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 Chemical Biology
of Natural Products
 Chemical Biology
of Natural Products

Edited by
David J. Newman, Gordon M. Cragg,
and Paul G. Grothaus
MATLAB® is a trademark of The MathWorks, Inc. and is used with permission. The MathWorks does not warrant
the accuracy of the text or exercises in this book. This book’s use or discussion of MATLAB® software or
related products does not constitute endorsement or sponsorship by The MathWorks of a particular pedagogical
approach or particular use of the MATLAB® software.

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Library of Congress Cataloging-in-Publication Data

Names: Newman, David J., 1939- | Cragg, Gordon M. L. | Grothaus, Paul.

Title: Chemical biology of natural products / [edited by] David J. Newman,
Gordon M. Cragg, and Paul Grothaus.
Description: Boca Raton : CRC Press, [2017] | Includes bibliographical
references and index.
Identifiers: LCCN 2017011281 | ISBN 9781439841938 (hardback : alk. paper) |
ISBN 9781315117089 (ebook)
Subjects: LCSH: Pharmacognosy. | Natural products.
Classification: LCC RS160 .C5227 2017 | DDC 615.3/21--dc23
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Contents
Preface.......................................................................................................................vii
Editors........................................................................................................................xi
Contributors..............................................................................................................xv

Chapter 1 Microbial Genome Mining for Natural Product Drug Discovery.........1

Richard H. Baltz

Chapter 2 Chemical Biology of Marine Cyanobacteria.......................................43

Lena Keller, Tiago Leão, and William H. Gerwick

Chapter 3 The Role of Combinatorial Biosynthesis in Natural Products

Discovery.............................................................................................87
Jeffrey D. Rudolf, Ivana Crnovčić, and Ben Shen

Chapter 4 Generation of New-to-Nature Natural Products through

Synthesis and Biosynthesis: Blending Synthetic Biology with
Synthetic Chemistry..........................................................................127
Christopher S. Bailey, Emily R. Abraham, and Rebecca J.M. Goss

Chapter 5 Terrestrial Microbial Natural Products Discovery Guided

by Symbiotic Interactions and Revealed by Advanced
Analytical Methods......................................................................161
Rita de Cássia Pessotti, Andrés Mauricio Caraballo-Rodríguez,
Humberto Enrique Ortega-Domínguez, and Mônica Tallarico Pupo

Chapter 6 Natural Products from Endophytic Microbes: Historical

Perspectives, Prospects, and Guidance..............................................189
Gary Strobel

Chapter 7 Novel Insights in Plant–Endophyte Interactions...............................205

Souvik Kusari, Parijat Kusari, and Michael Spiteller

Chapter 8 Microbial Coculture and OSMAC Approach as Strategies

to Induce Cryptic Fungal Biogenetic Gene Clusters.........................233
Georgios Daletos, Weaam Ebrahim, Elena Ancheeva,
Mona El-Neketi, Wenhan Lin, and Peter Proksch

v
vi Contents

Chapter 9 Natural Products of the Rhizosphere and Its Microorganisms:

Bioactivities and Implications of Occurrence...................................285
Maria C. F. de Oliveira, Jair Mafezoli, and A. A. Leslie Gunatilaka

Chapter 10 Novel Metabolites from Extremophilic Microbes Isolated from

Toxic Waste Sites...............................................................................333
Andrea Stierle and Don Stierle

Chapter 11 Deep-Sea Hydrothermal Vent Organisms as Sources

of Natural Products............................................................................387
Kerry L. McPhail, Eric H. Andrianasolo, David A. Gallegos,
and Richard A. Lutz

Chapter 12 Cone Snail Venom Peptides and Future Biomedical Applications

of Natural Products............................................................................425
Baldomero M. Olivera, Helena Safavi-Hemami,
Shrinivasan Raghuraman, and Russell W. Teichert

Chapter 13 Naturally Occurring Disulfide-Rich Cyclic Peptides from Plants

and Animals: Synthesis and Biosynthesis.........................................491
Simon J. de Veer and David J. Craik

Chapter 14 Synthesis and Target Identification of Natural Product–Inspired

Compound Collections......................................................................529
Luca Laraia and Herbert Waldmann

Chapter 15 On the Chemistry and Biology of the Marine Macrolides

Zampanolide and Dactylolide...........................................................555
Karl-Heinz Altmann, Simon Glauser, and Tobias Brütsch
Index.......................................................................................................................601
Preface
Chemical biology, biochemistry, and biological chemistry? So what, if anything,
are the similarities and differences? One easy definition of the first and last terms
would be that Chemical biology = The biology of chemicals, whereas Biological
chemistry = The chemistry of biology, and Biochemistry is the study of the chem-
istry of living systems.
This definition dilemma is further illustrated by the fact that there are eminent
universities across the United States where there are chemical biology programs in
the College of Chemistry (Berkeley, for example*), but also biochemistry in the same
university but housed in the College of Letters and Science†; and at the University
of Pennsylvania, the Chemistry Department has a Biological Chemistry Resource
Center‡ and a chemical biology postgraduate program,§ with a biochemistry pro-
gram included within the Medical School.¶ We should add that one of the editors
practiced as a biological chemist in the U.S. pharmaceutical industry 45 plus years
ago, ­studying the effect of small synthetic molecules on oxygenation of hemoglobin.
Today, he might well have been practicing chemical biology!
The Broad Institute, based in Cambridge, Massachusetts,** defines chemical biol-
ogy as “the science of small molecules in the context of living systems to discover
and to elucidate molecular pathways fundamental in cellular, developmental and
­disease biology.”††
Furthermore, a survey based on leading journals at the interface between chem-
istry and biology, conducted by the American Chemical Society (ACS), of 4000
scientists working at the interface of chemistry and biology, indicated that nat-
ural products did not feature in the top eight disciplines (bioorganic chemistry,
medicinal chemistry, molecular biology, enzymology, biophysics, biotechnology,
cell biology, and structural biology) selected as being linked to chemical biology.‡‡
Interestingly, when natural products was used as the lead term, the survey selec-
tions indicated that they were linked in decreasing order to medicinal chemistry,
bioorganic chemistry, chemical biology, plant science, and pharmacology, but not
to microbiology.
Attempting to separate the three disciplines mentioned in the first paragraph is
probably an exercise in frustration. However, what intrigues us about the Broad

* http://chemistry.berkeley.edu/ugrad/degrees/chembio.
† http://mcb.berkeley.edu/undergrad.
‡ https://www.chem.upenn.edu/content/penn-chemistry-biological-chemistry-resource-center.
§ https://www.chem.upenn.edu/content/graduate.
¶ http://www.med.upenn.edu/biocbiop/.
** https://www.broadinstitute.org/chembio-therapeutics.
†† http://www.broadinstitute.org/scientific-community/science/programs/csoft/chemical-biology/

chemical-biology-program.
‡‡ http://pubs.acs.org/bio/.

vii
viii Preface

Institute definition and the results of the ACS survey discussed above is the appar-
ently widespread lack of appreciation of the role played by natural products in the
area of chemical biology. Even more puzzling is the apparent failure to link natural
products to microbiology. We therefore decided that a volume highlighting the role
of natural products in chemical biology would be an enlightening undertaking and
that such a volume should include examples of all three methods of interrogating
Mother Nature in individual chapters.
The 15 chapters in this book range over the gamut of the definitions alluded to
above and serve to emphasize the dominant role played by microbes in the production
of bioactive metabolites. On the chemical biology front, they include the chemical
biology of cyanobacteria, combinatorial biosynthesis, including synthetic biology,
target identification from natural product inspired structures, and syntheses devised
around active natural product structures. Moving to secondary metabolites that may
be used in the future to probe biological systems or are themselves the products of
complex interactions, there are discussions covering materials from insect–microbe
symbioses, compounds from plant–endophytic microbes and rhizosphere interac-
tions, and the coculture of microbes to induce production of fungal metabolites. Also
covered are secondary metabolites from extremophilic sources, including toxic lakes
and deep-sea sediments and vent organisms, and a chapter covering genomic min-
ing of microbes to find novel bioactive natural products. Finally, moving closer to
biological chemistry and/or biochemistry, there are significant discussions of neuro-
toxins from venomous Conus species and the somewhat similar active cyclic sulfide-
bridged peptides from plants and animals.
There are many other very interesting topics at the interface of chemistry and
biology, in particular if one looks at the burgeoning reports related to the actual
sources of secondary metabolites in marine-related organisms, and perhaps in some
cases, in plants as well. What has now become quite evident is that the majority
of bioactive natural products described from the Porifera (sponges), and almost
certainly in other marine phyla as well, are almost certainly produced by as yet
uncultured microbes, whose secrets are now being revealed by the combination of
genomic analyses of single microbial cells coupled to very sophisticated physico-
chemical techniques.
As an example, the story of the pederin–mycalamide–onnamide locus is one
that even a few years ago would have been science fiction but is now recognized
as being correct. This story, in an abbreviated form, leading from the Paederus
beetle toxin, via a German entomologist’s suggestion that a microbe was involved,
through the work done by Piel and his collaborators on an as yet uncultured
Entotheonella species, was recently reported by one of the editors in an open-
access paper.*
That the investigation of biological phenomena now requires a multidisciplinary
approach, where chemists and biologists need to work together to uncover Mother

* Newman, D.J., The influence of Brazilian biodiversity on searching for human-use pharmaceuticals,
J. Braz. Chem. Soc., 2017, in press (http://jbcs.sbq.org.br/imagebank/pdf/160478RV_Biota.pdf).
Preface ix

Nature’s secrets, has become evident today, and it is our hope that the examples in
this book will further encourage scientists, be they chemical biologists, biochemists,
biological chemists, or just plain chemists and biologists, to work together in order
to further discover novel agents and their interplay, with the potential that some may
lead to new treatments for human diseases.
Our sincere thanks to all who have participated in this project!

