See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/289535259

Natural product discovery: past, present, and future

Article  in  Journal of Industrial Microbiology · January 2016

DOI: 10.1007/s10295-015-1723-5

CITATIONS READS

300 2,861

2 authors:

Leonard Katz Richard Baltz

University of California, Berkeley CognoGen
120 PUBLICATIONS   6,741 CITATIONS    169 PUBLICATIONS   7,653 CITATIONS   

SEE PROFILE SEE PROFILE

All content following this page was uploaded by Leonard Katz on 11 December 2017.

The user has requested enhancement of the downloaded file.

J Ind Microbiol Biotechnol (2016) 43:155–176
DOI 10.1007/s10295-015-1723-5
REVIEW
Natural product discovery: past, present, and future
Leonard Katz1 · Richard H. Baltz2 
Received: 13 November 2015 / Accepted: 14 December 2015 / Published online: 6 January 2016
© Society for Industrial Microbiology and Biotechnology 2016
Abstract  Microorganisms have provided abundant sources
of natural products which have been developed as com-
mercial products for human medicine, animal health, and
plant crop protection. In the early years of natural product
discovery from microorganisms (The Golden Age), new
antibiotics were found with relative ease from low-through-
put fermentation and whole cell screening methods. Later,
molecular genetic and medicinal chemistry approaches were
applied to modify and improve the activities of important
chemical scaffolds, and more sophisticated screening meth-
ods were directed at target disease states. In the 1990s, the
pharmaceutical industry moved to high-throughput screen-
ing of synthetic chemical libraries against many potential
therapeutic targets, including new targets identified from the
human genome sequencing project, largely to the exclusion
of natural products, and discovery rates dropped dramati-
cally. Nonetheless, natural products continued to provide key
scaffolds for drug development. In the current millennium, it
was discovered from genome sequencing that microbes with
large genomes have the capacity to produce about ten times
as many secondary metabolites as was previously recog-
nized. Indeed, the most gifted actinomycetes have the capac-
ity to produce around 30–50 secondary metabolites. With the
the Genomic Era. Dedicated to Professor Satoshi Ōmura for his
Special Issue: Natural Product Discovery and Development in
numerous contributions to the field of natural products.
* Richard H. Baltz
rbaltz923@gmail.com
1 are important and valuable agents: pharmaceuticals, her-
Synthetic Biology Engineering Research Center, University
of California-Berkeley, 5885 Hollis St. 4th Floor, Emeryville,
CA 94608, USA
2
CognoGen Biotechnology Consulting, 7636 Andora Drive,
Sarasota, FL 34238, USA
precipitous drop in cost for genome sequencing, it is now
feasible to sequence thousands of actinomycete genomes to
identify the “biosynthetic dark matter” as sources for the dis-
covery of new and novel secondary metabolites. Advances
in bioinformatics, mass spectrometry, proteomics, transcrip-
tomics, metabolomics and gene expression are driving the
new field of microbial genome mining for applications in
natural product discovery and development.
Keywords  Actinomycete · Antibiotic · Discovery ·
Genome mining · Natural product · Fungi · Secondary
metabolite · Streptomyces
Introduction
Natural products (NPs) represent a large family of diverse
chemical entities with a wide variety of biological activi-
ties that have found multiple uses, notably in human and
veterinary medicine and in agriculture [26, 36, 47, 85, 86].
They originate from bacterial, fungal, plant, and marine ani-
mal sources. The bacterial and fungal NPs described in this
review are products of “secondary metabolism”: molecules
that are not required for survival of the host under laboratory
conditions, but which undoubtedly provide some advantage
to the host in its native environment. Natural macromol-
ecules (DNA, RNA, and protein), their building blocks and
precursors, as well as intermediates of primary metabolism,
are typically excluded from the working definition of NPs.
NP discovery has been driven by the findings that they
bicides, insecticides, etc. Since the discovery of penicillin
more than 75 years ago, >23,000 NPs have been charac-
terized, the large majority of which are produced by bacte-
ria, mainly by the family Actinomycetaceae [25]. After the
13

