Fungal Iterative Type 1 PKSs

Fungal Iterative Type I PKSs

 

(i) Cloning and Expression of The Squalestatin Tetraketide Synthase. Squalestatin S1 is a potent inhibitor of squalene synthase and thus a potential anticholesterol drug. It consists of two separate polyketide chains (A and B, Figure 1). A cDNA library from Phoma sp. was probed with PKS domain fragments selective for C-MeT and KS domains.10 This led to the cloning of an iterative PKS with KS, AT, DH, CMeT, ER, KR and ACP domains. The PKS was heterologously expressed in Aspergillus oryzae to produce the tetraketide side chain 1 (Figure 1).11 We have named this PKS the squalestatin tetraketide synthase (SQTKS). The biosynthetic assembly of the tetraketide must be programmed. Methylation, for example, only occurs after rounds 1 and 2 of extension, KR and DH work in all rounds, but ER is also inoperative after the final extension. SQTKS is also similar to single extension modules of bacterial Type I PKS, the key difference being that SQTKS is iterative.

Fig.2

Figure 2. Squalestatin S1 and the biosynthesis of the tetraketide sidechain, showing: The biosynthetic origin of the carbon chains; the HPLC analysis and purification of 1 (blue crude chemical extract of WT A. oryzae, red extract of A. oryzae + pSQTKS, green purified 1); and the gene organisation of SQTKS, chemical structure of 1 and cryptic programme of SQTKS.

 

 (ii) The Fusarin and Tenellin Synthetases. These synthetases (FUSS and TENS)12,13 are unusual because they combine an iterative PKS with one module of a non-ribosomal peptide synthetase (NRPS) – we were the first to report such synthases in fungi which have subsequently shown via genomic sequencing programmes to be a rather common, if unanticipated, feature. The FUSS and TENS PKS consist of the catalytic domains: KS, AT, DH, C-MeT, KR and ACP. The PKS is fused to condensation (C), adenylation (A) and thiolation (T) domains which are terminated by an apparent reduction (R) domain. The PKS makes a programmed polyketide which becomes fused to an amino acid, selected and activated by the NRPS domain. The R domain then mediates ring formation and release (Scheme 2).

 

scheme2

 

Scheme 2. Fusarin and Tenellin Biosynthesis, showing the origin of the carbon atoms and the programme of the PKS

 

A rapid cloning strategy has been developed that allows the reassembly of very large PKS and PKS-NRPS genes in vectors based on those we have exploited for the expression of SQTKS. In the case of TENS, we have shown that fidelity of iPKS programming requires the presence of a trans-acting ER20 encoded by orf3. This results in high level and specific production of pretenellin-A 2. Absence of orf3 results in production of several acyl-tetramic acids with unreduced side chains in which the chain length and methylation patterns vary.14 Subsequent work using gene knock-out and gene silencing coupled with in vitro enzymology has allowed the role of all the tenellin genes to be defined,15 and the full pathway to be heterologously expressed.

 

1.        Design and Utility of Oligonucleotide Probes for Fungal Polyketide Synthases, T. P. Nicholson, C. M. Lazarus, B. A. M. Rudd, M. J. Dawson, T. J. Simpson and R. J. Cox, Chem. Biol., 2001, 8, 151.

2.        Rapid Cloning and Expression of a Fungal Polyketide Synthase gene Involved in Squalestatin Biosynthesis, R. J. Cox, F. Glod, D. Hurley, C. M. Lazarus, T. P. Nicholson, B. A. M. Rudd, T. J. Simpson, B. Wilkinson and Y. Zhang, Chem. Comm., 2004, 2260.

3.        Fusarin C Biosynthesis in Fusarium moniliforme and Fusarium venenatum, Z. Song, R.J. Cox, C.M. Lazarus and T.J. Simpson, ChemBioChem., 2004, 1196-1203.

4.        Biosynthesis of the 2-Pyridone Tenellin in the Insect Pathogenic Fungus Beauveria bassiana. K. L. Eley, L. M. Halo, Z. Song, H.  Powles , R.  J. Cox, A. M. Bailey, C. M. Lazarus, and T. J. Simpson, ChemBioChem, 2007, 8, 289-297.

5.         Authentic Heterologous Expression Of The Tenellin Iterative Polyketide Synthase Non-Ribosomal Peptide Synthetase requires co-expression with an enoyl reductase, L. M. Halo, J. W. Marshall, A. A. Yasaki, Z. Song, C. P. Butts, M.P. Crump, M. Heneghan, A.M. Bailey, T. J. Simpson, C. M. Lazarus and R.J. Cox, ChemBioChem, 2008, 9, 585-594.

6.        Late Stage Oxidations During the Biosynthesis of the 2-Pyridone Tenellin in the Entomopathogenic Fungus Beauveria bassiana. L. M. Halo, M. N. Heneghan, A. A.  Yakasai, Z. Song, K. Williams, A. M. Bailey, R. J. Cox, C. M. Lazarus and T. J. Simpson, J. Am. Chem. Soc., 2008, 130, 17988-17996.