David J. Newman
Gordon M. Cragg
Paul G. Grothaus
Editors
David J. Newman retired from the position of
chief of the Natural Products Branch (NPB) in the
Developmental Therapeutics Program at the National
Cancer Institute (NCI) in Frederick, Maryland, in
early January 2015. Born in Grays, Essex, United
Kingdom, in 1939, he received an MSc in synthetic
organic chemistry from the University of Liverpool in
1963. Following time as a synthetic chemist at Ilford,
Ltd., he joined the Agricultural Research Council’s
(ARC) Unit of Nitrogen Fixation at the Universities of
London and Sussex, as a research assistant in metallo-
organic chemistry, transferring to the microbial biochemistry group in early 1966 as a grad-
uate student and being awarded a DPhil in 1968 for work on microbial electron transport
proteins from Desulfovibrio. He moved to the United States in 1968 as a postdoc in the
Biochemistry Department at the University of Georgia, working on protein sequencing of
Desulfovibrio ferredoxins, and then in 1970, he joined Smith, Kline & French (SK&F) in
Philadelphia as a biological chemist. At SK&F, most of his work was related to antibiotic
discovery, and in 1985, when the antibiotic group was dissolved, he left SK&F. For the
next six years, he worked in marine and microbial discovery programs at Air Products,
SeaPharm, and Lederle, and then in 1991, he joined the NPB as a chemist responsible for
marine and microbial collection programs. He was given the National Institutes of Health
(NIH) Merit Award in 2003 for this work, and following Gordon M. Cragg’s retirement
from the position of chief of the National Products Branch of the National Cancer Institute
(NPB/NCI), at the end of 2004, he was acting chief until appointed chief in late 2006. He is
the author or coauthor of over 180 papers, reviews, and book chapters (and an editor, with
Gordon M. Cragg and David Kingston, of Anticancer Agents from Natural Products) and
holds 18 patents, mainly on microbial products. He is still associated with the NPB/NCI as
a special volunteer and also has a small consulting business to occupy his spare time!

Gordon M. Cragg obtained his undergraduate

training in chemistry at Rhodes University, South
Africa, and his DPhil (organic chemistry) from
Oxford University. After two years of postdoctoral
research at the University of California, Los Angeles,
he returned to South Africa to join the Council
for Scientific and Industrial Research. In 1966, he
joined the Chemistry Department at the University
of South Africa, and he transferred to the University
of Cape Town in 1972. In 1979, he returned to the
United States to join the Cancer Research Institute
at Arizona State University, working with Professor G.R. Pettit. In 1985, he moved to
the National Cancer Institute (NCI), National Institutes of Health (NIH), in Bethesda,

xi
xii Editors

Maryland, and was appointed chief of the NCI Natural Products Branch in 1989. He
retired in December 2004 and is currently serving as an NIH special volunteer. His
major interests lie in the discovery of novel natural product agents for the treatment of
cancer and AIDS, with an emphasis on multidisciplinary and international collabora-
tion. He has been awarded NIH merit awards for his contributions to the development
of the anticancer drug Taxol (1991), leadership in establishing international collabora-
tive research in biodiversity and natural products drug discovery (2004), contributions
to developing and teaching NIH technology transfer courses (2004), and dedicated
service to the NCI as a member of the PDQ Complementary and Alternative Medicine
Editorial Board (2010). In 1998–1999, he was president of the American Society of
Pharmacognosy and was elected to honorary membership in 2003 and named as a
fellow in 2008. In 2006, he was given the William L. Brown Award for Plant Genetic
Resources by Missouri Botanical Garden, which also named a recently discovered
Madagascar plant in his honor, Ludia craggiana. He has established collaborations
between the NCI and organizations in many countries, promoting drug discovery from
their natural resources. He has authored or coauthored over 180 papers, reviews, and
book chapters related to these interests.

Paul G. Grothaus earned a BSChem from

Creighton University in 1977 and his PhD from
Purdue University in 1983, where he completed
the first enantiospecific total synthesis of a tricho-
thecene mycotoxin, anguidine. His education was
followed by a postdoctoral stint at the University
of Washington, investigating the synthesis of ger-
macrolides. In 1984, he joined the Natural Products
Group in the Plant Sciences Division of Monsanto
Agricultural Company, where he investigated the
synthesis and structure–activity relationships of
agriculturally useful natural products. In 1988, he became the head of chemistry at
Hawaii Biotech, Inc., in Aiea, Hawaii, where he worked on drug discovery based on
both terrestrial and marine natural product leads. In 2002, he joined the Medicinal
Chemistry Department of Celera Genomics, Inc., in South San Francisco, California,
and rose to become an associate director of medicinal chemistry in 2005. Research
at Celera focused on the development of protease and kinase inhibitors and activity-
based probes for chemical proteomics studies.
At Celera, Dr. Grothaus led the chemistry group that designed and synthesized
irreversible and reversible inhibitors of Bruton’s tyrosine kinase (BTK), culminating
in the discovery of ibrutinib (CRA-32765/PCI-32765), a first-in-class, oral, once-
daily therapy that inhibits BTK, a key protein in the B-cell receptor signaling
complex. Following further preclinical and clinical development by Pharmacyclics,
Inc., Ibrutinib received three Oncology Breakthrough Therapy Designations by the
Food and Drug Association and was approved for the treatment of chronic lympho-
cytic leukemia/small lymphocytic lymphoma in 2014, mantle cell lymphoma in
2013, and Waldenstrom’s macroglobulinemia in 2015.
Editors xiii

In 2007, he joined the Natural Products Branch of the National Cancer Institute
in Frederick, Maryland, where he coordinates biomass collections, biological
screening of extracts, and collaborations with external natural product researchers.
Dr. Grothaus is the author or coauthor of 24 papers, reviews, and book chapters
and holds 5 patents.
Contributors
Emily R. Abraham Tobias Brütsch
School of Chemistry Department of Chemistry and Applied
University of St. Andrews Biosciences
St. Andrews, United Kingdom Institute of Pharmaceutical Sciences
ETH Zürich
Zürich, Switzerland
Karl-Heinz Altmann
Department of Chemistry and Applied Andrés Mauricio
Biosciences Caraballo-Rodríguez
Institute of Pharmaceutical Sciences Laboratory of Microbial Chemistry
ETH Zürich Department of Pharmaceutical
Zürich, Switzerland Sciences
School of Pharmaceutical Sciences of
Elena Ancheeva Ribeirão Preto
Institute of Pharmaceutical Biology and University of Sao Paulo
Biotechnology Ribeirao Preto, Brazil
Heinrich-Heine University
Düsseldorf, Germany David J. Craik
Institute for Molecular Bioscience
The University of Queensland
Eric H. Andrianasolo Brisbane, Queensland, Australia
Department of Marine and Coastal
Sciences Ivana Crnovčić
Center for Deep-Sea Ecology and Department of Chemistry
Biotechnology The Scripps Research Institute
Rutgers, The State University of Jupiter, Florida
New Jersey
New Brunswick, New Jersey Georgios Daletos
Institute of Pharmaceutical Biology and
Biotechnology
Christopher S. Bailey
Heinrich-Heine University
School of Chemistry
Düsseldorf, Germany
University of St. Andrews
St. Andrews, United Kingdom
Maria C.F. de Oliveira
Laboratory of Biotechnology and
Richard H. Baltz Organic Synthesis
CognoGen Biotechnology Post-graduate Program in Chemistry
Consulting Federal University of Ceará
Sarasota, Florida Fortaleza, Brazil

xv
xvi Contributors

Simon J. de Veer Rebecca J.M. Goss

Institute for Molecular Bioscience School of Chemistry
The University of Queensland University of St. Andrews
Brisbane, Queensland, Australia St. Andrews, United Kingdom