156
discovery of streptomycin by Selman Waksman and associ-
ates at Rutgers University in the 1940s, large NP discovery
efforts began in pharmaceutical companies, mainly in the
United States, Europe and Japan, and modest efforts fol-
lowed in isolated academic laboratories worldwide. Nota-
Ōmura at the Kitasato Institute (now Kitasato University)
ble exceptions include the academic laboratories of Satoshi
in Tokyo, the late Hamao Umezawa at the Institute of Anti-
biotics in Tokyo, the late Hans Zähner at the University of
Tübingen, and Hans Reichenbach at the Helmholtz-Zen-
trum für Infektionsforschung in Braunschweig. Working
independently, and in collaboration with others, these labo-
ratories identified more than 1000 novel NPs.
What has fascinated NP scientists is the chemical diver-
sity and complexity of NPs, examples of which are dis-
played in the compounds shown in Figs. 1, 2, 3 and 4. Not
only is there a large range of molecular weights (coumerin,
146 Da to daptomycin, 1620 Da), but many of the com-
pounds contain a significant number of stereo-specific car-
bon centers. The stereo-complexity of these molecules has
attracted synthetic chemists interested in developing routes
and reactions for total chemical synthesis, and biochem-
ists and molecular biologists interested in understanding
the enzymology, genetics, and regulation of biosyntheses.
Microbiologists interested in the discovery of microor-
ganisms that produce novel molecules and novel activities
were needed to isolate and identify the producing micro-
organisms and to test and quantify the antibacterial or anti-
fungal activities. Cell biologists tested for anti-cancer and
other pharmacological activities. The commercial require-
ment for high volume production attracted chemical engi-
neers, fermentation microbiologists and geneticists, and
isolation and analytical chemists. Organic chemists were
attracted to pharmaceutical and biotechnology companies
to develop semi-synthetic derivatives. This family of aca-
demic and industrial scientists created the field of natural
product research.
This review covers the strategies and methodologies
employed from the 1940s through the present time to dis-
cover new NPs. We describe the screening for activity
approaches employed for the first 30 years of NP discovery,
highlighting some of the important molecules discovered
during “The Golden Age”. We then discuss how the under-
standing of NP biosynthesis changed the approaches for NP
discovery. For example, sequencing efforts demonstrated
that entire NP biosynthetic gene sets were clustered, and
allowed the use of genetic probes for the search for new
NPs, enabled the mixing, matching, and substitution of NP
biosynthesis genes to generate novel “hybrid” NPs, as well
as novel derivatives that could not be produced by synthetic
chemistry. We end with a discussion of the impact of the
development of inexpensive, high-throughput Next Gen-
eration Sequencing and advanced bioinformatics tools on
13
J Ind Microbiol Biotechnol (2016) 43:155–176
the genomic approaches being taken for present and future
NP discovery, focusing on genome mining and silent gene
activation. The history of NP discovery, as we describe it,
is segmented into three overlapping periods, 1940s–1970s,
1970s–2000s, and 2000 and beyond. These periods were
chosen not because there were discrete breaks in the dis-
covery process but because significant advances during
these eras dramatically impacted the strategies employed to
discover new NPs.
This review is not intended to be comprehensive, but to
provide a basic understanding of the history of NP discov-
ery without a particular emphasis on molecule type or med-
ical field. Because of their overall value as drugs, however,
which heavily skewed NP discovery efforts in pharma-
ceutical companies towards specific medical applications
during the first 30 years of effort, we present a brief sum-
mary of the medical uses of NPs. Finally, because we both
participated in one or more aspects of NP discovery over
time, in addition to citing published material, we also use
unpublished, personal accounts of our experiences in this
perspective. For comprehensive reviews of the impact of
NPs on medicine, readers are guided to other reviews [36,
85, 86]. For insider accounts on NP discovery and develop-
ment programs in the Pharmaceutical Industry, readers are
referred to Feature Articles in SIM News [8, 112, 127].
Natural products as drugs
As of 2013, 1453 new chemical entities (NCEs) have been
approved by the US Food and Drug Administration, of
which ca. 40 % are NPs or NP-inspired (semi-synthetic NP
derivatives, synthetic compounds based on NP pharmaco-
phores, or NP mimics) [36, 68]. In the past 30 years, the
percentage of NP or NP-inspired NCEs has risen to approx-
imately 50 % of the total, and to ca. 74 % in the anti-tumor
arena [26, 47]. During the period 2008–2013, 25 NP or
NP-derived drugs were approved for use. Here we present
a brief summary of the many pharmaceutical applications
of NPs or NP-inspired compounds and a short overview of
their mechanisms of action or molecular targets. The struc-
tures of the NPs highlighted in bold are shown in Figs. 1, 2,
3 and 4.
The most prominent medicinal use of NPs has been in
the area of anti-infectives, notably anti-bacterial therapy
(Fig. 1). Dozens of NPs, and their second- and third-gener-
ation semisynthetic derivatives have been, and continue to
be used to treat Gram-positive or Gram-negative bacterial
infections in humans and animals. Briefly, azithromycin
or clarithromycin, both semi-synthetic derivatives of the
macrolide erythromycin, doxycycline, a second-genera-
tion tetracycline, amoxicillin, a semi-synthetic penicillin
derivative, and cephalexin, a derivative of cephalosporin
J Ind Microbiol Biotechnol (2016) 43:155–176 157

