Genomics and Transcriptomics of the Industrial Acarbose Producer Actinoplanes sp. SE50/110

155 Pages·2012·13.8 MB·English

Genomics and Transcriptomics of the
Industrial Acarbose Producer
Actinoplanes sp. SE50/110
Ph.D. Thesis
submitted to the
Faculty of Technology,
Bielefeld University, Germany
for the degree of Dr.rer.nat.
by
Patrick Schwientek
January, 2012
Referees:
Prof. Dr. Alfred Pu¨hler
Prof. Dr. Jens Stoye
Printed on non-aging paper according to DIN-ISO 9706.
Acknowledgments
This Ph.D. project was carried out in the period between January 2009 and January
2012 at the Center for Biotechnology (CeBiTec), Bielefeld University, Germany. It
constitutes an interdisciplinary collaboration between the CeBiTec’s Institute of Ge-
nome Research and Systems Biology, the CeBiTec’s Institute of Bioinformatics, and
the industrial partner Bayer HealthCare AG, Wuppertal, Germany. It is a pleasure
to thank the many people who made this thesis possible.
First and foremost, I would like to express my deep and sincere gratitude to my
supervisorsProf.Dr.AlfredPu¨hlerandProf.Dr.JensStoyefortheirstimulatingguid-
ance, invaluable support, and wide knowledge. It is due to their extensive experience
and foresight that this Ph.D. project went so smoothly and will forever remind me
of a challenging but also rewarding time. I am also deeply grateful to Dr. J¨orn Kali-
nowski, for his productive advice and with whom I enjoyed many fruitful discussions.
Furthermore, I wish to thank Prof. Dr. Karsten Niehaus for his expertise, help and
patience during electron microscopy.
This thesis would not have been possible without the help and support of several
experts from the wet-labs. I am especially thankful to Dr. Christian Ru¨ckert and Dr.
Rapfael Szczepanowski who were involved in the preparation and supervision of the
DNA-sequencing process. Moreover, I owe Sergej Wendler, Armin Neshat, Christina
Eirich, and Katharina Pfeifer great thanks for the strain cultivation and preparation
of the RNA-sequencing experiments. For enhancing my limited wet-lab competence
and for her help and expertise with high-GC PCRs, I am very thankful to Yvonne
Kutter. I also want to express my gratitude to Alexandra Tilker from the IIT GmbH
for her efforts in testing specialized PCR protocols and for her repeated acceptance
of sequencing orders after closing hour.
I am especially thankful to my cooperation partners from Bayer HealthCare AG,
Dr. Klaus Selber and Dr. Andreas Klein for their kind support, interesting discus-
sions, and delicious dinners. Furthermore, I wish to thank Dr. Bernd Weing¨artner
and Dr. Hermann Wehlmann for providing various information about Actinoplanes
cultivations and for providing isolated DNA for genome sequencing. I gratefully ac-
knowledge the funding by Bayer HealthCare AG that made my Ph.D. work possible.
I would like to thank the Cluster Industrial Biotechnology (CLIB2021) for finan-
cial support and for the organization of many training courses and activities that I
attended. I also want to thank my friends and fellow graduate students for making
this Ph.D. an active, communicative, and very enjoyable time of my life.
Intheend,Iwishtothankmyparentsfortheirgeneroussupportinallmypursuits,
especiallyduringthelastweeksofthisthesis. Lastly, Iwouldliketothankmypartner
Shokoufeh Ghezelbash for all her love and encouragement.
i

Abstract
Actinoplanes sp.SE50/110isknownasthewildtypeproducerofthealpha-glucosidase
inhibitor acarbose, a potent drug used worldwide in the treatment of type-2 diabetes
mellitus. As the global incidence of diabetes is rapidly rising, an ever increasing
demand for diabetes drugs, such as acarbose, needs to be anticipated. Consequently,
derived Actinoplanes strains with increased acarbose yields are being used in large
scaleindustrialbatchfermentation,whichwerecontinuouslyoptimizedbymutagenesis
andscreeningexperiments. However,beingappliedforover20years,thisconventional
optimizationstrategyhasnowreacheditslimitsandisgenerallysupersededbymodern
genetic engineering approaches, which require the genome sequence of the organism.