Weaam Ebrahim A.A. Leslie Gunatilaka

Institute of Pharmaceutical Biology and Natural Products Center
Biotechnology School of Natural Resources and the
Heinrich-Heine University Environment
Düsseldorf, Germany College of Agriculture and Life Sciences
and The University of Arizona
Tucson, Arizona
Faculty of Pharmacy
Department of Pharmacognosy Lena Keller
Mansoura University Center for Marine Biotechnology and
Mansoura, Egypt Biomedicine
Scripps Institution of Oceanography
Mona El-Neketi University of California, San Diego
Faculty of Pharmacy La Jolla, California
Department of Pharmacognosy
Mansoura University Parijat Kusari
Mansoura, Egypt Department of Biochemical and
Chemical Engineering
Technical University of Dortmund
David A. Gallegos Dortmund, Germany
Department of Pharmaceutical
Sciences Souvik Kusari
College of Pharmacy Department of Chemistry and Chemical
Oregon State University Biology
Corvallis, Oregon Institute of Environmental Research
Technical University of Dortmund
William H. Gerwick Dortmund, Germany
Center for Marine Biotechnology and
Luca Laraia
Biomedicine
Department of Chemical Biology
Scripps Institution of Oceanography
Max Planck Institute of Molecular
University of California, San Diego
Physiology
La Jolla, California
Dortmund, Germany

Simon Glauser Tiago Leão

Department of Chemistry and Applied Center for Marine Biotechnology and
Biosciences Biomedicine
Institute of Pharmaceutical Sciences Scripps Institution of Oceanography
ETH Zürich University of California, San Diego
Zürich, Switzerland La Jolla, California
Contributors
Wenhan Lin
State Key Laboratory of Natural and
Biomimetic Drugs
Peking University
Beijing, People’s Republic of China
Richard A. Lutz
Department of Marine and Coastal
Sciences
Center for Deep-Sea Ecology and
Biotechnology
Rutgers, The State University of
New Jersey
New Brunswick, New Jersey
Jair Mafezoli
Laboratory of Applied Phytochemistry
Post-graduate Program in Chemistry
Federal University of Ceará
Fortaleza, Brazil
Kerry L. McPhail
Department of Pharmaceutical
Sciences
College of Pharmacy
Oregon State University
Corvallis, Oregon
Baldomero M. Olivera
Department of Biology
University of Utah
Salt Lake City, Utah
Humberto Enrique
Ortega-Domínguez
Laboratory of Microbial Chemistry
Department of Pharmaceutical
Sciences
School of Pharmaceutical Sciences of
Ribeirão Preto
University of Sao Paulo
Ribeirao Preto, Brazil
xvii
Rita de Cássia Pessotti
Laboratory of Microbial Chemistry
Department of Pharmaceutical
Sciences
School of Pharmaceutical Sciences of
Ribeirão Preto
University of Sao Paulo
Ribeirao Preto, Brazil
Peter Proksch
Institute of Pharmaceutical Biology and
Biotechnology
Heinrich-Heine University
Düsseldorf, Germany
Mônica Tallarico Pupo
Laboratory of Microbial Chemistry
Department of Pharmaceutical
Sciences
School of Pharmaceutical Sciences of
Ribeirão Preto
University of Sao Paulo
Ribeirao Preto, Brazil
Shrinivasan Raghuraman
Department of Biology
University of Utah
Salt Lake City, Utah
Jeffrey D. Rudolf
Department of Chemistry
The Scripps Research Institute
Jupiter, Florida
Helena Safavi-Hemami
Department of Biology
University of Utah
Salt Lake City, Utah
Ben Shen
Department of Chemistry
Department of Molecular Medicine, and
Natural Products Library Initiative
The Scripps Research Institute
Jupiter, Florida
xviii Contributors

Michael Spiteller Gary Strobel

Department of Chemistry and Chemical
Biology
Institute of Environmental Research
Technical University of Dortmund
Dortmund, Germany
Andrea Stierle
Department of Biomedical and
Pharmaceutical Sciences
The University of Montana–Missoula
Missoula, Montana
Department of Plant Sciences
Montana State University
Bozeman, Montana
Russell W. Teichert
Department of Biology
University of Utah
Salt Lake City, Utah

Don Stierle Herbert Waldmann

Department of Biomedical and Department of Chemical Biology
Pharmaceutical Sciences Max Planck Institute of Molecular
The University of Montana–Missoula Physiology
Missoula, Montana Dortmund, Germany
 1 Microbial Genome
Mining for Natural
Product Drug Discovery
Richard H. Baltz

CONTENTS
1.1 Introduction........................................................................................................1
1.1.1 Importance of Natural Products and the Issue of Rediscovery..............1
1.1.2 Genome Mining Enhances Discovery and Circumvents Rediscovery....... 2
1.1.3 Scope of the Chapter..............................................................................2
1.2 The New Central Dogma of Genome Mining...................................................2
1.2.1 What Is Genome Mining and Why Is It Important?..............................2
1.2.2 What Are the Drivers for Successful Genome Mining?........................3
1.2.2.1 Primary Drivers.......................................................................3
1.2.2.2 Secondary Drivers.................................................................19
1.2.3 Strategies and Tactics for Genome Mining..........................................22
1.2.3.1 Mining Individual Microbes.................................................22
1.2.3.2 Mining Multiple Microbes....................................................25
1.3 Future Directions.............................................................................................30
1.3.1 Expanded Sampling for Gifted Microbes............................................30
1.3.2 Expanded Efforts to Obtain Finished Genomes for Gifted Microbes..... 31
1.3.3 Leveraging Genomics for Combinatorial Biosynthesis.......................32
References.................................................................................................................33

1.1 INTRODUCTION
1.1.1 Importance of Natural Products and the Issue of Rediscovery
Since the commercial development of penicillin and streptomycin in the 1940s
and 1950s, natural products (NPs) have played important, ever-expanding roles in
human medicine, animal health, and plant crop protection.1–4 In the early years of NP
discovery, it was relatively easy to discover new and novel antibiotics that were pro-
duced abundantly by many soil microorganisms, primarily actinomycetes.3,5–7 After
the most commonly produced antibiotics were discovered, it became progressively
more difficult to discover less-abundant antibiotics, and the previously ­discovered
antibiotics became a nuisance because of continuous rediscovery.5–7 Thus, as time
progressed from the 1950s through the 1980s, the problem of dereplication of known
compounds became evermore problematic, and the discovery process based on

1
2 Chemical Biology of Natural Products

low-throughput fermentation analysis became much less productive.3,6,8 In addi-

tion, NP discovery did not fit well with the new paradigm of the 1990s of high-
throughput screening of combichem and other chemical libraries against multiple
in vitro targets generated by microbial and human genomics projects.3

1.1.2 Genome Mining Enhances Discovery

and Circumvents Rediscovery

The first two complete genomic sequences of Streptomyces coelicolor and

Streptomyces avermitilis revealed that each organism encoded many more secondary
metabolite gene clusters (SMGCs) than were anticipated from their known second-
ary metabolomes.9,10 Many of the cryptic or silent gene clusters from these and other
streptomycetes have been investigated,11–14 and the observation of multiple cryptic
SMGCs has been generalized to many additional actinomycetes and to other cos-
mopolitan bacteria with large genomes.15 From these observations, the concept of
microbial genome mining emerged as a new paradigm for drug discovery.3,16–20 There
are a number of advantages to genome mining discussed in this chapter, not the least
of which is that it solves the confounding issues of rediscovery and dereplication of
known secondary metabolites (SMs).