Fig. 1  Structures of antibacterial natural products

C, are all commonly used as outpatient treatments of bac-
terial skin or respiratory infections. The macrolide tylosin
and its semi-synthetic derivative tilmicosin are used to treat
bacterial infections in animals. Macrolide antibacterial
agents related to erythromycin or tylosin act as translation
inhibitors by binding to the 50S ribosomal subunit, block-
ing translation of the nascent polyptide chain. They are
generally bacteriostatic for susceptible pathogens, but are
bactericidal for Streptococcus pneumonia. Tetracycline
also blocks translation by binding to the 50S ribosomal

13

158
Fig. 2  Structures of antifungal and antiparasitic natural products
subunit at a site distinct from the macrolides. Chloram‑
phenicol, a third ribosome binding agent is very active
against Gram-negative bacteria, but is no longer used clini-
cally, due to rare side effects. It binds to the A site of the
50S ribosome preventing the binding of aminoacyl-tRNA.
The bactericidal penicillins and cephalosporins covalently
bind to penicillin binding proteins in the bacterial cell wall
and block cell wall synthesis, leading to exposure of the
cell membrane to its osmotically unfavorable environment,
resulting in cell lysis. Clavulanic acid, an inhibitor of sev-
eral β-lactamases (which inactivate penicillins) is combined
with the semi-synthetic penicillin analog amoxicillin in
the compound Augmentin for use against penicillin-resist-
ant pathogens. Glycopeptides, such as vancomycin and
teicoplanin or their recently approved second-generation
structural relatives (e.g., telavancin, dalbavancin, and orita-
vancin) are used for Gram-positive infections that are not
readily treated by macrolides and tetracyclines. The mem-
brane active cyclic peptide daptomycin is used for hospi-
tal-based difficult to treat Gram-positive infections, includ-
ing methicillin-resistant Staphylococcus aureus (MRSA),
and the penems (e.g., meripenem, derived from thienamy‑
cin) are used to treat infections caused by Gram-negative
microorganisms. Natural penems are cephalosporin ana-
logs. Aminoglycosides, such as tobramycin, are used in
hospitalized patients carrying Gram-negative infections.
They interact with mRNA in the 30S–50S ribosome junc-
tion and cause mistranslation. Lipiarmycin (independently
identified from three different actinomycetes, and also
13
J Ind Microbiol Biotechnol (2016) 43:155–176
named clostomicin, tiacumicin and fidaxomycin), recently
approved by the FDA, is effective against Clostridium
difficile, the agent associated with severe colitis and diar-
rhea. This macrolide does not bind ribosomes, but inhibits
the β-subunit of RNA polymerase blocking transcription.
Rifampicin, a semi-synthetic analog of rifamycin, also
inhibits RNA polymerase and is used in combination with
other agents, including streptomycin, for the treatment of
tuberculosis.
Two different families of NPs are used to treat fungal
infections in humans. Polyene macrolides such as ampho‑
tericin B (Fig. 2) and nystatin bind to ergosterol creat-
ing holes in the fungal membrane and the echinocandins,
such as caspofungin, inhibit the fungal cell wall form-
ing enzyme glucan synthase. A large family of polyethers
including monensin, narasin, salinomycin, and lasalocid
are used in cattle feed as growth promoting agents or in
chicken feed to prevent coccidiosis. The antimicrobial and
antiparasidic effects of the polyethers are through their
activities as ionophores, chelating mono- or divalent cati-
ons. Ivermectin, a semi-synthetic derivative of avermectin,
is active against parasitic worms in humans and animals.
It is used to treat onchocerciasis (river blindness) and lym-
phatic filariasis (elephantiais) in humans. Artemisinin is
used to treat the malaria causing parasite Plasmodium fal-
ciparum. Artemisinin is derived from a plant, but is now
produced by the yeast Saccharomyces cerevisiae, based on
a successful synthetic biology approach to heterologously
express the NP pathway. Spinosad, an antiparasitic and
J Ind Microbiol Biotechnol (2016) 43:155–176 159

