The spectrum of control BL21 was smooth,while those of all recombinants emitted fluorescence around 635.5 nm(635.2–635.6 nm),verifying the successful expression of genes inE.coli,the synthesis of PCB and β-PC,and the attachment of PCB onto the apo-PC.Compared to the native fluorescence of wildβ-PC(λmax=644 nm)(Zhaoet al.,2007a),there was a blue-shift in the emission peak in the recombinant protein,possibly due to the change in chemical structure and/or the spontaneous PCB addition.

As shown in Fig.2A,the control strain BL21(solid line)had no emission peak but theλmaxof strain B(dashed line)was 635.6 nm.Fig.2B showed that theλmaxof strain BU(dashed line,635.6 nm)was the longest,followed by that of strain BS(dotted line,635.4 nm)and BUS(solid line,635.2 nm).Fig.2C indicated that theλmaxof B(dashed line,635.6 nm)was longer than that of BUS(solid line,635.2 nm).And Fig.2D showed that BT and B had the sameλmaxat 635.6 nm.

Fig.2 Fluorescence emission spectra of recombinant proteins.A,Comparison of fluorescence emission spectrum of BL21(solid line)and recombinant B(dashed line); B,Comparison of the spectrum of recombinant BUS(solid line),BU(dashed line)and BS(dotted line); C,Comparison of spectrum of B(dashed line)and BUS(solid line).D,Comparison of spectrum of BT(solid line)and B(dashed line).

Of the five recombinants,B had relatively high fluorescence intensity(Fig.2A),which was consistent with its deep blue-green color.Although such high fluorescence intensity was probably attributed to the high expression of β-PC rather than the high autocatalytic chromophorylation activity,the similar fluorescence emission of B and BUS(Fig.2C)strongly suggested that the β-PC fromA.platensishad autocatalytic chromophorylation activity inE.coli.Comparison of the fluorescent peak and intensity of BUS,BU and BS(Fig.2B)showed that BUS had the highest intensity,indicating that CpcS had the highest lyase activity when working together with CpcU.In addition,CpcS itself appeared to have a slight effect because the fluorescence peak of BS(635.4 nm)was between that of B(635.6 nm)and BUS(635.2 nm),thus a combination of B and BUS.The fluorescence intensity of BU was intermediate but its emission peaked was at 635.6 nm as well,suggesting that its fluorescence was caused by the autocatalytic chromophorylation activity of β-PC.Perhaps the expression of CpcU interfered with the production of CpcB,thus reducing the overall fluorescence.The situation for BT was similar to that for BU.Its fluorescence was probably induced by the CpcB activity since the emission peak of BT,BU and B was exactly the same(Fig.2D).According to Debreczenyet al.(1995),the fluorescence peak of PCB on cys-153 of β-PC chromophorylated by CpcT is shorter than that of PCB on cys-82 of β-PC chromophorylated by CpcS/U.It seems that the activity of CpcT fromSynechococcussp.PCC 7002 was not detectable in this experiment.

4 Discussion

The PC complex fromA.platensisholds potential applications in medicine and molecular biology; however,direct isolation from algal biomass has diverse restrictions.Thus,it is of practical significance to synthesize intact PBS inE.coli.AlthoughcpcBencoding holo-β-PC has been isolated early fromA.platensisFACHB 314,lyase genes(cpcS,cpcUandcpcT)and PCB-producing genes(hox1andpcyA)have not been cloned from this organism yet.Instead,we used genes fromSynechococcussp.PCC 7002 andSynechocystissp.PCC6803.Early studies had shown that these genes worked well in catalyzing chromophore attachment to PBS from other cyanobacterial species inE.coli(Zhaoet al.,2006; Geet al.,2009; Biswaset al.,2010),which allowed us to investigate the lyase activity of CpcS/U and CpcT in the synthesis of holo-β-PC fromA.platensisinE.coliusing the genes ofSynechococcussp.PCC 7002.

Once PCB was correctly ligated to apo-PC,its specific blue-green color and fluorescence spectrum should be detectable.InE.coli,the fluorescence intensity of recombinant PC depends not only on the expression level of apo-PC(plasmid copy number and promoter efficiency)(Biswaset al.,2010), but also on the quantity of PCBs attached to the apo-protein.For CpcB ofA.platensis,two lyases,i.e.,CpcS/U and CpcT,are needed because the stereochemistry of bilin attachment differs at the two attaching sites,β-82 and β-153(Shenet al.,2006).Results of sequence analyses showed that CpcS and CpcU were highly similar(about 53%),especially in N- and C-terminal regions containing conserved motifs(Shenet al.,2008).CpcT also clusters with the CpcS/U type of lyase(Scheer and Zhao,2008)but not with the lyases for α-PC(CpcE/CpcF).Based on amino acid sequence,CpcS merges into three clades(Shenet al.,2008).CpcS-I includes cyanobacteria such asSynechococcussp.PCC 7002 andSynechocystissp.PCC6803,which needs to form a heterodimer with CpcU to catalyse PCB attachment to β-PC and β-AP subunit(Shenet al.,2008); CpcS-II exists in a variety of marineSynechococcussp.,some of them produce PC with PEB chromophores(Ong and Glazer,1988); and CpcS-III includes cyanobacteria such asNostocsp.PCC 7120,which is able to ligate PCB to Cys-82 in a variety of PBPs as a monomeric lyase(Zhaoet al.,2006,2007b).In the present study,the genecpcScloned fromSynechococcussp.PCC 7002 belongs to CpcS-I and should work together with CpcU.This is consistent with our observation that BUS had the highest fluorescence intensity as compared to BU and BS individually.

