Neveen M. El-Metwally, Abd El-Nasser A. Khattab, Mona S. Shafei, Reda F. Allam, Yasser M. Ragab, Magdy A. Amin
Neveen M. El-Metwally1*, Abd El-Nasser A. Khattab2, Mona S. Shafei1, Reda F. Allam1, Yasser M. Ragab3, Magdy A. Amin3
1Chemistry of Natural and Microbial Products Department, National Research Centre, Dokki, Giza 12622, Egypt.
2Genetics and Cytology Department, National Research Centre, Dokki, Giza 12622, Egypt.
3Microbiology and Immunology Department, Faculty of Pharmacy, Cairo University.
Volume - 14,
Issue - 6,
Year - 2021
Polyhydroxybutyrate (PHB) is the most known degradable biopolymer, produced by some genera of bacteria under unfavorable growth conditions. Isolation and cloning of acetoacetyl-CoA reductase (phbB) and polyhydroxybutyrate synthase (phbC) genes from local isolate previously identified as Bacillus aryabhattai 6N-NRC (GenBank accession no. MH997667.1) was achieved. Suitable primers designed for the phbB and phbC PCR approach were used to clone the phbB and phbC genes. The phbB and phbC genes were successfully isolated, cloned and the PCR amplicon 744 bp and 1089 bp corresponding to phbB and phbC genes were identified, cloned with the pET-29a (+) carrying the phbB and phbC genes, transformed and expressed in Escherichia coli BL21. The amplification of the phbB and phbC genes using specific primers of pET-29a (+) plasmid was performed. The open reading frame of phbB sequence was found to be 99.06% identical to the sequence of acetoacetyl-CoA reductase of B. aryabhattai (GenBank accession no. CP024035.1), while the open reading frame of phbC sequence was found to be 87.18% identical to the sequence of polyhydroxybutyrate synthase of B. aryabhattai (Gen Bank accession no. CP024035.1) after DNA sequencing. The analysis of the recombinant proteins from E. coli BL21 recombinant colony by tricine-polyacrylamide gel electrophoresis clarified that the expressed phbB and phbC genes in E. coli BL21 strain showed distinct bands of intensity 26.3 KD and 37.5 KD, respectively.
Cite this article:
Neveen M. El-Metwally, Abd El-Nasser A. Khattab, Mona S. Shafei, Reda F. Allam, Yasser M. Ragab, Magdy A. Amin. Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli. Research Journal of Pharmacy and Technology. 2021; 14(6):3299-6. doi: 10.52711/0974-360X.2021.00574
Neveen M. El-Metwally, Abd El-Nasser A. Khattab, Mona S. Shafei, Reda F. Allam, Yasser M. Ragab, Magdy A. Amin. Cloning of Acetoacetyl-CoA reductase and Polyhydroxybutyrate synthase genes from the local isolate Bacillus aryabhattai 6N-NRC into Escherichia coli. Research Journal of Pharmacy and Technology. 2021; 14(6):3299-6. doi: 10.52711/0974-360X.2021.00574 Available on: https://rjptonline.org/AbstractView.aspx?PID=2021-14-6-64
1. Eerkes-Medrano D, Thompson RC, Aldridge DC. Microplastics in freshwater systems: a review of the emerging threats, identification of knowledge gaps and prioritisation of research needs. Water Research. 2015; 75: 63-82.
2. Jain R, Tiwari A. Biosynthesis of planet friendly bioplastics using renewable carbon source. Journal of Environmental Health Science and Engineering. 2015; 13(1): 1-5.
3. Lusher AL, Burke A, O’Connor I, Officer R. Microplastic pollution in the Northeast Atlantic Ocean: validated and opportunistic sampling. Marine Pollution Bulletin. 2014; 88(1-2): 325-333.
4. Pillai AB, Kumar AJ, Kumarapillai H. Enhanced production of poly (3-hydroxybutyrate) in recombinant Escherichia coli and EDTA–microwave-assisted cell lysis for polymer recovery. AMB Express. 2018; 8(1): 142.
5. Hang X, Zhang G, Wang G, Zhao X, Chen GQ. PCR cloning of polyhydroxyalkanoate biosynthesis genes from Burkholderia caryophylli and their functional expression in recombinant Escherichia coli. FEMS Microbiology Letters. 2002; 210(1): 49-54.
6. Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate (PHB) biosynthesis in Alcaligenes eutrophus H16. Identification and characterization of the PHB polymerase gene (phbC). Journal of Biological Chemistry. 1989; 264(26): 15298-15303.
