Biosynthesis and characterization of a recombinant eukaryotic allophycocyanin using prokaryotic accessory enzymes

Abstract Phycobiliproteins (PBPs) are colored fluorescent proteins present in cyanobacteria, red alga, and cryptophyta. These proteins have many potential uses in biotechnology going from food colorants to medical applications. Allophycocyanin, the simplest PBP, is a heterodimer of αβ subunits that oligomerizes as a trimer (αβ)3. Each subunit contains a phycocyanobilin, bound to a cysteine residue, which is responsible for its spectroscopic properties. In this article, we are reporting the expression of recombinant allophycocyanin (rAPC) from the eukaryotic red algae Agarophyton chilensis in Escherichia coli, using prokaryotic accessory enzymes to obtain a fully functional rAPC. Three duet vectors were used to include coding sequences of α and β subunits from A. chilensis and accessorial enzymes (heterodimeric lyase cpc S/U, heme oxygenase 1, phycocyanobilin oxidoreductase) from cyanobacteria Arthrospira maxima. rAPC was purified using several chromatographic steps. The characterization of the pure rAPC indicates very similar spectroscopic properties, λmax Abs, λmax Em, fluorescence lifetime, and chromophorylation degree, with native allophycocyanin (nAPC) from A. chilensis. This method, to produce high‐quality recombinant allophycocyanin, can be used to express and characterize other macroalga phycobiliproteins, to be used for biotechnological or biomedical purposes.

& Terry, 2009), bound to cysteine 82 by a heterodimeric lyase S/U . This pathway has been reported only for cyanobacteria. The subunits oligomerize as a trimers (αβ) 3 . The trimeric form of APC is the biologically functional state. This oligomeric state is necessary to provide the specific conformation and relative location of the chromophores to present the typical absorption and emission spectra of APC with λ max Abs = 651nm and λ max Em = 660 nm (MacColl, 2004;Samsonoff & MacColl, 2001). For its spectroscopic characteristic, purified APC is an important candidate for the biotechnological, pharmaceutical, cosmeceutical (Li et al., 2019;Pagels et al., 2019), and food industry (Dumay, Morançais, Munier, Le Guillard, & Fleurence, 2014). The recovery of allophycocyanin and other phycobiliproteins from natural sources requires large-scale cultures of cyanobacteria or considerable amount of eukaryotic algae to be processed. Nevertheless, the production of recombinant phycobiliproteins in E. coli would reduce the costs and time required to obtain them with the necessary quality for biotechnological purposes.
In the last decade, different protocols to obtain recombinant phycobiliproteins have been published in order to obtain molecular species with properties similar to the proteins purified from native organisms Liu et al., 2010). The production of α phycocyanin subunits with λ max Abs at 625 nm and λ max Em at 641 nm (Tooley & Glazer, 2002), subunits of αAPC (Hu, Lee, Lin, Chiueh, & Lyu, 2006;Liu et al., 2009), βAPC subunits of Synechocystis sp PCC6803 with λ max Abs at 611 nm and λ max Em at 642 nm (Chen, Lin, Li, Jiang, & Qin, 2013) are examples of these attempts. It has been reported also the obtaining of trimeric rAPC of Synechocystis sp PCC6803 in E.coli, by using multiple duet vectors (Liu et al., 2010) as well as from Synechococcus in E. coli . These vectors contained the sequences for the subunits α and β of the phycobiliproteins, the enzymes to produce phycocyanobilin and the subunits of a heterodimeric lyase for the covalent binding of the chromophore to the corresponding cysteine.
It has been reported that the isolated heterodimer (αβ) of a rAPC of Synechocystis sp PCC 6,803 obtained in E.coli has an absorption máximum at 615 nm, but when rAPC recovers its trimeric state (αβ) 3 , also recovers its absorption maximum at 650 nm (Liu et al., 2010).
There is not enough information on the metabolic pathway for the synthesis and binding of the phycobilin (PB) to the holo-APC in eukaryotic red alga. The whole genome of P. cruentum (Bhattacharva et al., 2013) and the plastid genome and transcriptome of A. chilensis (Hagopian, Silva Reis, & Kitayima, 2004;Vorphal et al., 2017) have been reported, but it was not possible to find the sequences of the enzymes of the pathway. In this article, we modify the methodology by using heterologous enzymes, to improve the chromophorylation of the trimers (αβ) 3 we used three duet expression vectors and a Histag only in the β subunits to avoid steric hindrance.
In eukaryotic red algae, APC is extracted in lower amount than the other phycobiliproteins because, as part of the core, it is associated to membranes through the linker core-membrane Tang et al., 2015). For biotechnological purposes, it is necessary that the recombinant APC (rAPC) be in a single oligomerization state as a trimer, highly chromophorylated, with the correct spectroscopic properties, and with a high yield after the purification process. In this article, we present an approach to obtain a trimeric allophycocyanin from a eukaryotic macroalgae in a prokaryotic system. To do that, three duet expression vectors were used, which contains coding sequences of A. chilensis allophycocyanin α and β subunits, and enzymes to obtain holo-APC (heterodimeric lyase cpc S/U, hemeoxygenase 1, phycocyanobilin oxido reductase) from the cyanobacteria Arthrospira maxima. The expression was accomplished, and the protein rAPC was purified and compared with native allophycocyanin from A. chilensis (nAPC) by absorption and emission spectroscopy, circular dichroism, and molecular sieve chromatography. This method leads to the production of rAPC with very similar properties to nAPC, which can be used for biotechnological purposes.

