Project Associates/Assistants
Priti Saxena
Rashmi Tickoo
Fabian Davamani
M Vijayalakshmi
Saurabh Mittal

PhD Students
Omita Mehta
Yogyata
Pooja Arora

Collaborators
Debasisa Mohanty

Our laboratory is interested in genome-based approaches to identify and exploit the microbial metabolic pathways that are involved in the biosynthesis of various natural products. The present focus is to understand the importance of various polyketide synthase gene clusters from Mycobacterium tuberculosis.

The genome sequence of M. tuberculosis has revealed a remarkable array of genes that are homologous to polyketide synthases. Polyketide synthases (PKSs) are a class of enzymes that are involved in the biosynthesis of secondary metabolites such as erythromycin, rapamycin, tetracycline, lovastatin and resveratrol. Our study attempts to understand and exploit the role of polyketide synthases in the biology of M. tuberculosis.

The objectives of the studies proposed are (i) identification and biochemical analyses of enzymes that are involved in the biosynthesis of metabolites, (ii) isolation and characterization of PKS gene products by heterologous expression of these genes in Streptomyces coelicolor and Escherichia coli, (iii) characterization of molecular mechanisms mediating the crosstalk between various polyketide synthases (PKSs) and fatty acid synthases (FASs) in M. tuberculosis, and
(iv) genetic and/or chemical knock-out of PKS genes to synthesize novel polyketides and to study the effects of these changes on mycobacterial pathogenecity.

Sequence comparison and analyses of Mycobacterium tuberculosis

In collaboration with Dr Mohanty’s group, we have developed automated web-based software to identify various domains in PKS genes. Our analysis has identified several unusual PKS genes in terms of their domain organization and arrangement. Several of these modular PKS genes have domains split into two independent polypeptide chains. Since there are no previous reports of such broken PKS genes, we have carried out detailed analysis to rule out the possibility of genome sequencing errors. We are presently investigating into one such complex PKS gene cluster from M. tuberculosis.

Cloning and heterologous expression of unusual PKS cluster

This cluster consists of six different open reading frames of which pks7 is one complete module having all three reductive domains i.e. KR, DH and ER. pks8, in contrast, is a partial module, as the ACP domain is absent from this protein. Interestingly, pks17 complements the rest of the domains by providing KR and ACP domains. pks9 is similar to several chain initiation modules, where active site cysteine residue is mutated to glutamine. These four PKS genes are flanked on either side by two chalcone synthases i.e. pks10 and pks11. In order to identify the polyketide metabolite biosynthesized by this cluster, we are following two-pronged strategy. We are expressing several of these proteins in E. coli, which would facilitate an easy access to these proteins, and would also provide an opportunity to explore substrate specificity of these proteins. We have now cloned and hyper-expressed four of these proteins in E. coli. PKS17 and PKS9 proteins have been expressed as hexa-histidine tagged proteins in the soluble form, whereas PKS10 and PKS11 have been expressed in soluble form as Nus-tagged fusion proteins. In an alternative approach we are expressing this gene cluster in Streptomyces, which should be able to support polyketide biosynthesis, both in terms of post-translation modification as well as availability of precursor substrates. In order to clone this 18kb cluster, a shotgun library of BAC Rv401 was constructed in Litmus 28 vector. PacI and XbaI restriction sites were engineered and all these fragments were spliced together by using appropriate restriction enzymes, which was then cloned in Streptomyces- E. coli shuttle vector. By using protoplast transformation, this recombinant shuttle plasmid was introduced into S. coelicolor CH999 strain. Our initial analysis has indicated the presence of atleast two proteins in the crude extract. We are in the process of confirming the expression of other proteins by western blot. Purified pks10, pks11, pks9 and pks17 proteins are being used to raise antibodies in rabbits. After confirming the expression of all six proteins in Streptomyces, we plan to isolate and characterize the metabolic product.

We have also initiated efforts to study this gene cluster in the host organism. Sequence analyses of this cluster in the genomes of various mycobacteria, i.e., M. tuberculosis, M. leprae, M. smegmatis (ongoing), and M. bovis (ongoing) have indicated that this cluster is completely conserved between M. tuberculosis and M. bovis. Whereas, this cluster has been reduced to pseudogenes in M. leprae genome, this cluster has not been detected in the ongoing M. smegmatis genome. By isolating RNA from M. bovis and by performing RT-PCR, we have confirmed that this cluster is transcriptionally active in M. bovis.

Crosstalk between FAS and PKS

We are examining a 50kb pps gene cluster to understand the molecular basis of the transfer of biosynthetic intermediates between PKS and FAS. This gene cluster is involved in the biosynthesis of a complex lipid phthiocerol dimycocerosate (PDIM), a lipid exclusively found in pathogenic mycobacterial species. PDIM is probably synthesized by the combined action of (i) the PKS-like ‘pps’ enzymes, (ii) an iterative mycocerosic acid synthase, mas, (iii) at least one acyl-CoA synthase, and (iv) membrane transport proteins such as mmpL7. We have cloned and expressed number of proteins from this cluster in E. coli. The acyl-CoA synthase proteins have been purified and presently we are investigating their substrate specificities. We have also expressed two PKS proteins PPSA and PPSE and a functionally unknown protein papA5 from this cluster. PPSA protein was purified and the apo-protein was post-translationally modified by using a heterologous phophopantetheinyl transferase from surfactin biosynthetic pathway. The acyl transferase domain of this protein was shown to be have specificity for malonyl-CoA.

Publications

Original peer-reviewed articles

1.     ¶Tsai SC, Miercke LJ, Krucinski J, Gokhale RS, Chen JC, Foster PG, Cane DE, Khosla C and Stroud RM (2001) Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc Natl Acad Sci USA. 98:14808-14813 (¶on deputation/work done elsewhere).

Reviews/Proceedings

1.     Gokhale RS and Tuteja D (2001) Biochemistry of polyketide synthases. In: Biotechnology (Eds. Reid and Rehm), WILEY-VCH Verlag, GmbH, Germany, 10:342-372.