7. Peoples OP, Sinskey AJ. Poly-beta-hydroxybutyrate biosynthesis in Alcaligenes eutrophus H16. Characterization of the genes encoding beta-ketothiolase and acetoacetyl-CoA reductase. Journal of Biological Chemistry. 1989; 264(26): 15293-15297.
8. Steinbüchel A, Schlegel HG. Physiology and molecular genetics of poly (β‐hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus. Molecular Microbiology. 1991; 5(3): 535-542.
9. Rehm BH. Polyester synthases: natural catalysts for plastics. Biochemical Journal. 2003; 376(1): 15-33.
10. Schubert P, Steinbüchel A, Schlegel HG. Cloning of the Alcaligenes eutrophus genes for synthesis of poly-beta-hydroxybutyric acid (PHB) and synthesis of PHB in Escherichia coli. Journal of Bacteriology. 1988; 170(12): 5837-5847.
11. Slater SC, Voige WH, Dennis DE. Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway. Journal of Bacteriology. 1988; 170(10): 4431-4436.
12. Liebergesell M, Steinbüchel A. Cloning and nucleotide sequences of genes relevant for biosynthesis of poly (3‐hydroxybutyric acid) in Chromatium vinosum strain D. European Journal of Biochemistry. 1992; 209(1): 135-150.
13. Schembri MA, Bayly RC, Davies JK. Cloning and analysis of the polyhydroxyalkanoic acid synthase gene from an Acinetobacter sp.: evidence that the gene is both plasmid and chromosomally located. FEMS Microbiology Letters. 1994; 118(1-2): 145-152.
14. Liebergesell M, Steinbüchel A. Cloning and molecular analysis of the poly (3-hydroxybutyric acid) biosynthetic genes of Thiocystis violacea. Applied Microbiology and Biotechnology. 1993; 38(4): 493-501.
15. Hustede E, Steinbüchel A. Characterization of the polyhydroxyalkanoate synthase gene locus of Rhodobacter sphaeroides. Biotechnology Letters. 1993; 15(7): 709-714.
16. Valentin HE, Steinbüchel A. Cloning and characterization of the Methylobacterium extorquens polyhydroxyalkanoic-acid-synthase structural gene. Applied Microbiology and Biotechnology. 1993; 39(3): 309-317.
17. Pieper U, Steinbüchel A. Identification, cloning and sequence analysis of the poly (3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber. FEMS Microbiology Letters. 1992; 96(1): 73-79.
18. El Rabey HA, Albureikan MO, Aly MM, Kabli SA, Schneider K, Nolke G. Isolation, cloning and sequencing of poly 3-hydroxybutyrate synthesis genes from local strain of Bacillus cereus mm7 and expressing them in E. coli. Journal of Investigative Genomics. 2017; 4(1): 7-14.
19. Lin JH, Lee MC, Sue YS, Liu YC, Li SY. Cloning of phaCAB genes from thermophilic Caldimonas manganoxidans in Escherichia coli for poly (3-hydroxybutyrate) (PHB) production. Applied Microbiology and Biotechnology. 2017; 101: 6419-6430.
20. Nayak DS, Singh B. Identification and isolation of acetoacetyl-CoA reducatase (phbB) gene involved in poly (β-hydroxybutyrate) biosynthesis in Ralstonia eutropha (IMTCC 1954 strain). International Journal of Current Microbiology and Applied Sciences. 2018; 7: 349-355.
21. Wu H, Chen J, Chen GQ. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli. Applied Microbiology and Biotechnology. 2016; 100(23): 9907-9916.
22. Deepa S, Bhuvanehswari, P, Kanimozhi K, Panneerselvam A. Utilization of industrial waste for the production of poly- beta- hydroxy butyrate (Phb) by Alcaligenes eutrophus. Research Journal of Science and Technology. 2013; 5(4): 421-424.
23. Alagunachiyar M, Jeevitha R, Handique M, Kesavan S.. Isolation and screening of poly hydroxy butyrate (PHB) producing bacteria from Jeppiaar Salt Pane, Chennai. Research Journal of Pharmacy and Technology. 2015; 8(9): 1276-1280.
24. Ojha N, Das N. Optimization and characterization of polyhydroxyalkanoates and its copolymers synthesized by isolated yeasts. Research Journal of Pharmacy and Technology. 2017; 10(3): 861-868.
25. Reddy AR, Kumar RB, Prabhakar KV. Isolation and identification of polyhydroxybutyrate (PHB) producing bacteria from sewage sample. Research Journal of Pharmacy and Technology. (2017); 10(4): 1065-1069.
26. Jaiganesh R, Tattwaprakash C, Kumar GR, Iyappan S, Jaganathan MK. Characterization and optimization of process parameter for maximum Poly (− β-) hydroxyl butyrate production by bacteria isolated from the soil. Research Journal of Pharmacy and Technology. 2019; 12(10): 4926-4930.
27. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 1989: 2nd ed.
28. Sambrook J and Russell DW. Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA. 2001: 3rd ed.
29. Schägger H, Von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry. 1987; 166(2): 368-379.
30. Wu W, Welsh MJ. Rapid Coomassie blue staining and destaining of polyacrylamide gels. BioTechniques. 1996; 20(3), 386-388.
31. Hussain N, Jim EY, Muhassinv PM, Sherin CS. Cloning of PA-1L gene from Pseudomonas aeruginosa in E. coli. Research Journal of Engineering and Technology. 2013; 4(4): 187-190.
32. Mishra N, Khan RH, Dwivedi B, Mishra SK. Cloning of Gene Coding Glyceraldehyde 3-Phosphate Dehydrogenase. Asian Journal of Pharmacy and Technology. 2014; 4(4): 184-188.
33. Sadhukhan K, Dholakia D, Bhadalkar AL. Genomic Studies of Symbiodinium spp. to understand Coral Reef Resilience in India-A review. Research Journal of Science and Technology. 2018; 10(2): 157-164.
34. Hadi HW. Genetic study on Bacillus cereus. Research Journal of Pharmacy and Technology. 2019; 12(3): 1301-1306.
35. Sutar DA, Jain BU, Kondawar M. A Review Article on Study of Cloning. Asian Journal of Research in Pharmaceutical Science. 2019; 9(2): 148-150.
36. Li R, Zhang H, Qi Q. The production of polyhydroxyalkanoates in recombinant Escherichia coli. Bioresource Technology. 2007; 98(12): 2313-2320.
37. Li H, Zhou S, Johnson T, Vercruysse K, Lizhi O, Ranganathan P, Phambu N, Ropelewski AJ, Thannhauser TW. Genome structure of Bacillus cereus tsu1 and genes involved in cellulose degradation and Poly-3-Hydroxybutyrate synthesis. International Journal of Polymer Science. 2017; 2017: 1-12.
38. Rehman RA. Cloning and Sequencing of the Polyhydroxybutyrate (PHB) Gene from locally isolated Bacillus spp. (Doctoral dissertation, University of the Punjab, Quaid-e-Azam Campus, Lahore, Pakistan). 2012.
39. Galehdari H, Alaei S, Mirzaei M. Cloning of poly (3-Hydroxybutyrate) synthesis genes from Azotobacter vinelandii into Escherichia coli. Jundishapur Journal of Microbiology. 2009; 2: 31-35.
40. Li ZJ, Cai L, Wu Q, Chen GQ. Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly (3-hydroxybutyrate) production. Applied Microbiology and Biotechnology. 2009; 83(5): 939-947.
41. Chien CC, Hong CC, Soo PC, Wei YH, Chen SY, Cheng ML, Sun YM. Functional expression of phaCAB genes from Cupriavidus taiwanensis strain 184 in Escherichia coli for polyhydroxybutyrate production. Applied Biochemistry and Biotechnology. 2010; 162(8): 2355-2364.
42. Clemente T, Shah D, Tran M, Stark D, Padgette S, Dennis D, Brückener K, Steinbüchel A, Mitsky T. Sequence of PHA synthase gene from two strains of Rhodospirillum rubrum and in vivo substrate specificity of four PHA synthases across two heterologous expression systems. Applied Microbiology and Biotechnology. 2000; 53(4): 420-429.
43. Desetty RD, Mahajan VS, Khan BM, Rawal SK. Isolation and heterologous expression of PHA synthesising genes from Bacillus thuringiensis R1. World Journal of Microbiology and Biotechnology. 2008; 24(9): 1769-1774.
44. Tomizawa S, Hyakutake M, Saito Y, Agus J, Mizuno K, Abe H, Tsuge T. Molecular weight change of polyhydroxyalkanoate (PHA) caused by the PhaC subunit of PHA synthase from Bacillus cereus YB-4 in recombinant Escherichia coli. Biomacromolecules. 2011; 12(7): 2660-2666.
45. Tanaka T, Shima Y, Ogawa N, Nagayama K, Yoshida T, Ohmachi, T. Expression, identification and purification of Dictyostelium acetoacetyl-CoA thiolase expressed in Escherichia coli. International Journal of Biological Sciences. 2011; 7(1): 9.-17.
46. Shi H, Kyuwa K, Takasu M, Shimizu, K. Temperature-induced expression of phb genes in Escherichia coli and the effect of temperature patterns on the production of poly-3-hydroxybutyrate. Journal of Bioscience and Bioengineering. 2001; 91(1): 21-26.