1.1.3 Scope of the Chapter

Microbial genome mining can be directed at members of the three domains of life:
Bacteria, Archaea, and Eukarya. Because of space limitations and interests of the
author, this chapter is focused on Bacteria with some discussion on Archaea. The
fungi from the domain Eukarya are important sources of NPs, and interested readers
are directed to recent publications relevant to fungal genome mining.21–25

1.2 THE NEW CENTRAL DOGMA OF GENOME MINING

1.2.1 What Is Genome Mining and Why Is It Important?
When we think of chemical biology, we often think in terms of the central dogma
of DNA to RNA to protein, or DNA replication, transcription, and translation
leading to enzyme function and cell biology. In the past, NPs were discovered
in the absence of knowledge of the extended central dogma of DNA to RNA to
protein (enzymes) to NPs.16 Microorganisms were isolated, fermentations were
carried out under different conditions, fermentation broths were screened for
antibacterial and other activities, active chemical agents were purified, and struc-
tures determined. Often, novel chemical scaffolds with important activities were
further embellished by chemical modifications, semisynthesis, mutasynthesis,
and in some cases, combinatorial biosynthesis.3 Now with inexpensive microbial
genome sequencing, and growing knowledge of SM biosynthetic enzyme func-
tions with linkages to the responsible genes located in SMGCs,26 it is possible to
predict new and novel SMGCs and possible structures bioinformatically,27–30 and
to predict or correlate associated SMGCs from metabolomics/proteomics data.31–41
Microbial Genome Mining for Natural Product Drug Discovery 3

The following is the new central dogma for genome mining: DNA sequence pre-
dicts new and novel chemical structures (genotype to chemotype); and new and
novel chemical structures predict associated DNA sequences (chemotype to
genotype).16,41 In the forward direction, DNA that predicts new or novel chem-
istry exerts enormous leverage for discovery because it eliminates the confound-
ing issues of rediscovery and dereplication that have stifled the productivity for
NP discovery in recent years. Although bioinformatics predictions from genome
sequences do not need inputs from the RNA and protein parts of the extended
central dogma (transcription and translation), bringing genome mining into a pro-
ductive discovery mode requires efficient transcription and translation of cryptic
SMGCs by using a variety of approaches.42

1.2.2 What Are the Drivers for Successful Genome Mining?

Successful Pharmaceutical or Biotechnology Company research programs start with
a vision and mission; then address these by developing strategies, tactics, and research
plans; and follow by executing the plans. The vision and mission for NP discovery
remain the same as 50 years ago: The vision is that NPs are important sources for
drug discovery and the mission is to discover and develop NPs for human medicine,
animal health, and plant crop protection. What has changed dramatically in the last
few years is the strategy and tactics that have engaged genomics and metabolomics
approaches to discovery. What is urgently needed to ensure success is refined strate-
gies and tactics based upon relevant new information (e.g., which microbes are the
most gifted for SM biosynthesis), and the development of appropriate research plans
that can be executed in time frames consistent with robust drug discovery.
To address the overall vision of revitalized NP discovery, I have organized the
various approaches and disciplines from the following standpoints: (i) what are
the most critical strategic issues and approaches (primary drivers), and (ii) what
are the key tactical approaches that directly support or enable the process to succeed
(secondary drivers).

1.2.2.1 Primary Drivers
1.2.2.1.1 Microbes Gifted for Natural Product Biosynthesis
At the front end of the genome mining process is the genomic sequence information
from microorganisms. It is well documented that not all microorganisms are created
equal when it comes to NP production.15,18,19,43–50 Culturable bacteria and archaea
have genomes ranging from <2 to >13 Mb, and the ones with the largest genomes
tend to encode large numbers of SMGCs.15 Across the culturable bacterial phyla,
the ability to encode multiple SMGCs is concentrated within the Actinobacteria,
Proteobacteria, Cyanobacteria, and Firmacutes.15,47–50 However, even among these
major culturable phyla, many species have small genomes, and are not suitable
choices for robust NP discovery.15 A recent program sponsored by the Department of
Energy—Genomic Encyclopedia of Bacteria and Archaea (GEBA-1)—has gener-
ated 1003 high-quality genome sequences of a wide range of bacterial- and archaeal-
type strains. The 974 bacterial species include many Proteobacteria, Firmacutes,
Bacteroidetes, Actinobacteria, and species from 17 other phyla.51 The distribution
4 Chemical Biology of Natural Products

25

20

15
% of total

10

0
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Genome size range

FIGURE 1.1 Distribution of genome sizes in bacterial-type strains sequenced in the GEBA-1
project.51 Horizontal axis, 1 = 0–1 Mb; 2 = 1–2 Mb; and so forth.

of bacterial genome sizes is summarized in Figure 1.1. Less than 2% of the bacterial
genomes are >8.0 Mb, and >80% have genomes ranging from <1.0 to 5.0 Mb. None
of the 29 archaeal-type strains has genomes >6 Mb (not shown). In contrast, most
bacteria gifted for SM production have genomes >8.0 Mb15 (see below).
The vast majority of microbes (>99%)52,53 are unculturable on typical microbio-
logical media, and these have been the source of much speculation and conjecture
about their potential for SM production.54–58 In the following sections, I discuss
the SM coding capacity of culturable and uncultured bacteria and archaea from a
variety of environmental sources. Accurate information on microbial SM coding
capacity is critical to inform future strategic and tactical approaches to revitalize
NP discovery.

1.2.2.1.1.1  Actinomycetes Microbes with very large genomes tend to be cos-

mopolitan, and encode diverse primary metabolic functions that facilitate growth and
reproduction on a wide range of nutrient sources.59 Many of these also encode SMs,
including antibacterial and antifungal agents among others. The most gifted micro-
organisms for SM production based on antiSMASH 3.0 analysis30 are actinomycetes
with genomes ranging from 8.0 to 12.7 Mb.15 Table 1.1 shows the SM coding capac-
ity for 18 actinomycetes with genomes >8.0 Mb that have been sequenced to comple-
tion. On average, they devote 1.64 Mb (16% of their coding capacity) to encode
35 SMs. The most gifted strains, Kutzneria albida, Streptomyces bingchenggensis,
and Streptomyces rapamycinicus, devote 2.5–3.09 Mb (>20% of coding capacity) to
encode 48–53 SMs. The 18 strains also encode an average of 17 SMs that employ
PKS, NRPS, or mixed NRPS/PKS biosynthetic mechanisms. The three most gifted
strains encode 24–30 of these pathways. In addition, the 18 actinomycetes encode an
average of 4.8 MbtH homologs and 3.6 phosphopantetheinyl transferases (PPTases).
MbtH homologs participate as chaperones in adenylation reactions carried out by
NRPS multienzymes, and PPTases are required to convert apo-ACPs and apo-PCPs
TABLE 1.1
SM Coding Capacity in Select Strains of Cultured Bacteria and Archaeaa
Microorganism Size (Mb) MbtH Genes PPTase Genes NRPS/PKS Clustersb Total Clusters Total SMs (Mb) % of Genome
Actinomycetes with large genomes
Streptomyces leeuwenhoekii DSM 42122 8.12 3 3 16 35 1.57 19.9
Saccharopolyspora erythraea NRRL 2338 8.21 3 3 16 36 1.53 18.6
Actinosynnema mirum DSM 43827T 8.25 7 2 18 30 1.59 19.2
Streptomyces collinus Tü 365 8.27 4 6 14 31 1.37 16.6
Streptomyces ambofaciens ATCC 23877 8.39 2 4 10 27 1.11 13.2
Streptomyces griseus NBRC 13350 8.55 8 3 17 40 1.71 20.0
Streptomyces coelicolor A3(2) 8.67 2 3 11 27 0.93 10.7
Actinoplanes missouriensis ATCC 14538T 8.77 2 2 6 11 0.46 5.3
Amycolatopsis orientalis HCCB10007 8.95 4 4 15 32 1.45 16.2
Streptomyces avermitilis MA-4680 9.03 4 7 17 37 1.51 16.7
Saccharothrix espanaensis DSM 44229T 9.36 6 1 15 35 1.66 17.7
Kutzneria albida DSM 43870T 9.88 9 3 24 48 2.50 25.3
Streptomyces hygroscopicus 5008 10.15 5 5 17 40 1.47 14.5
Amycolatopsis mediterranei U32 10.24 6 3 17 30 1.35 13.2
Streptosporangium roseum DSM 43021T 10.34 8 4 15 26 1.18 11.4
Streptomyces violaceusniger Tu4113 10.66 3 4 22 43 2.33 21.9
Streptomyces bingchenggensis BCW-1 11.94 4 5 30 53 2.60 21.7
Microbial Genome Mining for Natural Product Drug Discovery

Streptomyces rapamycinicus NRRL 5491 12.70 7 3 28 52 3.09 24.3

Average 9.47 4.8 3.6 17.1 35.2 1.64 16.0
Bacteroidetes/Chlorbi
Prevotella denticola F089 2.94 0 1 0 2 0.09 3.1
Elizabethkingia anophelis NUHP1 4.37 0 1 0 5 0.10 2.4
Bacteroides vulgaris ATCC 8462 5.16 0 1 0 1 0.05 0.9
5

(Continued)
 6

TABLE 1.1 (Continued)

