Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 3: RESULTSgenes that constitute the minimal PKS required to create the carbon skeleton of thepolyketide molecule (McDaniel et al., 1994). The PCR primers were designedaccording to the consensus sequences of 29 amino acid and 26 nucleic acidsequences derived from multiple sequence alignments of actinobacterial KS α genesavailable in the GenBank/EMBL databases at that time (Figures 20 and 21). Fromthese 26 KS α nucleic acid sequences 24 were from Streptomyces spp, and two fromnon-streptomyces species these were from a Actinomadura spp. and from aKibdelosporangium spp. The primers designed were used to amplify a 0.47 kbproduct corresponding to an internal fragment from within the KS α gene (Figure 19).The design strategy employed embedding a 19 base pair forward primer (act04f) inthe active site of the KS α gene at nucleic acid position 595-614 (numbers correspondto Streptomyces coelicolor GenBank accession number: X63449), the corresponding21 base pair reverse primer (act06r) was positioned at 1048-1069 flanking the KS αgene (Figures 20 and 21).Minimal Polyketide Synthase Gene Organizationβ-ketoacyl synthase KS α (1272 bp) β-ketoacyl synthase KS β (1227 bp)KSATKS AT ACPact04fact06rPCR product = 0.47 kbKS = ketosynthase AT = acyltransferase ACP = acyl carrier proteinFigure 19. Illustration depicting the conserved minimal PKS gene organization inType II PKS. Design strategy used to obtain a internal fragment within the KS α genefrom uncharacterised environmental actinobacterial isolates.Evaluation of the amino acid alignment of the act04f primer sequence, indicated thatthe 5’ region is variable (Figure 20 & Table 33). The act04f primer has the sequenceMVSTGC, in the amino acid sequence alignment with other actinobacteria it isevident that two degenerate codons occur at positions 1 and 4. Codon 4 is the criticalcodon at the 3’ end however three possible amino acids occur at that position acrossdifferent actinobacterial species these include Aspartic Acid (GAT/C), Alanine(GCA/T/C/G) and Tyrosine (TAT/C). In this case a degenerate primer covering allthese possible combinations would have been more appropriate. Partial nucleotide___________________________________________________________________________________71