Hence, the first part of this Ph.D. thesis dealt with the sequencing, assembly and
annotation of the complete genome sequence of Actinoplanes sp. SE50/110, the first
publicly available genome of the genus Actinoplanes. Due to its high GC-content of
71.32%andtheformationofstablesecondarystructuresthathinderedthesequencing
process,adaptedprotocolsweredevelopedwhichallowedtheestablishmentofthecom-
plete sequence. The final genome consists of a single circular chromosome with a size
of 9.4 Mb hosting about 8,400 genes. Besides the known acarbose biosynthetic gene
cluster sequence, several new non-ribosomal peptide synthetase-, polyketide synthase-
and hybrid-clusters were identified on the Actinoplanes genome. Another key finding
represents the discovery of a functional actinomycete integrative and conjugative ele-
ment,whichmightposeanelegantwayofgeneticallyaccessingtheorganism. Phyloge-
netic analysis of the core genome revealed a rather distant relation to other sequenced
species of the family Micromonosporaceae, whereas Actinoplanes utahensis was found
to be the closest species based on 16S rDNA comparison.
The second part of this work complemented the genomic information with tran-
scriptome experiments using RNA-sequencing technology. These analyses resulted in
thediscoveryofnon-codingRNAs, novelproteincodingsequences, andantisensetran-
scripts to known genes, which lead to an improved annotation of the Actinoplanes sp.
SE50/110 genome. Moreover, genome wide expression quantification provided – for
the first time – insights into the transcriptional landscape of the acarbose producer.
In this regard, differential expression testing between three different Actinoplanes cul-
tivations were also performed in order to elucidate the changes in gene expression
in response to varying growth-media compositions. It was found that the different
media had significant impact on growth rate and acarbose productivity, which was
clearly reflected on the transcriptional level. In particular, the acarbose biosynthe-
sis gene cluster happend to be highly up-regulated in maltose-containing media and
almostsilentintheglucose-containingmedium. Additionally,oneoftheidentifiednon-
ribosomal peptide synthetase gene clusters showed high expression, which resembled
the expressional pattern of the acarbose cluster across the analyzed conditions.
iii

Contents
1. Introduction 1
1.1. The genus Actinoplanes . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2. The strain Actinoplanes sp. SE50/110 . . . . . . . . . . . . . . . . . . 2
1.3. The secondary metabolite acarbose, its relevance, and mode of action 3
1.4. The biosynthesis of acarbose in Actinoplanes sp. SE50/110 . . . . . . 6
1.5. Industrial development and fermentation of acarbose . . . . . . . . . . 10
1.6. Bacterial genome sequencing approaches . . . . . . . . . . . . . . . . . 11
1.7. Bacterial genome annotation strategies . . . . . . . . . . . . . . . . . . 14
1.8. Means of bacterial transcriptome analysis . . . . . . . . . . . . . . . . 15
1.9. Motivation and aims of this thesis . . . . . . . . . . . . . . . . . . . . 19
2. Materials and Methods 21
2.1. Acquisition of the strain Actinoplanes sp. SE50/110 . . . . . . . . . . 21
2.2. Genomic DNA-sequencing methods . . . . . . . . . . . . . . . . . . . . 21
2.2.1. Cultivation of Actinoplanes sp. SE50/110 for DNA-sequencing 21
2.2.2. Isolation of genomic DNA from Actinoplanes sp. SE50/110 . . 22
2.2.3. PyrosequencingoftheActinoplanes sp.SE50/110genomicDNA
on the Genome Sequencer FLX . . . . . . . . . . . . . . . . . . 23
2.3. Genome assembly and mapping techniques. . . . . . . . . . . . . . . . 23
2.3.1. Genome assembly . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.3.2. Read mapping on the acarbose gene cluster . . . . . . . . . . . 23
2.4. Genome finishing methods . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4.1. ConstructionofafosmidlibraryfortheActinoplanessp.SE50/110
genome finishing . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.4.2. Polymerase chain reactions . . . . . . . . . . . . . . . . . . . . 24
2.4.3. SangersequencingofPCRproductsandterminalinsertsequences
from the Actinoplanes sp. SE50/110 fosmid library . . . . . . . 24
2.4.4. FinishingoftheActinoplanes sp.SE50/110genomesequenceby
manual assembly . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.5. Computational genome annotation . . . . . . . . . . . . . . . . . . . . 25