SM Coding Capacity in Select Strains of Cultured Bacteria and Archaea a
Microorganism Size (Mb) MbtH Genes PPTase Genes NRPS/PKS Clustersb Total Clusters Total SMs (Mb) % of Genome
Bacteroides fragilis YCH46 5.28 0 1 0 1 0.05 0.8
Bacteroides ovatus ATCC 8483 6.47 0 1 0 1 0.05 0.7
Cyanobacteria
Prochlorococcus marinus SS120 1.75 0 1 0 4 0.07 4.0
Pleurocapsa sp. PCC7319 4.99 0 2 5 11 0.33 6.7
Microcystis aeruginosa NIES-843 5.84 0 1 5 9 0.40 6.8
Cyanothece sp. PCC7424 6.55 0 1 3 10 0.28 4.2
Calothrix sp. PCC6303 6.96 0 2 2 12 0.41 5.9
Anabaena variabilis 7.11 0 2 6 14 0.59 8.3
Firmicutes—Bacilli
Lactobacillus rhamnosus GG 3.01 0 1 0 0 0.00 0.0
Lactobacillus casei BD-II 3.07 0 1 0 1 0.01 0.3
Lactobacillus plantarum WCFS1 3.31 0 3 1 2 0.08 2.4
Bacillus subtilis 168 4.22 1 2 5 17 0.65 15.4
Bacillus thuringiensis St. 97-27 5.24 1 3 1 5 0.23 4.4
Paenibacillus polymyxa SC2 5.73 0 3 7 12 0.68 11.9
Paenibacillus mucilaginosus 3016 8.74 0 3 10 16 0.94 10.8
Firmicutes—Clostridia
Ruminiclostridium therocellum DSM 1313 3.56 0 2 2 3 0.11 3.1
Clostridium kluyveri DSM 555 3.96 0 3 3 3 0.19 4.8
Clostridium pasteurianum BC1 4.99 0 2 1 2 0.07 1.4
Desulfitobacterium hafniense Y51 5.73 0 2 1 3 0.07 1.2
Clostridium beijerinckii NCIMB 8052 6.00 0 2 1 1 0.07 1.2
(Continued)
Chemical Biology of Natural Products
TABLE 1.1 (Continued)
SM Coding Capacity in Select Strains of Cultured Bacteria and Archaea a
Microorganism Size (Mb) MbtH Genes PPTase Genes NRPS/PKS Clustersb Total Clusters Total SMs (Mb) % of Genome
Proteobacteria—Alpha
Wolbachia endosymbiont 1.30 0 1 0 0 0.00 0.0
Acetobacter pasteurianus IFO 3283-01 3.34 0 1 0 3 0.06 1.8
Caulobacter crescentus CB15 4.01 0 1 0 4 0.09 2.1
Rhizobium leguninosarum WSM1689 6.90 1 3 1 5 0.13 1.9
Agrobacteriumm tumifaciens K84 7.27 0 3 3 8 0.24 3.3
Proteobacteria—Beta
Neisseria meningitidis MC58 2.27 0 1 0 5 0.13 5.7
Limnohabitans sp. 103DPR2 2.95 0 1 0 2 0.04 1.4
Comamonas testosteroni CNB-2 5.37 0 1 0 2 0.06 1.1
Burkholderia thailandensis E264 6.72 1 5 8 19 0.92 13.7
Burkholderia pseudomallei 1710b 7.31 3 3 11 27 1.03 14.1
Burkholderia gladioli BSR3 9.05 5 3 9 19 0.84 9.3
Proteobacteria—Gamma
Acinobacter johnsonii XBB1 4.08 0 1 0 3 0.07 1.7
Alteromonas mediterranea DE 4.48 0 1 0 1 0.01 0.2
Escherichia coli W 5.01 1 3 1 1 0.05 1.0
Aeromonas salmonicida A449 5.04 0 3 2 4 0.16 3.2
Microbial Genome Mining for Natural Product Drug Discovery

Azotobacter vinelandii DJ 5.37 2 2 4 8 0.30 5.6

Photorhabdus luminescens TT01 5.69 2 3 12 21 1.01 17.8
Pseudomonas syringae B728a 6.09 1 2 7 12 0.74 12.2
Pseudomonas fluorescens F113 6.85 1 2 3 9 0.35 5.1
(Continued)
7
 8

TABLE 1.1 (Continued)

SM Coding Capacity in Select Strains of Cultured Bacteria and Archaea a
Microorganism Size (Mb) MbtH Genes PPTase Genes NRPS/PKS Clustersb Total Clusters Total SMs (Mb) % of Genome
Proteobacteria—Delta
Desulfovibrio desulfuricans ATCC 27774 2.87 0 3 1 1 0.06 2.1
Desulfovibrio alaskensis G20 3.73 0 2 1 1 0.06 1.6
Geobacter metallireducens GS-15 4.01 0 1 0 4 0.11 2.7
Myxococcus fulvus HW-1 9.00 1 3 13 25 1.13 12.6
Myxococcus xanthus DK 1622 9.14 1 3 15 23 1.09 11.9
Sorangium cellulosum so ce56 13.03 1 3 10 32 1.21 9.3
Proteobacteria—Epsilon
Campylobacter jejuni ATCC 700819 1.64 0 1 0 0 0.00 0.0
Helicobacter pylori 26695 1.67 0 1 0 0 0.00 0.0
Nautilia profundicola AmH 1.68 0 1 0 0 0.00 0.0
Sulfurimonas autotrophica OK10 2.15 0 1 0 0 0.00 0.0
Arcobacter butzleri RM4018 2.34 0 1 0 0 0.00 0.0
Sulfurospirrilum barnesii SES-3 2.51 0 2 1 2 0.96 3.8
Archaea
Methanococcus jannaschii 1.66 0 0 0 0 0.00 0.0
Pyrococcus sp. NA2 1.86 0 0 0 1 0.01 0.5
Methanobacterium sp. MB1 2.03 0 1 1 2 0.09 4.4
Methanobrevibacter sp. YE315 2.27 0 0 4 4 0.23 10.0
Vulcanisaeta distributa DSM.14429 2.37 0 0 0 1 0.01 0.5
Sulfolobus islandicus 2.78 0 0 0 1 0.01 0.4
Methanobrevbacter ruminantium M1 2.94 0 1 2 2 0.10 3.4
Methanocella paludicola SANAE 2.96 0 1 1 1 0.05 1.5
Methanosarcina barkeri Fusaro 4.84 0 0 0 0 0.00 0.0
Methylobacterium extorquens AM1 5.51 0 2 2 7 0.21 3.5

a Table modified from Baltz.15

b NRPS, PKS, or mixed NRPS/PKS pathways.
Chemical Biology of Natural Products
Microbial Genome Mining for Natural Product Drug Discovery 9

to holo-enzymes required for polyketide and nonribosomal peptide assembly by PKS

and NRPS multienzymes.
There are many actinobacteria, including actinomycetes, with genomes ranging
from <3.0 to 8.0 Mb.51 Figure 1.2a shows the SM coding capacity of a sample of
completed actinomycete genomes ranging in size from 3.6 to 12.7 Mb.15 All encode
SMs, but none of the strains with genomes <8.0 Mb devotes >1.0 Mb to encode
SMGCs. The regression line drawn through the most productive strains may define
an upper limit to coding capacity for SMs, 0.31 Mb SM coding capacity per Mb

(a) 3.5

2.5
SMGCs (Mb)

2 Highly gifted

1.5
Gifted
1

0.5

0
0 2 4 6 8 10 12 14
Genome size (Mb)

(b) 3.5

2.5
SMGCs (Mb)

1.5

0.5

0
0 5 10 15
Genome size (Mb)

FIGURE 1.2 SM coding capacity versus genome size in select cultured bacteria. (a)
Actinomycetes. (b) Actinomycetes and other bacteria. Data from Baltz15 except that two addi-
tional myxobacterial genomes, Archangium gephyra DSM 2261 (12.5 Mb) and Chondromyces
crocatus (11.3 Mb), are added as stars.
10 Chemical Biology of Natural Products

above the X intercept of 3.0 Mb, below which no significant SMGC coding capac-
ity is predicted. This is consistent with data from an earlier study.46 Thus, the rela-
tionship of SM coding capacity (Y) is related to genome size (X) by the following
equation: Y = 0.31(X-3). From this simple relationship, it is clear that actinomycetes
with large genomes are the most likely to encode large numbers of SMs, including
many of PKS, NRPS, and mixed NRPS/PKS origin, the hallmarks of the most com-
mercially successful SMs historically.3 It is noteworthy that the medium size of 325
draft genomes of unspeciated Streptomyces strains at the time of this writing was
8.2 Mb (http://www.ncbi.nlm.nih.gov/genome), suggesting that many more gifted
Streptomyces sp. are already available for further analysis and genome mining.

