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ORIGINAL ARTICLE
Adv Biomed Res 2016,  5:50

Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli


Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan, Iran

Date of Submission01-Jun-2013
Date of Acceptance09-Dec-2014
Date of Web Publication16-Mar-2016

Correspondence Address:
Dr. Hamid Mir Mohammad Sadeghi
Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Sciences Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences, Isfahan
Iran
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Source of Support: This work was supported by Isfahan University of Medical Sciences (grant No 391170), Conflict of Interest: None


DOI: 10.4103/2277-9175.178790

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  Abstract 

Background: Poly3-hydroxyalkanoates (PHAs) are potential candidates for the industrial production of biodegradable plastics. Therefore, in the present study, expression and activity of one of the enzymes involved in the PHA synthesis, phaC2 (isolated from Pseudomonas aeruginosa PTCC1310), were investigated in Fad B deleted Escherichia coli.
Materials and Methods: The inserts obtained from recombinant pTZ57R plasmids were ligated into the pGEX-5x-1 expression vector and then transformed into Fad B deleted E. coli cells using the heat shock method. This protein was then expressed using isopropyl beta-d-thiogalactoside (IPTG) as an inducer. By changing expression conditions such as IPTG and glucose concentration, time and temperature of incubation with IPTG, the expression conditions were optimized.
Results: The optimum condition for the expression of this enzyme was: 1.5 mM IPTG, 1 mM glucose, incubated at 37°C for 2 hours.
Conclusion: We obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

Keywords: Fad B deleted E. coli, IPTG, phaC2, polyhydroxy alkanoate, protein expression


How to cite this article:
Abedi D, Moazen F, Akbari V, Mirzaalian F, Sadeghi HM. Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli. Adv Biomed Res 2016;5:50

How to cite this URL:
Abedi D, Moazen F, Akbari V, Mirzaalian F, Sadeghi HM. Optimization of the expression of phaC2 encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Fad B deleted Escherichia coli. Adv Biomed Res [serial online] 2016 [cited 2020 Oct 25];5:50. Available from: https://www.advbiores.net/text.asp?2016/5/1/50/178790


  Introduction Top


Biodegradable polymers offer a great solution to the environmental hazards caused by conventional plastics.[1],[2],[3],[4] One of these biodegradable plastics are biopolymers called poly-3-hydroxyalkanoates (PHAs) which behave similar to the synthetic plastics.[1],[2],[3],[4],[5] PHAs are a family of intracellular polyesters produced by a variety of microorganisms under stressful conditions such as sulfur or nitrogen limitation and after returning to normal conditions, cells would disintegrate these polymers.[1]

There are three groups of PHAs: Short chain length (SCL) PHAs, containing 3 to 5 carbon atoms in monomer units, medium chain length (MCL) PHAs containing 6 to 14 carbon atoms, and scl-co-mcl a repeat-unit monomers containing 3 to 14 carbon atoms.[1],[6] Most Pseudomonase strains are able to accumulate mcl-PHA and they have long generation time, ease of lysis, good understanding of their biochemical properties and genetics. Therefore, it would be advantageous to obtain large quantities of PHAs using recombinant  Escherichia More Detailscoli.[7],[8],[9]

PHA synthase are the most important enzymes involved in PHA biosynthesis. Four major classes have been recognized for PHA synthases. The class two PHA synthase has been found only in mcl-PHA-producing Pseudomonade strains. This system contains two PHA synthase genes, phaC1 and phaC2, which are separated by a phaZ gene (encoding for PHA depolymerase).[10]

In our laboratory, several isolates of Pseudomonas aeruginosa obtained from Iranian resources were examined for the presence of PHA synthase genes. One of those species (PTCC1310) contained these genes and thus used for further studies.

In previous studies the phaC2 gene was amplified from P. aeruginosa PTCC1310 by PCR, cloned and then expressed in E. coli BL21 (DE3). The results revealed that for increasing the yield of PHA production, mutant strains of E. coli that could block the oxidation of fatty acids and accumulate greater levels of PHA should be used. Therefore, the aim of the present study were to express the phaC2 gene in Fad B deleted E. coli (a mutant E. coli which blocks the oxidation of fatty acids and leads to more expression) and optimize the conditions for expression.