| DNA extraction and PCR conditions
The coding sequences of the six genes needed were amplified by PCR using KAPA HiFi polymerase. apcA and apcB were obtained using A. chilensis DNA as template. The purification of DNA from A.
chilensis was performed according to the literature (Ramakrishnan, Fathima, & Ramya, 2017). PCR were performed using the follow- shown the restriction sites for subcloning.
Each gene was cloned in TOPO-TA 2-1 vector (Thermo Fisher Scientific) and transformed into E.coli DH5α. Plasmid DNA was extracted with GeneJet mini plasmid kit (Thermo Fisher Scientific).

| Construction of the expression vectors
Expression vectors were constructed as follows: apcB and cpcU in their cloning vectors were digested with BamHI and SacI for apcB and NdeI and XhoI for cpcU and then ligated at the cloning site 1 and 2 in pETDuet-1 vector (novagen), respectively. apcA and cpcS were digested with NcoI and BamHI, and cpcS with NdeI and XhoI to be then ligated in the cloning site 1 and 2 of pCDFDuet-1 vector (Novagen), respectively. Finally, pcyA and ho1 were digested with NcoI and BamHI and hoI with NdeI and xhoI, they were cloned in pRS-FDuet-1 cloning sites 1 and 2 of, respectively. The expression vectors were sequenced at the Department of Ecology from Pontificia Universidad Católica de Chile and analyzed with Bioedit software to confirm the absence of mutations.

| In vivo heterologous expression of recombinant Allophycocyanin (rAPC)
30 ng of each expression vectors, pCDF-apcA-cpcS, pET-apcB-cpcU y pRSF-pcyA-ho1, were co-transformed in electrocompetent E coli BL21 x g for 20 min. The supernatant was used as input for the following purification steps.

| Purification of rAPC
The proteins were precipitated with ammonium sulfate (60% satura- a flux of 0.5 ml/min. All the chromatographic procedures were performed in an AKTA Prime (GE) system. The purified rAPC was concentrated in Amicon Ultra-15 50 K, to 0.5 mg/ml and stored at −20°C.
In parallel, nAPC was purified as reported previously (Dagnino-Leone, 2017) for comparative proposes. The chromophorylation degree was determined for nAPC and rAPC based on Glazer, 1988) in two independent experiments.

| Purification of rAPC
The purification of rAPC was performed from a pellet of 3.94 g of recombinant bacteria. Fractions of the semipurified rAPC from the ionic exchange chromatography account for 7 mg/L of bacterial culture, following its absorption at 651 nm. The following chromatography step with IMAC was also followed at 651 nm; two turquoise fractions were identified, the fraction retained in the column and the flow-through fraction. Both protein fractions were analyzed.
Even though both fractions have a λ max Abs close to 651 nm, only the retained fraction showed an identical spectrum with nAPC. This fraction accounts for 0.124 mg/L of bacterial culture. The characterization of this fraction is presented below.

| Characterization of rAPC
The purified rAPC showed the characteristic turquoise color. Its absorption and emission spectra are shown in Figure 1a. For both, nAPC and rAPC, their spectroscopic characteristics are very similar, with an absorption maximum at 651 nm, a shoulder at 620 nm and an emission maximum at 661 nm. Figure 1b  The degree of chromophorylation was close to 50%, (nAPC: 52%, rAPC: 57%). Figure A1 shows the spectra for the denatured nAPC and rAPC that were used for the calculations. rAPC and nAPC showed similar degree of chromophorylation. Table 1 shows a comparison of the characteristics of rAPC and nAPC.