1.2.2.1.1.2  Other Culturable Bacteria Table 1.1 also shows the SM coding

capacity for several culturable bacterial phyla.15 The bacterial and archaeal strains
with genomes <6.0 Mb generally devote <0.1 Mb of coding capacity to SMGCs,
including 0–3 NRPS/PKS clusters. Exceptions are strains of Bacillus, Paenibacillus,
Cyanobacteria, Azotobacter, and Photorhabdus. Among the 56 strains surveyed, 12
devote >0.5 Mb to SMGCs, including 7 that encode >1.0 Mb. The 10 most gifted bac-
terial taxa—Paenibacillus mucilaginosa, Burkholderia thailandensis, Burkholderia
pseudomallei, Burkholderia gladioli, Photorhabdus luminescens, Myxococcus
fulvus, Myxococcus xanthus, Chondromyces crocatus, Archangium gephyra, and
Sorangium cellulosum—encode 16–43 SMGCs, including 8–17 NRPS/PKS clus-
ters. Seven of these have genomes >8.0 Mb, including all five myxobacterial strains
(Table 1.1). The DNA coding capacity of the 56 bacterial species versus genome
size is combined with the data on actinomycetes in Figure 1.2b. The SMGC coding
capacity is 0 to very low in genomes <3.0 Mb.

1.2.2.1.1.3  Culturable Archaea In stark contrast to the actinomycetes with large

genomes and other gifted bacterial species, members of domain Archaea generally
have small genomes, mostly <3.0 Mb,51 and are nearly devoid of relevant SMGCs
(Table 1.1).15 There are no examples of important SMs produced by microbes from
the domain Archaea.3

1.2.2.1.1.4   Uncultured Bacteria and Archaea There has been a long-standing

conjecture dating back about two decades that uncultured microbes, which make up
>99% of the microbial world52 and encompass more than 50% of the known bacterial
phyla,53 may serve as inexhaustible sources of novel antibiotics and other SMs.54–58
Several companies, including TerraGen Diversity, Diversa, Ariad Pharmaceuticals,
and Cubist Pharmaceuticals, pursued cloning metagenomic DNA in the late 1990s
to early 2000s, but no novel NPs of clinical utility were discovered. More recently,
the Brady laboratory at Rockefeller University made impressive advances on tech-
nical aspects of metagenomic mining for NP pathways.60–66 One interesting result
potentially useful for drug development was the discovery of novel glycopeptide
antibiotics related to vancomycin.67,68 However, this discovery is not relevant to
cloning SMGCs from unculturable microbes, because many glycopeptide antibiot-
ics (e.g., vancomycin, balhimycin, teicoplanin, dalbavancin, A47934) are produced
by a wide range of readily culturable actinomycete genera. Importantly, additional
Another random document with
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 “This is the spirit, the spirit is present,
The spirit of this tapu!
The boy will be angry,
The boy will flame,
The boy will be brave,
The boy will possess thought.
Name this boy
That he may be angry, that he may flame,
To make the hail fall:
Dedicate him to fight for Tu;
Ward off the blow that he may fight for Tu.
The man of war jumps and wards off the blows.”

Here the ceremony terminated, and the assembly, as if inspired,

jumped up, and rushed to the fight, while the priest repeated the
following karakia, standing on some elevated spot, from which he
could command a view of the battle:

“The god of strength, let him be present;

Let not your breath fail you.”

After the battle was over the priest called those who survived, and
enquired of each if he had killed anyone, or taken any prisoners. All
who had been in battle before delivered up their weapons to him,
who deposited them in the house where they were kept. Those who
had fought for the first time were called and asked if they had killed
anyone. If the person addressed replied in the affirmative, the priest
demanded his mere—stone battle-axe—and broke it into pieces.
This was the invariable custom with young warriors when they had
imbued their hands in the blood of their enemies. The priest having
afterwards assembled them together, used the following words,
which were called the Haha:
 “This is the wind, the wind is feeding;
The wind descends,
The wind is prosperous,
The many sacred things of Tu.
The wind descends,
The wind is prosperous,
The living wind of Tu.”

The natives regard the wind as an indication of the presence of

their god, if not the god himself. After this ceremony the youths were
considered as men, though they were narrowly watched for some
time by the priest, and they were liable to be put to death if they
broke any of the sacred rules of the tapu. They could not carry a
load, cut their own hair, or plait a woman’s. If one of them was
discovered by the priest doing any of these things, he assumed his
authority, and pronounced the sentence of death by saying “Go
away, go away.” This so affected the person to whom it was
addressed, that it was quite sufficient to kill him.
There was another ceremony performed after fighting, which was
supposed to confer a benefit on all who had been engaged in the
battle, and were successful in killing or making slaves. It was called
he pureinga, which means a taking off of that sacredness which had
been put upon them before the fight, or, in other words, the taking off
the tapu:

“There is the wind;

The wind rests;
The wind is feeding;
The wind which gathers—
O wind subside!
O living wind!
O sacred wind of Tu!
Loose the tapu,
The god of strength;
Let the ancient gods dismiss the tapu,
O ... o ... o ... the tapu is taken away!”
 When they went to war, they were separated from their wives, and
did not again approach them until peace was proclaimed. Hence,
during a period of long-continued warfare, they remarked that their
wives were widows.
When a party attacking a pa had forced an entrance, they
generally killed all within it. At the time of the slaughter the victors
pulled off a lock of hair from each victim, and also from those they
saved as slaves, which they stuck in their girdles. When the carnage
was over, they assembled in ranks, generally three deep, each party
being headed by its own tohunga, to thank their gods, and also to
propitiate their favour for the future. When all the necessary
arrangements were made, they each gave the tohunga a portion of
the hair they had collected, which he bound on two small twigs;
these he raised above his head, one in each hand, the people doing
the same, except that they used twigs without any hair. They
remained in this posture whilst the priest offered a prayer for the
future welfare of the tribe. He then cast the twigs with the hair bound
to them from him, as did the warriors with theirs, and all joined in a
puha or war song. Then, standing quite naked, they clapped their
hands together and struck them upon their thighs in order to take off
the tapu from their hands which had been imbued in human blood.
When they arrived within their own pa, they marched slowly, and in
order, towards the house of the principal tohunga, who stood in his
waho tapu or sacred grove ready to receive them. As soon as they
were about one hundred yards from him, he called out, “Whence
comes the war party of Tu?” Whereupon he was answered by the
tohunga of the party. “The war party of Tu comes from the search.”
“From whence comes the war-party of Tu?” “The war party of Tu
comes from the stinking place.” “From whence comes the war party
of Tu?” “It comes from the south; it comes from the north; it comes
from the thicket where birds congregate; it comes from the
fortifications: it made speeches there; it heard news there.”
 New Zealand Arms.
When they got near the principal tohunga, the warriors gave the
remaining locks of hair to their own priests, who went forward and
presented them to the chief one: he offered them to the god of war,
with many prayers. They then performed the tupeke, or war dance,
and clapped their hands a second time. The slave of the tohunga
belonging to the war party then made three ovens, in which he
cooked a portion of the hearts of the principal warriors of the
conquered party. “When they were done, the chief tohunga took a
portion, over which he uttered a harakia, and then threw it towards
his god as an offering. Having eaten all the food of the three ovens,
he took the tapu off the warriors, and they were permitted to “tangi,”
or cry with their relations. The women came out armed, and if any of
the attacking party had been lost in the assault, they fell upon the
slaves and killed as many as they could. Among the Taupo tribes it
was not lawful for women and girls to eat human flesh, though this
restriction does not appear to have extended to other parts of the
island.
 Australian Weapons.
As we are now as close to Australia as we are likely for some time
to be, we may as well take a voyage over and see what sort of man
of war our dirty little friend the Bushman is. He is not at a loss for
weapons, nor for skill to use them. They may be enumerated as
follows:—The spear, nine or ten feet long, rather thicker than one’s
finger, tapered to a point, hardened in the fire and sometimes
jagged. The wammera or throwing stick, shows considerable
ingenuity of invention; about two and a half feet long, it has a hook at
one end which fits a notch on the heel of the spear, in whose
projection it acts, much like a third joint of the arm, adding very
greatly to the force. A lance is thrown with ease and accuracy sixty,
eighty, and a hundred yards. The waddy is a heavy knobbed club
about two feet long, and is used for active service, foreign or
domestic. It brains the enemy in the battle, or strikes senseless the
poor gin in cases of disobedience or neglect. In the latter instance a
broken arm is considered a mild martial reproof.

Throwing the Boomerang.

