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.
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).
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.