Cloning of acetyl-CoA acetyltransferase gene from Halomonas elongata BK-AG 18 and in silico analysis of its gene product

Polyhydroxybutyrate (PHB) is a biodegradable polymer that can be used as a subs tute for petrochemical plas cs. Bacteria accumulate PHB in their cells as carbon and energy reserves because of unbalanced growth condi ons. This study aimed to amplify phbA from the chromosomal DNA of Halomonas elongata BK-AG18, a PHB-producing bacterium that was previously isolated from the Bledug Kuwu mud crater of Central Java, Indonesia. The obtained phbA amplicon was 1176 bp. This fragment was cloned into a pGEM-T Easy cloning vector and used to transform Eschericia coli TOP10. The recombinant colonies were selected using blue-white screening, confirmed by size screening, reconfirmed by re-PCR, and sequenced. When puta ve phbA sequences were aligned with H. elongataDSM2581 chromosome using BLASTN, this sequence showed 99% iden ty. The deduced amino acid sequences of this clone showed 100% iden ty to PhbA of H. elongata DSM2581, sugges ng that the obtained cloned fragment is a phbA gene. The 3D structure predicted by I-TASSER showed that PhbA of H. elongata BK-AG18 had a high similarity to the acetyl CoA acetyltransferase structure of Ralstonia eutropha H16. PhbA of H. elongata BK-AG18 possesses three cataly c residues, namely Cys88, His348, and Cys378.

Plastic materials are widely used for various human activities.This massive plastic usage has caused serious environmental problems because of their non-biodegradability (Singh 2014).Common plastics are manufactured from non-renewable fossil fuels and their increasing demand will speed up fossil fuel depletion (Colwill et al. 2012).Therefore, the alternative and more environmentally friendly plastics are needed.Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can be used as plastics, synthesized and accumulated by bacteria as intracellular carbon storage granules in response to nutrient limitations (Dietrich et al. 2013).Polyhydroxybutyrate (PHB) is the most common type of PHA that accumulating in bacteria and archaeal cells (Muangsuwan et al. 2015).
The biosynthetic pathway of PHB in bacteria includes three reactions catalyzed by three different enzymes, namely acetyl-CoA acetyltransferase, beta-ketoacyl-ACP reductase, and PHB polymerase.The acetyl-CoA acetyltransferase, encoded by phbA gene, catalyzes the condensation of acetyl-CoA molecules to produce acetoacetyl-CoA molecule.

Bacterial strain, plasmid, and chemicals
H. elongata BK-AG18 local strain was used as a source of chromosomal DNA.E. coli TOP10 was used as the host strain for gene cloning.These bacteria were obtained from the Biochemistry Laboratory of the Institut Teknologi Bandung.The pGEM-T Easy (Promega) was used as cloning vector.Oligonucleotide primers were ordered from Macrogen, South Korea.KAPA Taq Ready Mix PCR Kit, T4 DNA ligase and DNA ladder were purchased from Promega.Sodium chloride from Amresco and yeast extract, tryptone, bacto agar from Deben Diagnostics Ltd. were used as Luria-Bertani medium's ingredients.Ampicillin and tetracyclin from Bioline were used as antibiotics to screen a recombinant clone.Isopropyl- β-D-galactopyranoside (IPTG) and 5-bromo-4-chloro-3indoyl-β-D-galactoside from Promega were also used to screen positive recombinant clones.

Primer design
The phbA primers were designed based on phbA full nucleotide sequences from H. elongata DSM2581 in Gen-Bank database by the authors.Some forward and reverse primer candidates were designed and the parameters of % GC, Tm, the absence of hairpin, dimer, and loop structure were analyzed using Oligo Analyzer.Fast PCR was performed to see the mispriming probability.

Culturing condi on, genomic DNA isola on and amplifica on of phbA gene
H. elongata BK-AG18 was grown in a modified high glucose medium (HM) as described by Hertadi et al. (2017).Chromosomal DNA of H. elongata BK-AG18 was isolated using Klijn method (Klijn et al. 1991).Amplification of phbA gene was carried out by PCR by the folowing primers: phbAF(5'-ATGTCAGAAGTGGTGATCGTC-3') as forward primer and phbAR (5'-TCAGCGCTCGATGGCCAG-3') as re-verse primer.PCR were conducted on an automatic thermal cycler (BIORAD) for one cycle predenaturation at 95°C for 4 min, 34 cycles of denaturation at 94°C for 30 s, annealing at 61°C for 30 s, and extension at 72°C for 90 s.The reaction was completed by final extension at 72°C for 5 min.The phbA amplicon was confirmed by electrophoresis on 1% (w/v) agarose gel.

