Isolation, Cloning and Sequencing of Poly 3-Hydroxybutyrate Synthesis Genes from Local Strain of Bacillus cereus mm7 and Expression them in E. coli

Abstract

Poly 3-hydroxybutyrate (PHB) is the most known degradable biopolymers, produced by some genera of bacteria under unfavorable growth conditions. Isolation, cloning, and expression of PHB genes from PHB producing Bacillus cereus was achieved. A highest Poly 3-hydroxybutyrate producer bacterium was selected and identified as Bacillus cereus MM7, based on morphological and physiological characters in addition to 16S rRNA gene sequences. The four genes of Bacillus cereus MM7 that involved in Poly 3-hydroxybutyrate production were successfully amplified using specific primers which were designed depending on the sequence alignment of the known PHA biosynthetic genes from Bacillus cereus, cloned in E. coli DH5α and completely sequenced. Moreover, the expression of the four genes responsible for PHA biosynthetic in E. coli XL1-Blue and E coli MJ 109 was assayed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis. In the current study Bacillus cereus was able to synthesize PHB. PHB genes were isolated, cloned and expressed in E. coli DH5α. The PHB encoding proteins were analyzed in SDS-PAGE and the PHB corresponding protein bands were analyzed using mass spectrometry to nominate their amino acid sequence. Keywords: Pre cultureThe pre culture was prepared in 250 ml Erlenmeyer flasks containing 50 ml of nutrient broth [16]. Each flask was inoculated with a loop of the tested cultures and incubated in a 130 rpm speed rotary shaker at 30˚C for 24h.

Enhanced production of poly(3-hydroxybutyrate) in recombinant Escherichia coli and EDTA–microwave-assisted cell lysis for polymer recovery

“…In another study, Tomizawa et al ( 2011 ) have engineered a recombinant construct harbouring phaR and phaC from Bacillus cereus complemented with phaA and phaB from R. eutropha to yield 7.44 g/L of PHB. Recently, El Rabey et al ( 2017 ) have reported the construction of a recombinant system exclusively using the PHB synthesis genetic background from B. cereus , but PHB accumulation was not mentioned. A few other studies on PHB production by recombinant E. coli incorporating genetic elements from Gram negative bacteria have reported a maximum yield of 0.21 g/L and 2.02 g/L (Arifin et al 2011 ; de Almeida et al 2010 ).…”