Introduction

Poly (hydroxyalkanoates) (PHAs) are natural biopolymers produced by many microorganisms. These polymers are accumulated by cells as stor-age materials under stress conditions such as ni-trogen limitation and excess supply of carbon sources (1-3).
PHAs have a wide range of applications be-cause of their thermo-plasticity and degradability properties. Controlled drug release systems, scaf-fold materials in tissue engineering, absorbable surgical sutures, prosthesis and bioimplants are some of their uses in medicine (4). Due to the growing needs for these biopolymers in industrial applications, there is great interest to produce them in large amounts through recombinant DNA technology.
Although many wild-type microorganisms are known to produce PHAs, natural PHA producers have a long generation time, are often hard to lyse, and contain pathways for PHA degradation. There are several advantages of using recom-binant E.coli for the production of PHAs such as fast growth, ease of lysis, very good understand-ing of their genetics and biochemical properties, and broad substrate utilizing abilities (5-8).
PHA synthases are the key enzymes of PHA biosynthesis. In our previous study, the PHA bio-synthesis genes of P.aeruginosa PTCC 1310 (pha C1 and phaC2 genes) were cloned and sequenced (9). The aim of this study was to express these genes and optimize the conditions for their ex-pression in E.coli.The optimization of conditions for phaC genes expression can serve as an im-portant stage for establishing PHA production in E.coli.

Materials and Methods

Bacterial strains and growth conditions
The bacterial strains used were E.coli BL(DE3) and Pseudomonas citranolleise (P.citranolleise) (DSMZ 11735). E.coli was grown at 37°C in Lu-ria-Bertani medium (LB). P.citranolleise was grown in Minimal Salt Medium (MSM) contain-ing 1.5% glucose. When needed, ampicillin and Isopropyl-b-D thiogalactopyranoside (IPTG) were added to the medium.

Construction of expression plasmids
Two plasmids, pTZPHAC1 and pTZPHAC2, which contained the PHA synthase genes (phaC1 and phaC2) from P. aeruginosa PTCC 1310, were constructed from genomic DNA as described pre-viously. (9) These plasmids were extracted and di-gested with restriction enzymes. The NdeI and BamHI enzymes were used to digest pTZPHAC1 and for digestion of pTZPHAC2 NdeI was used (BamHI was not used because it digests the phaC2 sequence). The obtained phaC1 and phaC2 inserts were ligated into pET15b (Novagen, USA) ex-pression vector digested with NdeI, BamHI and NdeI, respectively. These recombinant vectors were transformed into competent E.coli BL21 (DE3) cells using the CaCl2 method (10).

Expression of phaCgenes and purification of His-tagged enzymes
For the expression of PHA synthase, a positive cell clone was inoculated into 5 ml of LB medium (100 μg/ml Amp). An overnight culture was used to inoculate (1% v⁄v) 50 ml of fresh LB medium in 250 ml flasks and incubated at 37°C to an OD600nm of 0.4-0.6. Then the expression of phaC genes was induced by the addition of IPTG to a final concentration of 1 mM. The expressed enzymes were purified with Ni-NTA His Bind Resin (Novagen, USA) as described by the manufac-turer.
Briefly, 1 ml of each sample was collected and centrifuged after an appropriate growth period. The pellets were resuspended in 200 µl of buffer A and clarified by centrifugation at 15000 g for
10 min and the supernatant was taken for the next steps. Fifty l of the 50% 1X Ni-NTA His Bind Resin was added to each sample and mixed gently for 30 min at room temperature. After centrifuga-tion of samples, the supernatant was removed and the pellet was washed two times with 250 µl of buffer C. Finally, the His-tagged enzyme was eluted with buffer E and 20 µl of each sample was electrophoresed on a 12% polyacrylamide gel using SDS-PAGE.

Optimization of conditions for phaC genes expression
IPTG concentration, time and temperature of incubation with IPTG were optimized for phaC genes expression in E.coli BL21 (DE3) cells. For optimization of enzyme expression, IPTG concen-tration at four levels (0.5, 1, 1.5 and 2 mM), three different times of incubation with IPTG (1, 2 and 3 hrs) and the temperature of incubation with IPTG at three levels (25, 37 and 39°C) were ex-amined. Each assay was repeated three times for pET-phaC1 and pET-phaC2 and one time for pET15b as the negative control (a total of 252 ex-periments). Expressed protein samples were ana-lyzed on a 12% SDS-PAGE gel and the intensity of bands were quantified and compared using SPSS 10 version and differences at p<0.05 were considered as significant.

