title: Molecular cloning, characterization and expression of a gene encoding phosphoketolase from Termitomyces clypeatus.
abstract: A phosphoketolase (pk) gene from the fungus Termitomyces clypeatus (TC) was cloned and partially characterized. Oligonucleotide primers specific for the phosphoketolase gene (pk) were designed from the regions of homologies found in the primary structure of the enzyme from other fungal sources related to TC, using multiple sequence alignment technique. The cDNA of partial lengths were amplified, cloned and sequenced in three parts by 3' and 5' RACE and RT-PCR using these oligonucleotide primers. The full length ds cDNA was constructed next by joining these three partial cDNA sequences having appropriate overlapping regions using Overlap Extension PCR technique. The constructed full length cDNA exhibited an open reading frame of 2487 bases and 5' and 3' UTRs. The deduced amino acid sequence, which is of 828 amino acids, when analyzed with NCBI BLAST, showed high similarities with the phosphoketolase enzyme (Pk) superfamily with expected domains. The part of the TC genomic DNA comprising of the pk gene was also amplified, cloned and sequenced and was found to contain two introns of 68 and 74 bases that interrupt the pk reading frame. The coding region of pk cDNA was subcloned in pKM260 expression vector in correct frame and the protein was expressed in Escherichia coli BL21 (DE3) transformed with this recombinant expression plasmid. The recombinant protein purified by His-tag affinity chromatography indicated the presence of a protein of the expected size. In vivo expression studies of the gene in presence of different carbon sources indicated synthesis of Pk specific mRNA, as expected. Phylogenetic studies revealed a common ancestry of the fungal and bacterial Pk. The TC is known to secrete several industrially important enzymes involved in carbohydrate metabolism. However, the presence of Pk, a key enzyme in pentose metabolism, has not been demonstrated conclusively in this organism. Cloning, sequencing and expression study of this gene establishes the functioning of this gene in T. clypeatus. The Pk from TC is a new source for commercial exploitation.
authors.affiliation: Department of Biotechnology, Visva-Bharati University, Santiniketan 731235 West Bengal, India.
authors.forename: Prabuddha
authors.initials: P
authors.lastname: Sarkar
authors.affiliation: Department of Biotechnology, Visva-Bharati University, Santiniketan 731235 West Bengal, India. Electronic address: amit.roy@visva-bharati.ac.in.
authors.forename: Amit
authors.initials: A
authors.lastname: Roy
mesh_terms: D000446:Aldehyde-Lyases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D005800:Genes, Fungal; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D055434:Termitomyces / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development
chemical_list: D018076:DNA, Complementary; D004271:DNA, Fungal; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C022367:phosphoketolase
keywords: Fungal phosphoketolase; Intron; KF690709; Overlap Extension PCR technique; Termitomyces clypeatus