| D ISCUSS I ON
Phycobiliproteins have an enormous biotechnological potential, their applications go from food colorant to biomedical uses because they possess antioxidant and antitumorous properties. They are also used in photodynamic therapies as fluorescent probes because their spectroscopic characteristics. Allophycocyanin is the most simple phycobiliprotein, it possesses only one phycocyanobilin molecule per subunit attached to the peptide backbone, and its native functional oligomer is a trimer (De Marsac, 2003;MacColl, 1998MacColl, , 2004. Phycobiliproteins from red algae are much less studied than cyanobacterial. An exception is the red microalgae Porphyridium cruentum (Bermejo, Ruiz, & Acien, 2007;Bermejo, Talavera, & Alvarez-Pez, 2001;Nagy, Bishop, Klotz, Glazer, & Rapoport, 1985), but for eukaryotic macro algae the studies of phycobiliproteins are only a few (Galland-Irmouli et al., 2000;Lüder, Knoetzel, & Wiencke, 2001). Allophycocyanin is the less abundant in eukaryotic phycobilisomes (Glazer, 1988) fact that presents a problem for the study and biophysical characterization of this protein.
In this work, we have obtained an eukaryotic recombinant allophycocyanin, rAPC, from A. chilensis using prokaryotic accessory enzymes (heterodimeric lyase S/U, hemeoxigenase 1 and phycocyanobilin oxido reductase) from A. maxima in E.coli with their spectroscopic and biochemical properties comparable to the purified native allophycocyanin. The expression system designed is based on the literature but with changes in order to obtain a fully functional protein. We designed an expression system to produce equivalent number of copies for α and β subunits. This was confirmed by the SDS-PAGE (Figure 1) in which the intensity of the bands stained with Coomassie blue was also similar. The expression system was also selected to obtain a higher number of copies for the enzymes responsible for the synthesis and binding of the chromophores by using the vector pRSF which has a replication time 5 times faster than the vector that contained the α and β subunits of rAPC. This is important because the objective was to obtain a high degree of chromophorylation. Another important difference was the addition of a His-tag only to the N-terminal of β subunits, instead of the N-terminal of α subunits  or to the N-terminal of both subunits as described in Liu et al., (2010). The structural information we had on APC (Dagnino-Leone, 2017) was used in order to have less effect in the oligomerization state as a trimer. Figure 5 shows the molecular model of A. chilensis rAPC trimer, the position of the His-tag is indicated and it was designed to eliminate the steric hindrance that could be produced in the organization of the subunits, and it would account for the trimeric oligomerization state obtained with this protocol. In previous reports , a different combination of three duet vectors were used; in (Liu et al., 2010) the authors included the six necessary genes in two expression vectors, inserting F I G U R E 3 Circular dichroism spectra of nAPC and rAPC. Normalized Far-UV CD spectra for both protein are shown. The spectra for both protein are very similar, with the features of proteins with mainly helices as secondary structure elements F I G U R E 4 Thermal stability characterization of nAPC and rAPC. Normalized thermal denaturation plots, followed by CD ellipticity at 222 nm for both proteins, are shown. nAPC is more stable than rAPC protein, displaying Tm values of 64 and 56°C, respectively cpcS and cpcU in tandem in the cloning site 2 of pCDF vector and also ho1 and pcyA in the cloning site 2 of vector pRSF. In both cases, lower chromophorylation efficiency (27%) was reported, compared with the expected for a native protein. In our case, we reached similar chromophorylation degree for the recombinant protein in comparison with the native one using the same methodology.
Experimental information about the oligomeric state of nAPC in other red alga was not available even though in (Murakami, Mimuro, Ohki, & Fujita, 1981) the authors reported a trimeric state for the functional APC purified from Anabaena cylindrica. Our results from molecular sieve chromatography point to a trimeric state for nAPC from A. chilensis and for rAPC. This result agrees with the models obtained by X-ray crystallography which report (αβ) 3 as the biological unit (Dagnino-Leone, 2017), (Brejc, Ficner, Huber, & Steinbacher, 1995;McGregor, Klartag, David, & Adir, 2008;Murray, Maghlaoui, & Barber, 2007;Schmidt, Krasselt, & Reuter, 2006). The circular dichroism spectrum for nAPC as well as for rAPC agrees with the secondary structure reported for the crystallographic structures of APC for Agarophyton chilensis (PDB ID: 5TJF; Bhattacharva et al., 2013), and also agrees with other APC structures from cyanobacteria reported at the PDB (Brejc et al., 1995;McGregor et al., 2008;Murakami et al., 1981;Murray et al., 2007), with predominance of helical structures revealed by the two minima at 222 and 208 nm.  (Maksimov et al., 2014).
The spectrum of nAPC and rAPC at denaturing conditions (8 M urea, pH 2) and the relationship between the concentrations of phycocyanobilin chromophore allows calculating the degree of chromophorylation of nAPC and rAPC. These values were the same with an estimated value of 52% and 57%.  report that for monomers, the chromophorylation rate is 40% and for the trimer totally chromophorylated is 6.4%.
To this point, the A. chilensis rAPC showed very similar properties with nAPC, as it is shown on Table 1.
We were able to produce 7 mg/L of recombinant rAPC, but only 0.124 mg/L corresponds to a functional trimeric conformation. Biswas et al (10) reported the production of 5 to 12.4 mg of rAPC from Synechococcus sp. strain PCC 7,002, but they did not report the amount of functional protein for comparison . More experiments are needed to fine tuning the protein expression of A. chilensis rAPC.
Changing temperature and induction time would allow optimize the production of functional A. chilensis rAPC in E. coli.
In summary, we have obtained a recombinant eukaryotic allophycocyanin in its trimeric and functional conformation, by using a cyanobacterial enzymatic accessory system. rAPC has very similar properties with nAPC, and it is completely functional for biotechnological and/or biomedical purposes. In addition, this system would allow the study of the biophysical characteristics of the other subunits with different spectroscopic properties, present in the core of the phycobilisome of Agarophyton chilensis, such as α II and β 18 . The system also will allow the production of other recombinant phycobiliproteins from other red macroalga for biotechnological purposes.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analyzed during this study are included in this published article.