  Materials and Methods Top


Bacterial strains and growth conditions

The bacterial strains used were Fad B deleted E. coli and P. aeruginosa PTCC 1310. Fad B deleted E. coli was grown at 37°C in Luria-Bertani medium (LB). Pseudomonasaeruginosa was grown in minimal salt medium (MSM) containing 1.5% glucose. When needed, ampicillin, kanamycin and isopropyl-β-D thiogalactopyranoside (IPTG) were added to the medium. Fad B deleted E. coli is resistant to kanamycin so the plates were supplemented with this antibiotic.

Construction of the expression plasmids

pTZ57R plasmid containing the PHA synthase gene (phaC2) from P. aeruginosa PTCC1310 was used for this study.[11] At first, this plasmid was digested with restriction enzymes (Xho I for 45 min in 37°C and then Eco RI added for 30 min at 37°C). After digestion with Eco RI and Xho I restriction enzymes, the obtained phaC2 inserts were ligated into the pGEX-5x-1 expression vector. For this digestion, the first XhoI enzyme was added for 45 min at 37°C and after that Eco RI enzyme was added for 30 min at 37°C. This recombinant vector was transformed into competent Fad B deleted E. coli cells using the CaCl2 method.[12] We used fast digest enzymes in this study.

Expression of phaC2 genes

For the expression of PHA synthase, a positive clone was inoculated into 5 ml of LB medium (100 µg/ml amp). After an overnight culture, bacterial growth (1% v/v) was added to 50 ml of fresh LB medium in 250 ml flasks and incubated at 37°C until OD 600 nm of 0.4–0.6 was reached. By adding IPTG to a final concentration of 1 mM, the expression of phaC2 was induced.

Optimization of conditions for phaC2 gene expression

For optimization of the enzyme expression, IPTG concentration at four levels (0.5, 1, 1.5, 2 mM), four different times of incubation with IPTG (1, 2, 3 and 4 hrs), the temperature of incubation with IPTG at three levels (25, 30, 37°C) and glucose concentration at three levels (0.5, 1, 2 mM) were examined. Expressed protein samples were analyzed on a 12% SDS-PAGE gel.

Detection of PHA accumulation

The accumulation of PHA in bacteria was detected with Nile-red.[13] Recombinant pGEX as well as pGEX and P. aeruginosa (as the positive control) were investigated for PHA production. E. coli cells were spread on an LB plate supplemented with sodium-gluconate (w/v 0.5%), Nile-red (0.05 µg/ml), IPTG (0.1 mM) and ampicillin (100 µg/ml). P. aeruginosa 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 plates were incubated at 37°C for 24 hours. After that, plates which contained rpGEX revealed an orange color under UV light at 302 nm.


  Results Top


The phaC2 gene was ligated to the pTZ57R vector and for confirming the presence of the insert, double digestion with Eco R1 and Xho 1 restriction enzymes was performed. Gel electrophoresis of this digested products revealed that the expected band in 1770 bp was present. Subsequently, the phaC2 sequence was digested out and ligated into pGEX-5x-1 expression vector and the clones containing this insert were identified using PCR [Figure 1]. rpGEX also was digested with Xho I and Eco RI and the correct band was revealed by gel electrophoresis (1770 bp), so the phaC2 gene was correctly ligated to the vector [Figure 2].
Figure 1: Colony PCR of rpGEX. Presence of 1770 bp band indicated that this colony contained the recombinant plasmid (lane 2). Lane1: molecular weight marker

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Figure 2: rpGEX digestion with XhoI and Eco RI, the insert was detected as 1770 bp band (lane 2). Lane 1: molecular weight marker

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The functional expression of PHA synthase was assessed by Nile-red. The PHA enzyme was expressed by adding IPTG. As demonstrated in [Figure 3], both P. aeruginosa and E. coli containing rpGEX revealed an orange color under the UV light at 302 nm. So Nile-red plate assay revealed functional expression of PHA enzymes by recombinant Fad B deleted E. coli.
Figure 3: P. aeruginosa and E. coli containing rpGEX revealed an orange color under UV light