2.5.1. PredictionofcodingsequencesontheActinoplanessp.SE50/110
genome sequence . . . . . . . . . . . . . . . . . . . . . . . . . . 25
2.5.2. FunctionalannotationoftheidentifiedCDSontheActinoplanes
sp. SE50/110 genome . . . . . . . . . . . . . . . . . . . . . . . 25
2.5.3. Phylogenetic analyses . . . . . . . . . . . . . . . . . . . . . . . 26
2.6. RNA-sequencing and analysis . . . . . . . . . . . . . . . . . . . . . . . 27
2.6.1. Cultivation of Actinoplanes sp. SE50/110 for RNA-sequencing 27
v
2.6.2. Total RNA isolation from Actinoplanes sp. SE50/110. . . . . . 28
2.6.3. Preparation of cDNA libraries and high-throughput sequencing 29
2.6.4. Determination of cell dry weights of Actinoplanes cultures . . . 30
2.6.5. Quantification of acarbose in the supernatant of Actinoplanes
cultures by HPLC and UV detection . . . . . . . . . . . . . . . 30
2.6.6. Bioinformatic analysis of RNA-seq results . . . . . . . . . . . . 30
2.7. Gas-chromatographic analysis of the anti-self-annealing additive . . . . 32
3. Results 33
3.1. Solving the high-GC problem for Actinoplanes sp. SE50/110 genome
sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1.1. Analysis of gap regions resulted from standard PE sequencing . 34
3.1.2. ThegapsintheActinoplanessp.SE50/110acarbosegenecluster
are due to an extremely low read coverage . . . . . . . . . . . . 37
3.1.3. The gaps in the acarbose gene cluster are characterized by sec-
ondary structure formation . . . . . . . . . . . . . . . . . . . . 39
3.1.4. Adapted sequencing conditions solved the high-GC problem . . 43
3.2. The complete genome sequence of Actinoplanes sp. SE50/110 . . . . . 44
3.2.1. AssemblyoftheActinoplanes sp.SE50/110draftgenomesequence 44
3.2.2. Finishing of the draft genome sequence . . . . . . . . . . . . . 45
3.2.3. Annotation of the complete genome sequence . . . . . . . . . . 46
3.3. Discoveries of the Actinoplanes sp. SE50/110 genome . . . . . . . . . . 48
3.3.1. General features of the Actinoplanes sp. SE50/110 genome . . 48
3.3.2. Phylogenetic analysis of the Actinoplanes sp. SE50/110 16S
rDNA reveals highest similarity to Actinoplanes utahensis . . . 51
3.3.3. Comparative genome analysis indicates 50% singletons in the
Actinoplanes sp. SE50/110 genome. . . . . . . . . . . . . . . . 52
3.3.4. ThehighqualitygenomesequenceofActinoplanes sp.SE50/110
corrects the previously sequenced acarbose cluster. . . . . . . . 52
3.3.5. Severalgenesoftheacarbosegeneclusterarealsofoundinother
locations of the genome. . . . . . . . . . . . . . . . . . . . . . . 54
3.3.6. Trehalose synthesis in Actinoplanes sp. SE50/110 . . . . . . . . 56
3.3.7. TheActinoplanes sp.SE50/110genomehostsanintegrativeand
conjugative element . . . . . . . . . . . . . . . . . . . . . . . . 56
3.3.8. Four putative antibiotic production gene clusters were found in
the Actinoplanes sp. SE50/110 genome sequence . . . . . . . . 59
3.4. RNA-sequencing of the Actinoplanes sp. SE50/110 transcriptome . . . 62
3.4.1. CultivationofActinoplanessp.SE50/110fortranscriptomeanal-
ysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.4.2. Improving the Actinoplanes genome annotation by RNA-seq . 64
3.4.3. ExpressionanalysisofActinoplanessp.SE50/110growninthree
different cultivation media . . . . . . . . . . . . . . . . . . . . . 77
4. Discussion 91
4.1. Establishment of the complete Actinoplanes sp. SE50/110 genome se-
quence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
4.2. Annotation of the Actinoplanes sp. SE50/110 genome sequence . . . . 92
4.3. New insights related to the acarbose metabolism . . . . . . . . . . . . 93
4.3.1. Acarbose re-import after exclusion of acbHFG . . . . . . . . . 93
4.3.2. Putative formation of component C by trehalose synthases . . 93
4.4. The actinomycete integrative and conjugative element pACPL . . . . 94
4.5. The putative antibiotic gene clusters of Actinoplanes sp. SE50/110 . . 94
4.6. Transcriptome analyses of Actinoplanes sp. SE50/110 . . . . . . . . . 95
4.6.1. Improvement of genome annotation by RNA-seq . . . . . . . . 96
4.6.2. Differential expression testing by RNA-seq . . . . . . . . . . . . 97
4.6.3. Short assessment of computational methods for bacterial RNA-
seq analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
5. Conclusions and Outlook 99
. Bibliography 101
A. Appendix 133
A.1. Supplementary figures . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
A.2. Supplementary tables . . . . . . . . . . . . . . . . . . . . . . . . . . . 135