Detection and Expression of Biosynthetic Genes in Actinobacteria ...

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BERVANAKIS, G.Chapter 1: INTRODUCTIONIn this study a fragment from the KS gene was targeted to be amplified by PCR, as itis a common gene present in all modular PKS clusters (Figure 7). Amplification ofshort DNA fragments from modular PKS clusters has provided a accurate approach inmatching them to a PKS domain. Furthermore, it has been estimated that sequencing300 – 500 bases provides sufficient amino acid sequence identity to identify afragment as part of a modular PKS gene (Santi et al., 2000). The KS gene is highlyconserved containing between 60-75% identity and 75-84% similarity at the DNAsequence level over the whole domain in the erythromycin PKS and avermectin PKS(MacNeil et al., 1993). The KS gene contains a region that encodes a highlyconserved motif GPXXXXXTACSS which is required for the formation of a thioesterlinkage to the growing acyl chain (Motamedi et al., 1997). Another SMBG of type IPKSs which could be targeted includes the AT gene, which incorporates starter units.The AT gene contains 33-54% identity and 54-71 % similarity at the DNA sequencelevel over the whole domain between erythromycin PKS and avermectin PKS(MacNeil et al., 1993).An example of a typical type I PKS is that of erythromycin PKS, which is encodedby three genes, designated eryAI, eryAII, and eryAIII (Figure 7A). The three genesencode polypeptides that are over 3300 amino acids in length named DEBS(deoxyerythronolide B synthase). DEBS is composed of modules that containcombinations of KS, AT, KR, DH, ER, and ACP, as well as a thioesterase (TE) at theend of the DEBS3 module (Katz, 1997). The KS-AT-(DH-ER-KR)-ACP modularorganization is a common feature found in most type I PKS genes (Figure 7).Similarly to the DEBS, rapPKS consists of three multifunctional proteins designatedRAPS1 (8,563 amino acids), RAPS2 (10,222 amino acids) and RAPS3 (6,260).However these polypetides contain more than two modules each, as compared to onlytwo found in DEBS (Figure 7B).The loading module consists of three domains. Thefirst domain is Lig (ligase), the second is a ER and thirdly an ACP. The first domaincatalyzes the conversion of a carboxylic acid to an active acyl derivative for thetransfer to the ACP. The final product is attached to the ACP as a thioester for thetransfer to the KS domain for the first chain extension module. Chain termination isperformed by the pipecolate incorporating enzyme encoded by the gene rapP(Schwecke et al., 1995)._____________________________________________________________________15
BERVANAKIS, G.Chapter 1: INTRODUCTIONA) eryAI (DEBS1) eryAII (DEBS2) eryAIII (DEBS3)B) rapA (RAPS1)rapB (RAPS2)rapC (RAPS3)Figure 7. Modular organization of two polyketide synthases. (A) Gene cluster oferythromycin consisting of three genes eryAI, eryAII, eryAIII, which encode 6-deoxyerythronolide-B synthase [DEBS] (B) Gene cluster of rapamycin containing thethree genes rapA, rapB and rapC which encode the rapamycin polyketide synthase[RAPS] (adapted from Katz, 1997).1.3.1.4 Biosynthetic Gene Clusters of Type II Polyketide SynthasesA characteristic feature of type II PKS genes is their high amino acid sequencehomology and conserved gene organization (Figure 9). As part of the geneorganization three core open reading frames (ORFs) of requisite genes known as the“minimal” PKS need to be expressed for in vivo aromatic polyketide biosynthesis tooccur (Figure 8A). The minimal PKS include the bifunctional domainketosynthase/acyltransferase (KS/AT), chain length factor (CLF) and acyl carrierprotein (ACP) (McDaniel et al, 1994). Additional ORFs encoding ketoreductase_____________________________________________________________________16
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BERVANAKIS, G. APPENDIX 1Appendix 1
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