Fig. 3  Structures of anti-tumor natural products

13

160
Fig. 4  Structures of natural products with various bioactivities
insecticidal agent comprised of a mixture of the polyke-
tides spinosyn A and spinosyn D, is approved for the topi-
cal treatment of head lice in humans, and as an important
insecticide active against a wide range of lepidopterous and
dipterous insect pests in agriculture.
A large number of NPs or NP-inspired agents have been
found to exhibit anti-tumor activity [26, 47]. Approved
drugs from bacterial sources include the anthracycline
mitomycin C (Fig. 3) and the mixed polyketide-nonribo-
somal peptide (PK-NRP) bleomycin, both of which act
by causing breaks in DNA, though via different chemical
mechanisms; the di-cyclic peptide actinomycin D, a tran-
scription inhibitor; doxorubicin, a semi-synthetic derivi-
ative of the anthracycline polyketide daunorubicin, that
acts by intercalating into DNA and blocking the enzyme
topisomerase II; and romidepsin, a histone deacetylase
inhibitor. Anti-tumor terpenes from plant sources include
vincristine, vinblastine and paclitaxel. These agents block
mitosis by binding to microtubules. Other anti-tumor
agents from plants are the topoisomerase II inhibitor etopo-
side, a semi-synthetic agent related to podophyllotoxin;
the topoisomerase I inhibitor camptothecin, a quinoline
alkyloid, as well as its water-soluble semi-synthetic deriva-
tive topotecan; and the recently approved translation inhibi-
tor omacetaxine mepesuccinate, a natural ester of the alka-
loid cephalotaxine. Other recently approved anti-cancer
drugs include the tetrapeptide epoxyketone carfilzomib,
a semi-synthetic derivative of epoxomicin, and eribulin,
13
J Ind Microbiol Biotechnol (2016) 43:155–176
a macrocylic ketone analog of the NP halichondrin B.
Salinosporamide A is a proteosome inhibitor not yet
approved as an anti-cancer agent. Many NPs that exhibit
anti-tumor activities are too cytotoxic to be administered
as drugs by themselves. A number has been conjugated to
monoclonal antibodies for the purpose of target-specific
delivery. NPs present in approved antibody–drug-conju-
gates (ADCs) include the ansamycin polyketide maytan‑
sine and the acylpeptide dolastatin. The formerly approved
ADC containing calicheamicin, an enediyne polyketide
was recently withdrawn due to severe toxic side effects.
However, many more enediynes have been discovered by
focused genome mining [101], thus providing a wide range
of chemical warheads for potential drug development.
An important advance in the treatment of hypercholes-
terolemia was the development of the class of NPs known
as statins, e.g., compactin (Fig. 4), lovastatin, and semi-
synthetic derivatives simvastatin, atorvastatin, pravasta-
tin. These compounds inhibit the mammalian and fungal
enzyme HMG-CoA reductase in the cholesterol biosynthe-
sis pathway. They continue to be among the highest rev-
enue generating pharmaceuticals worldwide. The bacterial
polyketides rapamycin and FK506 (tacrolimus), and the
fungal cyclic peptide cyclosporine A are immunosupres-
sants that are widely used after organ transplantation to sup-
press rejection. Fingolimod, a drug used to treat multiple
sclerosis, was developed from the fungal compound myri‑
ocin, an inhibitor of serine palmitoyltransferase, by using
J Ind Microbiol Biotechnol (2016) 43:155–176 161
structure–activity studies to determine the pharmacophore.
Canagliflozin, a semi-synthetic derivative of the flavanoid
phlorizin, found in many plants, is used to treat type 2 dia-
betes, and capsaicin, derived from chili peppers, is used
topically in over-the-counter creams to reduce minor aches
and pains. In addition, many plant-derived NPs, e.g., cou‑
marin, are using in food as flavorings or as neutraceuticals.
Many of the examples described above represent FDA
approved NPs or NP-inspired compounds. More than 100
other NPs or NP-inspired synthetic or semi-synthetic deriva-
tives originally obtained from bacterial, fungal, plant, and
marine sources are currently in clinical trials as anti-infective,
anti-cancer, or other pharmaceutical agents. For a comprehen-
sive list of NP-based compounds currently in clinical trials, the
reader is directed to the excellent review by Butler et al. [30].
Each of the NPs described exhibits a distinct mechanism of
action on their respective targets, although compounds within
a given structural class with the same pharmacological activ-
ity share binding sites in a common target. Erythromycin and
