Expression of pederin biosynthesis enzymes from an uncultivated symbiont

1
Expression of pederin biosynthesis enzymes from
an uncultivated symbiont Katrin Zimmermann, Jörn
Piel Max-Planck-Institute for Chemical Ecology,
Department of Bioorganic Chemistry,
Hans-Knöll-Straße 8, 07745 Jena

1. Introduction The polyketide pederin is found
in beetles of the genera Paederus and Paederidus.
This substance is a highly potent antitumor
compound with IC50 values in the subnanomolar
range for some tumor model systems 1. The
beetles use pederin as a defense agent against
predators such as spiders 2. Former studies
showed that the true pederin producer is a
Pseudomonas sp. that lives in symbiosis with the
beetles 3. Since this bacterium is so far
uncultivatable, the analysis of the pederin genes
must be conducted by heterologous gene
expression. Pederin is assembled by a type
I polyketide synthase (PKS), the genes of which
are located on three different regions of the
symbiont genome 4,5. The pederin system belongs
to the recently described trans-AT-type group of
PKSs 5. In contrast to regular PKSs, the
acyltransferase domains are lacking in every
module and are complemented in trans by
individual AT proteins 6. Interestingly,
although malonyl-CoA seems to be the only AT
substrate for pederin biosynthesis, the pederin
cluster encodes two external ATs. In addition,
the pederin PKS harbors several novel catalytic
domains that warrant a closer investigation.
4. Expression of acyltransferases The AT genes
were amplified by PCR and cloned into several
expression vectors. However, expression in any of
these plasmids failed to give detectable or
soluble protein in E. coli. After modifying the
first ten codons according to the preferred codon
usage of E. coli, both ATs could finally be
expressed as soluble MBP fusion proteins from
pMalc2x and purified by affinity chromatography.
The identity of the proteins was confirmed by
MALDI-TOF analysis.
PedD
PedC
M...marker un...uninduced sample SN...supernatant
after centrifugation of sonicated
culture (soluble proteins) P...pellet after
centrifugation of sonicated culture
(insoluble proteins) e...elution fraction of
purification of MBP fusion protein
M un SN P el
M un SN P
el
83,32 kDa
80,97 kDa
5. Expression of acyl carrier proteins All three
types of ACPs were amplified by PCR and cloned
into pMalc2x. Expression of the holo fusion
proteins was carried out under coexpression of
the phosphopantetheinylase gene sfp from the
vector pREP4. Soluble fusion proteins were
obtained in all cases and analyzed by MALDI-TOF.
M...marker un...uninduced sample SN...supernatant
after centrifugation of sonicated
culture (soluble proteins) P...pellet after
centrifugation of sonicated culture
(insoluble proteins) e...elution fraction of
purification of MBP fusion protein
holo fusion proteins PedI2 52,14 kDa PedI3
63,63 kDa PedN 52,05 kDa

6. Expression of the putative oxidoreductase
PedB Almost all trans-AT PKS systems have an
FMN-dependent oxidoreductase gene with unknown
function in common, which is often fused with an
AT gene. This suggests that such genes are
involved in the early steps of polyketide
biosynthesis, such as precursor supply. To study
the homologue in the pederin system (PedB),
overexpression of the gene was attempted. Small
amounts of soluble MBP fusion protein could be
obtained after codon modification and expression
from pMALc2x.
Proposed pederin biosynthesis pathway. Routes A
and B represent two different possible pathways.
The pederin cluster encodes two external ATs and
three distinct types of acyl carrier proteins
(ACPs).
M...marker un...uninduced sample SN...supernatant
after centrifugation of sonicated
culture (soluble proteins) P...pellet after
centrifugation of sonicated culture
(insoluble proteins) e...elution fraction of
purification of MBP fusion protein
2. Goals The aim of this project is to
overexpress both ATs and different types of ACP
partners, along with other interesting enzymes of
pederin biosynthesis. These enzymes are then
functionally analyzed to obtain insights into the
peculiar enzymology of trans-AT-PKSs.
7. Summary and outlook Both ATs, all three types
of ACPs and the oxidoreductase PedB could be
overexpressed from pMALc2x and purified.
Preliminary experiments showed that cleavage of
the fusion proteins yields intact enzymes. The
availability of these proteins now sets the stage
to study fundamental biosynthetic processes of
trans-AT PKSs, a so far little studied group of
enzymes with novel features. These functional
analyses could answer several questions related
to pederin biosynthesis, such as Why are two AT
genes instead of one in the cluster? What is the
function of the didomain and the external ACP?
What is the role of the conserved oxidoreductase?
By using radioactively labeled acyl-CoA
substrates and Fourier transform mass
spectroscopy in enzymatic assays, we are
currently addressing these issues.
3. The enzymology of ATs and ACPs In polyketide
biosynthesis, the ATs transfer their acyl-CoA
substrates to ACP domains or enzymes. For
analysing the acyltransferases in enzymatic
assays, an expression of possible ACP partners is
therefore necessary. Notably the pederin cluster
contains different ACPs monodomain ACPs (pedI2
e.g.), a repeated ACP (pedI3) and a
monofunctional, external ACP (pedN). It would
therefore be interesting to know whether the two
ATs are functionally identical or whether they
have different ACP or substrate specificities. To
obtain functionally overexpressed ACPs in E.
coli, they must be converted to the holo proteins
by a phosphopantetheinyl transferase.
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M., and Hui, D. 2004. ChemBioChem 593-98 6
Cheng, Y.-Q., Tang, G.-L., and Shen, B. 2003.
PNAS 1003149-3154