Detection and Expression of Biosynthetic Genes in Actinobacteria ...

BERVANAKIS, G.Chapter 3: RESULTS3.1.3 PCR Screening Assay for dTDP-Glucose SynthaseSecondary metabolites such as aminoglycosides often contain deoxygenated hexosesugar components that are essential for biological activity (Liu & Thorson, 1994). Acommon early enzyme dTDP-glucose synthase involved in the biosynthesis of alltypes of 6-deoxyhexose was chosen to design PCR primers for screening assays. ThePCR primers were designed according to the consensus sequences derived frommultiple sequence alignment of 15 actinobacterial dTDP-glucose synthase genesequences available in the GenBank and EMBL databases. From these 15 sequences,13 were from Streptomyces spp., and two from non-streptomyces these included 1from a Micromonospora sp. and 1 from an Actinoplanes sp (Table 40).Table 40: dTDP-glucose synthase genes with their respective nucleic acid and aminoacid sequences used in the design of the strD01f and strD01r PCR primers.Class / Gene Secondary Metabolite /(SMBG)Deoxyhexose(dTDP –glucosesynthase)ActinobacteriaAmino AcidAccessionNumberNucleicAcidAccessionNumberAcarbose (acbA) Actinoplanes sp. SE50/110 CAA77210 Y18523Fortimicin (fotD) Micromonospora olivasterospora AAD31892 AF144041Oleandomycin (oleS) Streptomyces antibioticus AAD55453 AF05579Mithramycin (mtmD) Streptomyces argillaceus CAA71846 Y10907Oleandomycin (orf8) Streptomyces antibioticus Tü99 AAF59934 AF237894Blensomycin (blmD) Streptomyces bluensis AAD28517 AF126354Tylosin (urdG) Streptomyces fradiae AAA21343 U08223Streptomycin (strD) Streptomyces glaucescens GLA.0 CAA07386 AJ006985Streptomycin (strD) Streptomyces griseus CAA68514 Y00459Kanamycin (knaD) Streptomyces kanamyceticus AAD31891 AF144040Granaticin (gra-orf16) Streptomyces violaceoruber AAA99940 L37334Nogalamycin (snogJ) Streptomyces nogalater AAF01820 AF187532Paromomycin (prmD) Streptomyces rimosus f.AAD31893 AF144042paromomycinusSpectinomycin (spcD) Streptomyces spectabilis AAD31796 AF128272Avilamycin (aviD)Streptomyces viridochromogenes CAA72714 Y11985Tü57The 24 base pair forward primer (strD01f) at position 326 – 350 nucleotides (numberscorrespond to S. griseus GenBank accession number: Y00459) and the 20 base pairreverse primer (strD01r) at position 677 – 697 nucleotides (Figure 26). FASTAsequence similarity searches of the GenBank/EMBL with the designed primersrevealed matching dTDP-glucose synthase gene sequences from other actinobacterialspecies, ranging from 87 – 100 %. PCR screening experiments using the designedprimers amplified the predicted 0.37 kb product size in the two actinobacterial species_____________________________________________________________________88