tylosin both bind to the 23S rRNA in domain V of the bacterial
ribosome, but the third sugar on tylosin also binds to domain
II of 23S rRNA. The study of drug mechanism of action has
revealed that similar pharmacological activities can be obtained
from compounds of dissimilar structure. For example, rapa-
mycin and cyclosporine A are both immunosuppressants, but
have different targets and mechanisms of action. Other com-
pounds, such as tetracycline and daunorubicin, have high struc-
tural similarity but exhibit different pharmacological effects:
antibacterial via binding to ribosome and anti-tumor via DNA
intercalation, respectively. Finally some compounds have more
than one pharmacological effect. For example, in addition to its
antibacterial activity, erythromycin exhibits binding to motilin
receptor in the human gut and is used clinically for treatment
of diabetic gastroparesis, as well as to stimulate gastrointesti-
nal motor activity when needed in patients following surgery.
Because erythromycin is a large compound, the pharmacoph-
ores responsible for the different properties are composed of
different sub-sets of atoms of the molecule. These examples
illustrate the variety of binding targets and pharmacological
effects NPs have and underscore not only the values they have
brought as medicines, but also as agents that have been used to
deepen our understanding of fundamental biology.
Table 1 presents a list of 100 important NPs from diverse
microbial sources and with diverse biosynthetic mecha-
nisms. Most have use in human or animal medicine, or in
agriculture. Several are important research tools, which
will be further discussed below.
Strategies and methodologies for NP discovery
Over the past seven decades, natural product discovery has
been in a process of evolution; strategies were relatively
simple during the first 30 years, then grew more diverse
and complex, accelerated by advancements in science and
technology in the next two decades. Overall efforts in NP
discovery have decelerated with the waning interest from
the pharmaceutical industry over the past twenty-years. In
more recent times, inexpensive microbial genome sequenc-
ing has opened totally new strategies for secondary metab-
olite drug discovery. We recount some of the strategies and
approaches in the following sections.
The first 30 years (1940s–1970s): phenotypic
screening
Although the development of penicillin for wide use dur-
ing WWII demonstrated success at bringing a NP to mar-
ket, it was the systematic screening of actinomycetes
obtained from soil by the Waksman group at Rutgers in
the 1940s leading to the discoveries of actinomycin, strep-
tothricin, and, most notably, streptomycin, that prompted
pharmaceutical companies to begin screening extracts of
actinomycetes and fungi for activities primarily against
pathogenic bacteria. The process required acquisition of
microorganisms, fermentation, product isolation, and test-
ing of fermentation broths and purified compounds against
test organisms. For testing, companies used bioassays for
phenotypic screening. In general, a phenotypic screen
employs a cell line (bacteria, yeast, eukaryotic cells or tis-
sues in culture, etc.) and a method to determine a response
to an applied test compound, without prior knowledge of its
mechanism of action. The easiest read-out is cell inhibition
or death, and, in the first 30 years this was used to iden-
tify more than 1000 NPs that had antibacterial or antifungal
activity, including dozens that were ultimately approved as
drugs. Early cancer drugs were also detected in a pheno-
typic screen using stable cancer cell lines cultivated first in
experimental animal models and later, in vitro.
Acquisition of microorganisms
Acquisition of actinomycetes and fungi was facilitated
by the abundant numbers of these microbes in soil. At Eli
Lilly and Company, Abbott Laboratories, and other phar-
maceutical companies, employees were encouraged to col-
lect soil samples when they traveled around the world on
business or vacation. At Abbott, a large map of the world
was kept on the wall of the soil screening laboratory with
more than 2000 pins stuck at the locations from which soils
were collected. Every continent, and almost every country
had at least one pin. In many cases, new microbes produc-
ing important compounds could be tracked back to the
fessor Satoshi Ōmura of the Kitasato Institute collecting a
originating soils. The most famous example involved Pro-
13