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The effects of incubation time and temperature, IPTG concentration and glucose concentration were analyzed by SDS-PAGE which revealed that the target 84-KD protein was expressed as is demonstrated in [Figure 4] and [Figure 5]. The maximum production of PHA synthase enzymes occurred when 1.5 mM IPTG and 1 mM glucose were used at 37°C for 2 hours.
Figure 4: Lane 1 is rpGEX with 0.5% glucose, lane 2 is rpGEX with 1% glucose, lane 3 is rpGEX with 2% glucose. Recombinant protein expression in these experiments was induced with 1.5% IPTG. Lane 4 is uninduced rpGEX, lane 5 is protein molecular weight marker, lane 6 is rpGEX induced at 25°C, lane 7 is rpGEX induced at 30°C, lane 8 is rpGEX induced at 37°C. Recombinant protein expression in these experiments was induced by 1.5% IPTG. As it is demonstrated, lanes 3 and 8 have sharper bands in comparison to others

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Figure 5: Lane 1 is uninduced rpGEX, lane 2 is protein molecular weight marker, lane 3 is rpGEX induced with 2 % IPTG for 2 hrs, lane 4 is rpGEX induced with 1% IPTG for 2 hrs, lane 5 is rpGEX induced with 1% IPTG for 3 hrs, lane 6 is rpGEX induced with 1% IPTG for 4 hrs ,lane 7 is rpGEX induced with 1.5% IPTG for 1 hrs, lane 8 is rpGEX induced with 1.5% IPTG for 2 hrs, lane 9 is rpGEX induced with 1.5% IPTG for 3 hours. As it is demonstrated in the figure lane 8 has a sharper band as compared to others

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  Discussion Top


Escherichia coli strains have been widely used for the expression of recombinant proteins. Fad B deleted E. coli cells have been the host organism of choice for the expression of certain enzymes because of their ability to block the oxidation of fatty acids and accumulate greater levels of PHA.[14]

In many studies, optimization of conditions for the expression of recombinant proteins has been performed. Wen and his coworkers investigated the effect of temperature and concentration of IPTG on the expression of PET-ILC-HIL-2-MGM. They found that maximum protein expression occurred when 0.3 mM IPTG was used at 42°C.[15] In another study, Qun et al. expressed two PHAC genes in E. coli and the effect of IPTG concentration on PHA monomer composition was evaluated.[16] In the present study we investigated four factors: The concentration of IPTG, temperature, glucose concentration and time of induction of the target protein. Our results show that the optimum conditions for the expression of the phaC2 gene are 1.5 mM IPTG and 1 mg glucose for 2 hrs at 37°C. In comparison with other studies, optimum incubation time for PHA synthase enzyme expression was shorter and IPTG concentration was greater than in those studies.

In a study conducted by Abedi et al., phaC1 and phaC2 genes were expressed in E. coli BL21(DE3).[17] Perivious studies showed that caltivating E. coli with fatty acids as sole carbon source resulting in accumulation higher amounts of PHA.[18] In the present study, we used a mutant strains of E. coli (Fad B deleted) in order to block the oxidation of fatty acids and accumulate greater levels of PHA. In agreement with our results, the functional expression of phaC1 in E. coli fad and fab mutants were reported.[19]

In summary, we obtained functional expression of the phaC2 gene and investigated various conditions that could influence the expression of protein to optimize production of PHA synthase enzymes. This would allow us to study PHA production in large quantities.