Cloning and recombinant clone selec on
The amplicon of phbA gene was ligated into pGEM-T Easy using T4 DNA ligase and transformed into competent E.coli TOP10 cells using heat shock method (Sambrook and Russell 2001).The transformants were bluewhite selected on Luria Bertani solid medium supplemented with100 µg/ml ampicillin, 20 ug/ml X-gal, and 0.1 M IPTG.The recombinant plasmids were confirmed by size screening and re-PCR.Recombinant plasmids DNA were isolated from the transformants with Geneaid High Speed Plasmid Mini Kit (Geneaid Biotech Ltd., Taiwan).

Sequencing of phbA gene
Determination of phbA nucleotide sequences was performed by 1stBASE, Malaysia with dye terminator dideoxy Sanger Method using universal primer of T7 and SP6 promoter.

Cataly c residues and three dimensional structure predic on of PhbA
PhbA amino acid sequence was deduced from phbA gene sequences.Prediction of PhbA catalytic residues was performed using Sequence Annotated by Structure (SAS) and Catalytic Site Atlas (CSA).On the other hand, the prediction of PhbA 3D structure was conducted using I-TASSER.

Chromosome isola on and gene amplifica on
The chromosome of H. elongata BK-AG18 was successfully isolated.Using this chromosome and the designed  primers, the performed PCR reaction has produced a phbA gene as shown by a single band of the amplicon on agarose gel electrophoresis (Figure 1).

Cloning and recombinant clone selec on
The 1.2 kb obtained amplicon was inserted into the pGEM-T Easy vector and transformed into E.coli TOP10.The transformants will survive in LB-ampicillin medium, and the recombinant clones will appear as white colonies.On the other hand, the transformants with plasmid without insert will appear as blue colonies (Figure 2a).The positive recombinant clones were confirmed by size screening (Figure 2b) and re-PCR (Figure 2c).As presented in Figure 2b and 2c, the recombinant plasmid has larger size compared to the non-recombinant plasmid and the re-PCR produces 1.2 kb fragment as expected.

Sequencing of phbA gene in pGEM-phbA
The sequence of 1176 bp phbA gene and its deduced amino acids is shown in Figure 3.The BLASTN analysis indicated that this phbA gene showed 99% identity with the whole genome of H. elongata DSM2581 (Figure 4a).The BLASTP analysis on the deduced amino acid of this gene, PhbA, showed 100% identity with acetyl-CoA acetyltrans-ferase from H. elongata DSM2581 (Figure 4b).Literature studies stated that PhbA super family contains three catalytic residues: Cys89, His348, and Cys378 as shown in Zooglea ramigera (Modis and Wierenga 2000).The PhbA-deduced amino acids from H. elongata BK-AG18 also has Cys88, His348, and Cys378, confirming that the isolated gene is phbA.The PhbA 3D structure predicted by I-TASSER showed 68.72% identity to acetyl-CoA acetyltransferase structure from Ralstonia eutropha H16 (RePhbA).The modelling 3D structure of PhbA predicted by I-TASSER with its catalytic residues can be seen in Figure 5.
In RePhbA, Cys89 has a function as covalent catalyst, His349 as general base, while Cys378 was a second nucleophile (Kim and Kim 2014).The superposition showed that Cys88, His348 and Cys379 residues in our PhbA structure have the same positions with Cys89, His349 and Cys378 from RePhbA.

Conclusions
The designed primers had successfully amplified phbA gene that code acetyl-CoA acetyltransferase from H. elongata BK-AG18 chromosomal DNA.This phbA gene showed 99% identity to the whole genome of H. elongata DSM2581.The deduced amino acid sequences of this gene showed 100% identity with acetyl-CoA acetyltransferase of H. elongata DSM2581.The three amino acid on PhbA catalytic sites are Cys88, His348, and Cys378 which is similar to the PhbA catalytic sites of Zooglea ramigera, confirming that the phbA gene isolated is coding for PhbA protein superfamily.

FIGURE 1
FIGURE 1 Electropherogram of PCR result.M: Generuler 1 kb DNA Ladder, 1: phbA amplicon.The obtained amplicon size was approximately 1.2kb which is similar to the size of the phbA gene from H. elongata DSM2581 from GenBank.

FIGURE 3
FIGURE 3The nucleo de sequences of phbA gene and its deduced amino acid sequences.