The stone tomahawk is employed in cutting opossums out of their
holes in trees, as well as to make notches in the bark, by inserting a
toe into which, the black can ascend the highest and largest gums in
the bush. One can hardly travel a mile in New South Wales without
seeing these marks, old or new. The quick eye of the native is
guided to the retreat of the opossum by the slight scratches of its
claws on the stem of the tree. The boomerang, the most curious and
original of Australian war implements, is, or was, familiar in England
as a toy. It is a paradox in missile power. There are two kinds of
boomerang, that which is thrown to a distance straight ahead, and
that which returns on its own axis to the thrower. “I saw,” says Mr.
Mundy, “a native of slight frame throw one of the former two hundred
and ten yards and much further when a ricochet was permitted. With
the latter he made several casts truly surprising to witness. The
weapon after skimming breast high, nearly out of sight, suddenly
rose high into the air, and returning with amazing velocity towards its
owner, buried itself six inches deep in the turf, within a few yards of
his feet. It is a dangerous game for an inattentive spectator. An
enemy or a quarry ensconced behind a tree or bank safe from spear
or even bullet, may be taken in the rear and severely hurt or killed by
the recoil of the boomerang. The emu and kangaroo are stunned
and disabled, not knowing how to avoid its eccentric gyrations.
Amongst a flight of wild ducks just rising from the water, or a flock of
pigeons on the ground, this weapon commits great havoc. At close
quarters in fight the boomerang, being made of very hard wood with
a sharp edge, becomes no bad substitute for a cutlass.
The hieleman or shield, is a piece of wood about two and a half
feet long, tapering to the ends with a bevelled face not more than
four inches wide at the broadest part, behind which the left hand
passes through a hole perfectly guarded. With this narrow buckler
the native will parry any missile less swift than the bullet.
In throwing the spear after affixing the wammera, the owner
poises it, and gently shakes the weapon so as to give it a quivering
motion which it retains during its flight. Within fifty or sixty paces the
kangaroo must, I should conceive, have a poor chance of his life.
 Hurling the Spear.

The spear is immeasurably the most dangerous weapon of the

Australian savage. Many a white man has owed his death to the
spear; many thousands of sheep, cattle, and horses have fallen by it.
Several distinguished Englishmen have been severely wounded by
spear casts; among whom I may name Captain Bligh, the first
governor of New South Wales, Sir George Grey, and Captain
Fitzgerald, the present governors of New Zealand and Western
Australia, and Captain Stokes, R.N., long employed on the survey of
the Australian coasts. The attack by the blacks upon the Lieut.-
Governor of Swan River, occurred so lately as December 1848. In
self-defence he was compelled to shoot his ferocious assailants just
too late to save himself, being seriously hurt by a spear passing
through his thigh.
Our artist, Mr. Harden S. Melville, while attached to the Australian
exploring expedition, in H.M.S. “Fly,” had a narrow escape from
making a disagreeably close acquaintance with one of these
formidable barbed war tools. The ship’s boat had put ashore at a
spot where there was a congregation of native huts, though not a
solitary human inhabitant could be distinguished. With a spirit,
however, which evinced more devotion to the cause of science than
to the usages of polite society, our friend must needs penetrate to
the interior of one of the kennel-like abodes, though to effect this
purpose he had to crawl on all fours. Whether he found anything to
repay him for his pains I don’t recollect; I only know that he had
barely scrambled to the perpendicular, with his back to the bush,
when the seaman who was with him, with laudable promptitude,
called his attention to an interesting object in the distance. It was a
native—the owner of the house Mr. Melville had so unceremoniously
ransacked, no doubt—and there he stood with his spear nicely
adjusted to the wammera and all a-tremble for a cast. The instant,
however, that our artist (who I may tell the reader is a perfect giant)
turned his face instead of his back to the native, the spear was
lowered and the danger at an end.
 Australian Duel.
Lax as is the native Australian’s morality still he has his code of
honour and should one of its articles be infringed he will not be
content to lay wait for the aggressor and drive a spear through his
back, or strike him dead with his boomerang while he is safe
concealed and secure from observation; no, he must have “the
satisfaction of a gentleman,” he must call his man “out,” and compel
him to be murdered or commit a murder. So in this respect the
bushman, “the meanest specimen of humanity,” is as respectable an
individual as many a noble born and highly educated Englishman,
who lived in the reign and basked in the friendship of the “first
gentleman in Europe.” He shows himself even more respectable; for
whereas gentlemen of a past generation would meet and fire bullets
or dash and stab at each other with naked swords about ever so
trifling a matter, as a dispute about the cut of a coat or the character
of a sweetheart, the bushman never appeals to the honourable
institution of duelling, except an enemy be guilty of the heinous
offence of denying that he has a thick head. “He no good, his scull
no thicker than an emu’s egg-shell.” If a bushman brook such an
insult as this he is for ever the scoff and jest of all who know him; but
the chances are that he will not brook the insult; he will send a friend
to the slanderer to bid him bring his stoutest “waddy,” that it may be
shivered on the thick head of the warrior he had traduced.
The combatants meet and a select party of friends are invited to
see fair. The weapons are the familiar “waddys,” and the men stand
opposite each other with their heads bare. There is no tossing for
position or any other advantage; indeed there is no advantage to be
gained excepting who shall have first “whack,” and that is always
allowed to the challenger. The man who is to receive first whack, if
he is a person of experience, knows the hard and soft parts of his
cranium and takes care so to manœuvre that the former shall be
presented to the up-raised club. Down comes the weapon with a
thud that makes the recipient’s teeth chatter, but beyond that he has
sustained no inconvenience, and now he straightens his back and
grins, for it is his turn. His opponent lowers his head as he had done
and a loud hollow noise follows, which the man’s friends hail with
delight, as it indicates that though his skull may be dented it is not
yet cracked. And so the duel proceeds, whack for whack, until one
mightier than before, or on a “sore place,” stretches one of them on
the grass.
 CHAPTER XXIV.