 
  References Top

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Lee SY, Chang HN. Production of Poly (hydroxyalkanoic acid). Adv Biochem Eng Biotechnol 1995;52:27-58.  Back to cited text no. 1
    
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Khanna S, Srivastava AK. Recent advances in microbial polyhydroxyalkanoates. Process Biochem 2005;40:607-20.  Back to cited text no. 2
    
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Braunegg G, Lefebvre G, Genser KF. Polyhydroxyalkanoates, biopolyesters from renewable resources: Physiological and engineering aspects. J Biotechnol 1998;65:127-61.  Back to cited text no. 3
    
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Ramsay J, Ramsay B. Polyhydroxyalkanoates, separation, purification and manufacturing methods. In: Flickinger MC, Drew SW, editors. Encyclopedia of Bioprocess Technology. United states: John Wiley and Sons; 1999. p. 2040-50.  Back to cited text no. 4
    
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Aldor IS, Keasling JD. Process design for microbial plastic factories: Methabolic engineering of polyhydroxyalkanoates. Curr Opin Biotechnol 2003;14:475-83.  Back to cited text no. 5
    
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Solaiman DK, Ashby RD. Genetic characterization of the poly (hydroxyalkanoate) Synthases of various Pseudomonas oleovorans strains. Curr Microbiol 2005;50:329-33.  Back to cited text no. 6
    
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Valentin HE, Broyles DL, Casagrande LA, Colburn SM, Creely WL, DeLaquil PA, et al. PHA production, from bacteria to plants. Int J Biol Macromol 1999;25:300-6.  Back to cited text no. 8
    
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Qi Q, Rehm BH, Steinbuchel A. Synthesis of poly (3-hydroxyalkanoates) in Escherichiacoli expressing the PHA synthase gene phac2 from Pseudomonas eaeruginosa. Comparison of phaC1 and phaC2. FEMS Microbiol Lett 1997;157:155-63.  Back to cited text no. 9
    
10.
Solaiman DK. PCR cloning of Pseudomonas resinovorans polyhydroxyalkanoate biosynthesis genes and expression in Escherichiacoli. Biotechnol Lett 2000;22:789-94.  Back to cited text no. 10
    
11.
Abedi D, Najafabadi AJ, Sadeghi HM, Vallian S. Cloning and partial sequencing of phaC1 and phaC2 genes encoding poly (3-hydroxyalkanoate) synthases from Pseudomonas aeruginosa PTCC1310. Biotechnology 2007;6:497-504.  Back to cited text no. 11
    
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Sambork J, Russell DW. Molecular cloning a laboratory Manual. 3rd ed. NewYork: Cold Spring Harbor Laboratory Press; 2001.  Back to cited text no. 12
    
13.
Serafin LS, Lemos PC, Levantesi C, Tandoi V, Santos H, Reis MA. Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating micro organisms. J Microbiol Methods 2002;51:1-18.  Back to cited text no. 13
    
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Terpe K. Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl MicrobiolBiotechnol 2006;72:211-22.  Back to cited text no. 14
    
15.
Wen Q, Ma L, Wang X. Culture condition optimization of engineered E. coli BL21/pET_11c/hIL_2_mGM_CSF. Nan Fang Yi Ke Da Wue Bao 2006;26:418-20,24.  Back to cited text no. 15
    
16.
Ren Q, Beilen JB, Sierro N, Zinn M, Kessler B, Witholt B. Expression of PHA polymerase genes of Pseudomonaseputida in Escherichia coli and its effect on PHA formation. Antonie Van Leeuwenhoek 2005;87:91-100.  Back to cited text no. 16
    
17.
Abedi D, Beheshti M, Najafabadi AJ, Mirmohammadsadeghi H, Akbari V. Optimization of the expression of genes encoding poly (3-hydroxyalkanoate) synthase from Pseudomonas aeruginosa PTCC1310 in Escherichiacoli. Avicenna J Med Biotechnol 2012;4:47-51.  Back to cited text no. 17
    
18.
Langenbach S, Rehm BH, Steinbuchel A. Functional expression of the PHA synthase gene phaC1 from Pseudomonas aeruginosa in Escherichiacoli results in poly (3-hydroxyalkanoate) synthesis. FEMS Microbiol Lett 1997;150:303-9.  Back to cited text no. 18
    
19.
Hein S, Paletta JR, Steinbuchel A. Cloning, characterization and comparison of the Pseudomonas medonica polyhydroxyalkanoate synthase phaC1 and phaC2. Appl Microbial Biotechnol 2002;58:229-36.  Back to cited text no. 19
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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