Investigation of polyketide biosynthetic pathways in the sponge Theonella swinhoei and the beetle Paederus fuscipes [Elektronische Ressource] / vorgelegt von Tu Anh Nguyen

Tu Anh Nguyen

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Investigation of polyketide biosynthetic
pathways in the sponge Theonella swinhoei
and the beetle Paederus fuscipes


Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms-Universität Bonn


vorgelegt von
Tu Anh Nguyen
aus
Ha Noi, Viet Nam



Bonn (2009)



















Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der
Rheinischen Friedrich-Wilhelms-Universität Bonn.














Gutachter: 1. Prof. Dr. Jörn Piel
2. Prof. Dr. Gabriele M. König
3. Prof. Dr. Christa E. Müller
4. Prof. Dr. Hans-Georg Sahl


Tag der Promotion: 7. 12. 2009
Abstract

Onnamides are natural products that possess strong antitumor activity and were isolated from the
marine sponge Theonella swinhoei. Previous data suggested that an as-yet unculturable symbiotic
bacterium living in association with the sponge is the actual onnamide producer. In order to gain
insight into the biosynthetic pathway of onnamides in nature, their biosynthetic genes had to be
identified. Onnamides are encoded by the onnamide (onn) gene cluster, which contains giant trans-
AT polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes. PKSs and NRPSs
are constituted of modules, each module consisting of domains. In trans-AT PKSs, the
acyltransferase (AT) domains are present as discrete genomic regions, outside of the multimodular
enzymatic core.

OH O COOHOH O COOH
NN HH
CH O3 OH OOH O HH
O N HNNHO N HNNH 22
OCHOCH3 3
O O O NHO OO NH

Onnamide A Onnamide B

For the purpose of covering the entire gene cluster, onn genes were cloned from the metagenomic
sponge DNA consisting of DNA of the sponge and of its complex symbiotic community. A portion
of the onn cluster was previously identified by screening a fosmid library constructed from the
metagenomic DNA but was found to encode a truncated PKS. The present study deals with the
isolation of the remaining onn genes from the 400,000 clone library of the sponge T. swinhoei.
Based on the genomic region isolated previously, an iterative primer walking strategy was applied,
which enabled cloning of the remaining regions of the onn gene cluster. According to a recently
developed method for the prediction of substrate specificities of trans-AT PKSs, the isolated gene
cluster is responsible for synthesis of onnamide B. Isolation of the entire cluster was hampered by
the finding that the metagenomic DNA contained numerous variants of the onnamide PKS. We
hypothesize that these PKS variants belong to different symbiont strains that jointly generate the
structurally diverse library of onnamides present in T. swinhoei. Based on the phylogenetic analysis
of ftsZ, a non-PKS taxonomic marker gene located immediately downstream of the onn genes,
i evidence was obtained that the onnamide producer is a member of the bacterial phylum Chloroflexi.
In addition to these studies, the adenylation (A) domains of two NRPS modules of the onn gene
cluster were expressed to provide the basis for functional studies on onnamide biosynthesis.

The onnamide genes represent the first biosynthetic gene cluster that as isolated from a sponge.
With the onn genes in hand, not only fundamental insights into the biosynthetic pathway of
onnamides can be obtained but also strategies can be developed for the sustainable production of
rare marine drug candidates by microbial heterologous expression systems.

Previous studies by Piel et al. in 2004 provided evidence for the presence of another trans-AT PKS
cluster in the total DNA of T. swinhoei. This hypothesis was corroborated by a more detailed
bioinformatic analysis conducted in the present study on short PCR products amplified from the
sponge metagenome. To isolate the cluster, specific primers were used to screen again the fosmid
library of the sponge. From this screening, two positive fosmids were identified and completely
sequenced. Analysis of this sequence verified that the newly isolated genes belong to an as-yet
uncharacterized polyketide. According to the analysis, some structural features of this polyketide
were predicted. However, frame-shifts present in several genes suggested that the pathway is not
functional and likely represents an evolutionary relic. Nevertheless, the results confirmed that PKS
functions can be predicted even when starting from a short partial sequence amplified by PCR.


Pederin

pPD7E4 is a cosmid that was previously isolated from the genome of an uncultivated Pseudomonas
symbiont of the beetle Paederus fuscipes. The complete sequence showed that this cosmid
contained a small gene cluster with both PKS and non-PKS genes. In particular, one of the non-PKS
genes was found to be a halogenase gene. This finding suggests that the gene cluster from pPD7E4
does not belong to the known ped genes responsible for pederin biosynthesis, but rather encodes
biosynthesis of a halogenated compound. In nature, organohalogens often possess bioactivities.
ii Therefore, in an attempt to identify the compound, the cosmid pPD7E4 was heterologously
expressed applying the homologous recombination. After several genetic modifications, the
heterologous expression of pPD7E4 inside Pseudomonas putida KT2440 was successful at the
RNA level, setting the stage for the characterization of the unknown compound.

























iii Contents

Abstract.................................................................................................................................. i
Contents........ iv
List of Figures........................................................................................................................x
List of Tables ..................................................................................................................... xiv
Acknowledgements .......................................................................................................... xvii
Chapter 1 ...............................................................................................................................1
Introduction...........................................................................................................................1
1.1 Natural products ........................................................................................................................ 1
1.2 Polyketides and nonribosomal peptides .................................................................................... 3
1.3 Polyketide synthases ................................................................................................................. 5
1.3.1 PKS structure.................................................................................................................. 5
1.3.2 Polyketide synthesis ....................................................................................................... 6
1.3.3 PKS classification 8
1.3.3.1 Types of PKSs 8
1.3.3.2 Hybrid PKS-NRPS.................................................................................................. 10
1.3.3.3 Cis and trans-AT PKSs........................................................................................... 10
1.4 Nonribosomal peptide synthases............................................................................................. 11
1.5 The symbiont hypothesis.........................................................................................................14
1.5.1 Insects and their symbionts .......................................................................................... 15
1.5.2 Sponge symbiosis and secondary metabolites.............................................................. 15
1.5.2.1 Microbial sponge symbiosis.................................................................................... 15
1.5.2.2 Sponge secondary metabolites................................................................................ 17
1.6 Pederin family ......................................................................................................................... 19
1.6.1 The beetle P. fuscipes and the previous genetic work on pederin ................................ 21
iv 1.6.2 The sponge T. swinhoei and the previous genetic work on onnamides........................ 23
1.7 The metagenomic approaches in natural product research...................................................... 26
1.7.1 Difficulties in natural product exploitation .................................................................. 26
1.7.2 Metagenomics............................................................................................................... 27
Chapter 2 ....................