Two PCB attachment sites,β-82 and β-153,of β-PC interact with lyase CpcU/S and CpcT,respectively.In β-PC,PCB-Cys-84(82)commonly absorbs at a longer wavelength than PCB-Cys-155(153)and serves as the predominant fluorescing chromophore(Debreczenyet al.,1995).Theλmaxof BT,635.6 nm,was unlikely caused by CpcT since it is not shorter than theλmaxof BUS,635.2 nm.Here inE.coli,recombinant CpcS/U seemed to function well although the activity of CpcT was not detectable.

Recombinant B showed relatively high intensity of fluorescence,suggesting that CpcB is able to attach PCB onto itself spontaneously or by specific autocatalytic activity.Because CpcU and CpcT alone showed no lyase activities in theE.coliwhile BU and BT showed certain fluorescence,we speculated that this fluorescence came from CpcB itself rather than the lyase activity.Spontaneous addition of PCB has been observed at all binding sites(Cys-α-84,Cys-β-84,and Cys-β-155)of PC,but the process was found slow and generally led to a mixture of products,in contrast to the correct alignment previously seen for autolytic attachment(Arcieroet al.,1988; Zhaoet al.,2006).Recombinant B,BU and BT had the sameλmax,indicating that the fluorescence emission was caused by a mixture of spontaneous products.In fact,it was consistent with autocatalytic attachment of CpcB as previously demonstrated in other studies(Zhaoet al.,2004; 2007b).However,spontaneous and/or autocatalytic chromophore binding is unlikely to occurin vivobecause biliprotein synthesis is a major metabolic pathway in cyanobacteria and can cause low concentrations of chromophores for other uses(Scheer and Zhao,2008).The fluorescence of BUS would therefore more likely reflect the activity of lyase pathways rather than spontaneous addition of PCB.

In addition,results from the present study showed that the fluorescence emission peak recombinants was around 635.5 nm,with an 8–9 nm blue-shift from the native(λmax=644 nm).Such a blue-shift could be related to the change in chemical structure of PBPs and/or spontaneous PCB addition.PC is composed of two subunits(αβ)but usually exists as a trimer(αβ)3or a hexamer(αβ)6in wild-type strains(Sidler,1994; Schmidtet al.,2007).In the present study,only β-PC was expressed inE.coliand thus no integrated PC could be constructed.Since the absorption and emission peaks of monomers are more strongly blue-shifted than trimers(MacCollet al.,1980),the peak of successfully transformed,expressed,and chromophorylated PCB-CpcB should be shorter than the natural PC.

In this study,the lyases showed a relatively low catalytic rate(CpcS/U)or even no detectable activity(CpcT).A possible reason is that the heterologous lyase genes were used.A similar phenomenon was observed in previous work(Biswaset al.,2010).According to his results,the chromophorylation levels of PC inE.coliwere much lower(only 17%–37%)when the lyase genes and the apo-protein genes were cloned from different Cyanobacteria even very closely related(SynechococcusPCC 7002 andSynechocystisPCC6803 respectively)than when those genes were all from one species(SynechococcusPCC 7002).In contrast to CpcT,CpcS-I is a nearly universal bilin-Cys-84-PBP lyase that catalyzes the regiospecific covalent attachment of PCB to cysteine 84 of PecB,CpcB,ApcA,ApcB and even catalyzes phycoerythrobilin(PEB)to both subunits of C-phycoerythrin(Zhaoet al.,2006,2007).CpcS likely has a broader specificity for substrates than CpcT does,being consistent with their amino acid sequence similarity among species.The amino acid similarity of CpcS ofSynechococcusPCC 7002(ABV80281.1)andSynechocystisPCC6803(BAA17117.1)was 64%,and that between CpcS ofSynechococcusPCC 7002(ABV80281.1)and CpcS-II ofNostocPCC7120(Q8YZ70.1)was 49%.The amino acid sequence similarity of CpcT ofSynechococcusPCC 7002(ABY63662.1)andSynechocystisPCC6803(P74371.1)was 55%,while that ofSynechococcusPCC 7002 andNostocPCC7120(Q8YZ40.1)was as low as 27%.Thus,the transcripts would have different activities in a heterologous host due to their differences in solubility,optimal temperature,and/or the gene expression level(Biswaset al.,2010).This possibly explains the relatively low activity of CpcT compared to CpcS inE.coli.

InE.coli,cpcBfromA.platensisFACHB314 was correctly expressed and successfully chromophorylated with relatively low efficiency,as was evidenced by the specific fluorescence emission peak of PC.The low activity of the co-expressed lyases is possibly related to the use of heterologous components from taxonomically divergent species.To overcome this problem,we will continue the attempt to clone the lyase and PCB-producing genes ofA.platensisby referring to the published whole genome sequence of a closely related strain,A.platensisNIES-39(Fujisawaet al.,2010).Once the homologous components are obtained,we will further investigate the target gene expression levels and activities for PC production in theE.colisystem.

Acknowledgements

This work was supported by the National Science and Technology Major Project of China(2008ZX08001-004).

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