Stage III Project - Grant Elmes

SGM Summer Student - Rosie Stentiford

Supervisor Dr Russell Cox
Second Assesor Professor T. J. Simpson

Background

Polyketides are a class of naturally occurring molecules which often have important biological properties. For example oxytetracycline and erythromycicn are important commercial antibiotics. Generally theses compounds are too complex for total synthesis to be a viable way of obtaining large quantities for commercial exploitation. Fermentation of bacterial or fungal strains followed by extraction and purification are preferered by pharmaceutical companies.

Genes coding for polyketide production have been found in many fungal and bacterial species. However it is difficult to confirm that a particular set of genes is involved in the production of a particular polyketide because many organisms posess more than a single set of polyketide genes. In order to identify what a particular polyketide synthase gene produces this project will investigate the possibility of inserting individual genes into a bacterium which is known to produce no polyketides (Streptomyces coelicolor CH999). The genes are expected to be translated into proteins in the usual way and the proteins should then synthesise the relevent polyketide. Extraction and identification of the polyketide will then be performed. This is the first step in an important long-term effort to produce novel antibiotics through genetic modification of PKS genes.

Programme

Streptomyces species have been shown recently to be suitable hosts for the expression of heterologous genes (e.g from fungi and other bacteria). In particular, the expression of recombinant fungal polyketide synthase (PKS) genes has been demonstrated. PKS enzymes require post-translational modification in order to affix a 4'-phosphopantetheine prosthetic group to the acyl carrier protein sub-domain, catalysed by so-called holo-ACP synthase. Expression of 6-methyl salicylic acid synthase (6MSAS) by S. coelicolor CH999 leads to high production levels of the metabolite, indicating correct post translational modification by this bacterium.

This project will assess expression of a recombinant putative norsolorinic acid synthase gene from the fungus Aspergillus parasiticus and putative minimal actinorhodin genes from the bacteria Streptomyces coelicolor. The bacteria S. coelicolor CH999 which produces no polyketides will be used as the host organism (S. lividans has been used successfully for 6MSAS in this laboratory but results are confused by background natural polyketide production). Standard microbiological methods to grow a number of strains of S. coelicolor CH999 containing expression vectors for the putative PKS genes, as well as similar control strains carrying a 6MSA expression vector. Extraction of the culture broths will be followed by HPLC and NMR analysis. Synthetic standards of 6MSA, norsolorinic acid and SEK4/4b (produced by the act genes) are available for comparative purposes. The relevent genetic constructs have been synthesised and are ready for expression.

References

. D. J. Bedford, E. Schweizer, D. A. Hopwood and C. Khosla, J. Bacteriol., 1995, 177, 4544-4548.

. R. J. Cox, T. S. Hitchman, K. J. Byrom, I. S. C. Findlow, J. A. Tanner, J. Crosby and T. J. Simpson, FEBS Letters, 1997, 405, 267-272.

. R. A. Stentiford, Summmer Project 1997.

. Fungal PKS by Dr Colin Lazarus Dept. Botany, bacterial PKS by Dr Anne-Lise Matharu and Dr R. J. Cox, School of Chemistry.