Detection of PHA accumulation
To confirm the expression of functional PHA synthase enzymes by E.coli BL21 (DE3) cells car-rying the phaC genes, the accumulation of PHA in bacteria was detected with Nile red (11). The E.coli BL21 (DE3) cells carrying the phaC gene, E.coli BL21 (DE3) without the phaC gene (as the nega-tive control) and P.citranolleise (as the positive control) were investigated for PHA production. E.coli BL21 (DE3) cells were streaked on an LB plate supplemented with sodium-gluconate (0.5% w/v), Nile red (0.05 µg/ml), IPTG (0.1 mM) and ampicillin (100 µg/ml). P.citranolleise was grown on MSM supplemented with sodium-gluconate (0.5% w/v), glucose (1.5% w/v) and Nile red
(0.05 µg/ml). The agar plates were incubated at 37°C for 24 hr.

Results

Expression and purification of phaC synthase
The PHA synthase enzymes were expressed by the addition of IPTG, then the cell suspension was centrifuged. The pellets were added to Ni-NTA His Bind resins. After purification, samples were analyzed by SDS-PAGE, which revealed that the target protein of 62 kD was expressed (Figure1).

Optimization of conditions for phaC gene expression
The effect of incubation temperature and IPTG concentration on SDS-PAGE band intensity of phaC1 and phaC2 were investigated and the re-sults are shown in figure 2. The maximum pro-duction of the PHA synthase enzymes occurred when 1 mM IPTG was used at 37°C. The effects of incubation time and IPTG concentration on SDS-PAGE band intensity of phaC1and phaC2 were evaluated (Figure 3). Maximum production of PHA synthase enzyme was observed when 1 mM IPTG was used for 2 hr.

Detection of PHA accumulation
Nile red plate assay revealed functional expres-sion of PHA synthase by recombinant E.coli BL21 (DE3) cells (Figure 4).

Discussion

Optimization of conditions for the expression of recombinant proteins has been performed in many studies. Bai et al evaluated the effect of con-centration of IPTG and cell density on the level of expression of catalase (12). In another study per-formed by Wen and his coworkers, the effect of temperature and concentration of IPTG on the ex-pression of PET-ILC-HIL-2-MGM was investi-gated (13). They found that maximum protein ex-pression occurred when 0.3 mM IPTG was used at 42°C. Yang et al optimized four factors, including the initial cell density, concentration of IPTG, incubation temperature and induction time during the induction of the target proteins (14). According to their results, the expression of superoxide dis-mutase was optimal when E.coli cell density was OD600nm=0.6, IPTG concentration was 0.125 mM and incubation at 37°C was for four hours. In another research performed by Qun et al, two phaC genes from P.putida were expressed in E.coli and the effect of IPTG concentration on PHA content and PHA monomer composition was evaluated (15).
In the present study we investigated three fac-tors: the concentration of IPTG, temperature, and time of induction of the target proteins. Our re-sults showed that the expression of phaC1 and phaC2 with 1 mM of IPTG for 2 hr at 37°C was the optimum condition for the expression of these genes. In comparison with other studies, optimum incubation time for PHA synthase enzyme expres-sion was shorter and IPTG concentration was greater than those studies.
Using a Nile red plate assay, the accumulation of PHA in bacteria was detected which confirmed the expression of functional PHA synthase en-zymes in E.coli BL21(DE3) cells. To increase the yield of PHA production, using mutant strains of E.coli that could block the oxidation of fatty acids and accumulate greater level of PHA is also re-commended (16).

Conclusion

In this study, PhaC genes from an Iranian isol-ate, P.aeruginosa, were expressed in E.coli and their expression was optimized. According to our findings, the concentration of IPTG, incubation temperature and incubation time during the induc-tion with IPTG can affect the expression of these proteins. Therefore, it is now possible to continue our studies in producing these enzymes in larger quantities for industrial production of biodegrade-able polymers.

Acknowledgement

This work was part of Dr Maryam Beheshti’s PharmD thesis and was supported by the Isfahan University of Medical Sciences (grant no. 185085).