Expression of Type III PKS genes in S. coelicolor

Anyarat Thanapipatsiri1,2, Juan-Pablo Gomez-Escribano2, Jan Claesen2,

Mervyn Bibb2 and Arinthip Thamchaipenet1

1Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand; 2Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom

Type III polyketide synthases (PKSs) are small homodimeric proteins found in plants, fungi and bacteria1. Bacterial Type III PKSs are responsible for assembling Coenzyme A-linked acetate-derived substrates to give different length Type III polyketides. The first bacterial Type III PKS, RppA, found in S. griseus, catalyses the formation of 1,3,6,8 tetrahydroxynaphthalene (THN) from five molecules of malonyl-CoA2

At this stage, S. coelicolor M1152 [∆act ∆red ∆cpk ∆cda rpoB (C1298T)], a derivative of strain M145 from which four antibiotic biosynthetic gene clusters have been deleted and into which a mutation has been introduced that pleiotropically enhances the level of secondary metabolite production, was successfully engineered by PCR targeting to remove all three native Type III PKS genes (SCO1206, SCO7221 and SCO76713) providing a clean background for heterologous expression. SCO7221 and SCO7671 were sequentially in-frame deleted from S. coelicolor M1152 leaving a 81 bp scar sequence (unmarked). An apramycin resistance cassette was then introduced to replace SCO1206 to make the triple mutant. This triple mutant constitutes a Type III PKS “superhost”.

To test the efficacy of the expression vector, SCO7221, encodes germicidins production (Song et al., 20064), was cloned into a multi-copy constitutive expression vector pIJ86 (AprR, ermE*p) and the resulting construct was introduced into the single SCO7221 deletion mutant. HPLC analysis of the SCO7221 overexpression strain, the SCO7221 deletion mutant and the parental strain M1152 confirmed high levels of germicidins production in the overexpression strain.

Type III PKSs of actinobacteria available in Genbank and the John Innes Centre (JIC) genome databases were analysed bioinformatically and their evolutionary relationships determined. The comparison revealed that Type III PKSs can be classified into nine subgroups. The 14 genes (both characterised and uncharacterised) from those nine subgroups were selected for cloning and expression. So far, 11 genes, from 6 different subgroups, have been cloned into the expression vector pIJ86-Kan (KanR, ermE*p) and introduced into the Type III PKS “superhost”. The recombinant strains will then be analysed by HPLC and LC-MS.

References

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