Caffre warfare—Great cry and little carnage—A Caffre war chant—

War song of the renowned Cucutle—A Griqua Pitsho—An African
council of war—The chiefs speech—The chief accused of apathy
—A reproof to the kidney-eaters—Death before dishonour—
Archery in Eastern Africa—Fan bowmen—War weapons of
cannibal Fans—War knives and brain hatchets—The women
warriors of Dahomey—The king’s fingers—King Gezo likened to a
hen—Amazon parables—Pretty picture of an Abyssinian warrior
—Omen birds—A non-believer in English gunnery—The sceptic
convinced—A potent candle end—Savage metallurgy—The king
and the blacksmith—Le Vaillant turns bellows mender.
urning to Southern Africa, we find that among the Caffres
the trade of war is conducted with a method and
precision seldom found among savages. The most
common causes of warfare are, what is proper tribute to
the chief, grazing privileges, and territorial boundaries;
no body of men, however, ever fall upon another body of their
inimical countrymen without certain formalities are observed, with a
view to warn the enemy what he may shortly expect, and to prepare
himself accordingly. Bearing in their hands the tail either of a lion or a
panther, ambassadors are sent to enquire whether the other side still
persist in their obstinacy; if so, the tails are flourished threateningly,
which is equivalent to a declaration of war.
The declaration made, all the vassal chiefs with their dependants
are summoned to assemble. Everyone must implicitly obey this
mandate, and follow his leader; whoever does not, is in danger of
having his whole property confiscated. As soon as the army is
collected at the habitation of the king, a number of deer are killed,
that the warriors may be strengthened for the fight by eating
abundantly of their flesh; at the same time they dance, and deliver
themselves up entirely to rejoicing. The king presents the most
distinguished and the most valiant among the Caffres with plumes of
feathers from the wings of a sort of crane, and these they wear upon
their heads as marks of honour. These plumes are regarded as
official badges, and those wearing them are looked on as officers;
and it is expected that every man so distinguished will not only
manœuvre his company, but, spear or club in hand, head it and do
battle with the leading warrior on the opposing side. If a leader shirks
his duty, he is condemned by the Caffre law to an ignominious death.
Among the followers, too, whoever forsakes his leader is slain as
soon as captured.
When the army moves, it takes with it as many deer as are
deemed necessary for its support; and when the stronghold is
approached, the “tail-bearers” are once more sent forward to give a
last notice of the intended attack, repeating the motives which have
given occasion to the war. If the enemy declares that at present he is
not quite prepared,—that he has not yet collected his fighting men;
or that it would be much more convenient if the other party would
wait till the blacksmiths had made a few more assagies and
sharpened the old ones,—the attacking party is content to squat
down and kill and eat their bullocks and smoke their pipes till the
enemy notifies his readiness to begin. A wide open place, without
bushes and without rocks, is chosen as the field of battle, to avoid all
possibility of an ambush, which is considered as wholly degrading.
The two armies, raising a loud war cry, approach in two lines till
they are within seventy or eighty paces of each other. They now
begin throwing their assagies, at the same time endeavouring to turn
aside those of the enemy. The king or commander-in-chief, whoever
he may be, remains always in the centre of the line, and takes an
active part in the fight. Some of the inferior commanders remain near
him, the rest remaining at the heads of their divisions. By degrees
the two bands approach nearer and nearer to each other, till at
length they come hand to hand, when the spears are thrown aside,
reliance being placed on the clubs to decide the fortunes of the day.
Should night surprise the combatants, hostilities are suspended,
the chiefs of either party meeting and endeavouring to bring about a
treaty of peace; but should this be found impracticable, the fight
commences again in the morning. If one of the armies takes to flight,
the commander alone is blamed: everything depends on his
personal bravery; and his falling back is the signal for the whole body
to do the same. A flying enemy is immediately pursued; and above
all things the conquerors seek to possess themselves of their women
and children and cattle. If the vanquished party agrees to submit, his
submission is immediately accepted, on condition that he
acknowledges his conqueror from that time forward as his sovereign,
and solemnly promises obedience to him. When this is done, the
captured women and children are sent back, as well as part of the
cattle taken, it being a household maxim among these people that
“we must not let even our enemies die with hunger.”
In these Caffre fights, however, the loss of life is never very
considerable; the assagie is the principal weapon, and with it the
Caffre is a not very certain marksman. To see the dancing and
yelling, and the air thick with spears, one would suspect the
bloodiest carnage; but it will often happen that after a few hours’
battle, in which say two thousand are engaged, it is a great chance if
more than about twenty on each side are slain and about double that
number wounded.
Caffre warfare, too, is merciful, as well from deliberation as from
ignorance; and one falling unarmed into the hands of the enemy is
seldom or never put to death; the women and children equally have
nothing to fear for their lives. For this reason, women are sometimes
employed as ambassadors, when there is danger that matters have
been pushed too far, and that a male negotiator may be put to death
before he has time to explain his errand.
“The Basutos and the Caffres,” says Mr. Cassalis, “are
passionately fond of a kind of war-dance, at which the women are
only present to aid by their songs and cries. A circle is formed by
some hundreds of robust men, having the head adorned with tufts
and plumes, and a panther’s skin thrown over the left shoulder. The
signal is given, the war-song commences, and the mass moves
simultaneously as if it were but one man. Every arm is in motion;
every head turns at once; the feet of all strike the ground in time with
such force that the vibration is felt for more than two hundred yards.
Every muscle is in movement; every feature distorted; the most
gentle countenance assumes a ferocious and savage expression.
The more violent the contortions, the more beautiful the dance is
considered. This lasts for hours; the song continues as loud and the
frantic gestures lose none of their vigour. A strange sound is heard
during the short intervals when the voices are silent in accordance
with the measure; it is the panting of the dancers, their breath
escaping with violence, and sounding afar off like an unearthly death
rattle. This obstinate prolongation of so fatiguing an exercise arises
from the challenges made to each other by the young men, which
are even sent from one village to another. The question is, Who can
keep up the longest? The gain of an ox depends upon a few more
leaps. Dancers have been seen to fall down dead upon the spot;
others receive injuries which are difficult to cure. There is another
war-dance which is less fatiguing. In this they form themselves in a
straight line, and then run forward singing as if they were about to
attack an enemy. When they have reached a certain distance, they
halt, some men leave the ranks, fence from right to left, and then
return to their comrades, who receive them with great acclamations.
As soon as the line is again unbroken they return in the same
manner to their starting point.
Besides war dances the savages of this region have war songs, of
which the two following will serve as samples:—

“Goloane is going to fight;

He departs with Letsie.
He runs to the enemy,
Him against whom they murmur,
Him whom they will never obey.
They insult his little red shield;
And yet it is the old shield
Of the ox of Tane.
What has not Mosheth just said?
Cease to defy Goloane the veteran.
However this may be, there are horses coming;
Goloane brings back from the battle
A grey horse and a red one;
These will return no more to their masters.
The ox without horns will not be restored.
To-day war has broken out
More fiercely than ever;
It is the war of Butsani and the Masetelis.
A servant of Mohato,
Goloane has hurled a piece of rock;
 Goloane has hurled a piece of rock;
He has hit the warrior with the tawny shield.
Do you see the cowardly companions of this overthrown warrior
Standing motionless near the rock?
Why can their brother not go and take away
The plumes with which they have adorned their heads?
Goloane, thy praises are like the thick haze
Which precedes the rain:
Thy songs of triumph are heard in the mountains;
They go down to the valleys
Where the enemy knelt before
The cowardly warriors!... They pray!...
They beg that food may be given to them—
They will see who will give them any.
Give to our allies,
To the warriors of Makaba;
To those whom we never see come to attack us.
Goloane returns lame from the strife;
He returns, and his leg is streaming;
A torrent of dark blood
Escapes from the leg of the hero.
The companion of Rantsoafi
Seizes an heifer by the shoulder;
It is Goloane, the son of Makao,
Descendant of Molise.
Let no one utter any more insolence!
Ramakamane complains—
He groans—he says that his heifer
Has broken his white shoulder.
The companion of the brave
Goloane has contended with Empapang and Kabane.
The javelin is flung!
Goloane avoids it skilfully,
And the dart of Rabane
Is buried in the earth!”

Here is another in which a warrior having fought his country’s

battles thinks it not unbecoming to be his own trumpeter:

“I am Cucutle!
The warriors have passed singing,
The hymn of the battle has passed by me;
It has passed, despising my childhood,
 p , p g y ,
And has stopped before the door of Bonkauku.
I am the black warrior.
My mother is Boseleso!
I will rush as a lion,
Like him that devours the virgins
Near the forests of Fubasekoa.
Mapatsa is with me—
Mapatsa, the son of Tele—
We set off singing the song of the Trot.
Ramakoala, my uncle, exclaims:
Cucutle, where shall we fight?
We will fight before the fires of Makoso.
We arrive....
The warriors of the enemy, ranged in a line,
Fling their javelins together;
They fatigue themselves in vain:
The father of Moatla rushes into their midst,
He wounds a man in the arm
Before the eyes of his mother,
Who sees him fall,
Ah! Where is the head of the son of Sebegoane?
It has rolled to the middle of his native town.
I entered victorious into his dwelling,
And purified myself in the midst of his sheepfold:
My eye is still surrounded with the clay of the victory.
The shield of Cucutle has been pierced;
Those of his enemies are intact,
For they are the shields of cowards.
I am the white thunder
Which growls after the rain!
Ready to return to my children,
I roar: I must have prey!
I see the flocks and herds escaping
Across the tufted grass of the plain;
I take them from the shepherd with the white and yellow shield.
Go up on the high rocks of Macate;
See the white cow run into the midst of the herd.
A Makose will no longer despise my club;
The grass grows in his deserted pens,
The wind sweeps the thatch
From his ruined huts;
The humming of the goats is the only noise that is heard
In his town, once so gay.
 Tired, and dying with thirst, I went to the dwelling of Entele;
His wife was churning delicious milk,
The foam of which was white and frothy
Like the saliva of a little child.
I picked up a piece of a broken pot
To drink out of the vessel,
Which I soon left empty.
The white cow that I conquered
Has a black head;
Her breast is high and open—
It was the nurse of the son of Matayane—
I will go and offer it to my prince.
The name of my chief is Makao,
And Makao is Makoo:
I swear it by the striped ox
Of Mamasike!”

During Mr. Moffat’s missionary sojourn among the natives of

Southern Africa it frequently fell to his lot to become pleader and
arbitrator in most important public matters. Once, when among the
Griquas, the neighbouring tribe of Mantatees threatened war; and
the fiery Griquas were eager to accept the challenge. The English
missionary, however, was against the whole business, and did not
hesitate so to express himself at the war council.
Orders were sent off to the different towns and villages that a
pitsho, or parliament, be convened on the following day. As subjects
of great national interest were to be discussed, all were in motion
early. About 10 a.m. the whole body of armed men, amounting to
about one thousand, came to the outskirts of the town, and returned
again to the public fold, or place of assembly, some singing war-
songs, others engaged in mock fights, with all the fantastic gestures
which their wild imaginations could invent. The whole body took their
seats lining the fold, leaving an arena in the centre for the speakers.
 African Arms.
A few short extracts from some of the speeches will serve to show
the manner in which these meetings are conducted. Although the
whole exhibits a very grotesque scene, business is carried on with
the most perfect order. There is but little cheering, and still less
hissing, while every speaker fearlessly states his own sentiments.
The audience is seated on the ground, each man having before him
his shield, to which is attached a number of spears; a quiver
containing poisoned arrows is hung from the shoulder; and a battle-