162 J Ind Microbiol Biotechnol (2016) 43:155–176

Table 1  Diverse natural products produced as secondary metabolites by bacteria or fungi

Secondary metabolite Biosynthetic origin Major use Producing organism Type of organism Pubmed citationsa

Acarbose Glycoside Antidiabetic (HM) Actinoplanes sp. Actinomycete 1878

Actinomycin NRPS Antitumor (HM) Streptomyces anulatus Actinomycete 24,705
Actinorhodin PKS II Antibacterial (RT) Streptomyces coelicolor Actinomycete 448
Adriamycin PKS II Antitumor (HM) Streptomyces peucetius Actinomycete 59,850
Albomycin Peptidyl-nucleoside Antibacterial Streptomyces sp. ATCC Actinomycete 130
700974
Amphomycin NRPS Antibacterial Streptomyces canus Actinomycete 97
Amphotericin B PKS I Antifungal (HM) Streptomyces nodosus Actinomycete 20,386
Antimycin NRPS-PKS I Research tool; Piscicide Streptomyces (multiple Actinomycete 4558
sp.)
Apramycin Aminoglycoside Antibacterial (AH, RT) Streptoalloteicus hindu- Actinomycete 303
stanus
Ascomycin NRPS-PKS I Immunomodulator Streptomyces hygro- Actinomycete 255
(HM) scopicus
Avermectin (Ivermectin)b PKS I Anthelmintic (HM, AH) Streptomyces avermitilis Actinomycete 741 (6208)b
Bialphos NRPS Herbicide (CP) Streptomyces viridochro- Actinomycete 182
mogenes
Bleomycin NRPS-PKS I Antitumor (HM) Streptomyces verticillus Actinomycete 17,239
Calcimycin (A23187) PKS I Calcium ionophore (RT) Streptomyces chartru- Actinomycete 11,796 (16,702)
ensis
Candicidin PKS I Antifungal (HM) Streptomyces (multiple Actinomycete 291
sp.)
Capreomycin NRPS Antitubercular (HM) Saccharothrix mutabilis Actinomycete 545
Carbomycin PKS I Antibacterial (HM) Streptomyces halstedii Actinomycete 166
Cephalosporin NRPS Antibacterial (HM) Cephalosporium acre- Fungus 45,337
monium
Cephamycin NRPS Antibacterial (S) Streptomyces cla- Actinomycete 3462
vuligerus
Chloramphenicol Shikimate modification Antibacterial (HM) Streptomyces venezuelae Actinomycete 38,609
Chlorobiocin Aminocoumarine Antibacterial (S) Strteptomyces roseo- Actinomycete 96
chromogenes
Chloroeremomycin NRPS Antibacterial (HM) Amycolatopsis orientalis Actinomycete 39 (247)
(oritavancin)
Clavulanic acid (aug- Other Antibacterial (HM) Streptomyces cla- Actinomycete 6348 (12,346)
mentin) vuligerus
Compactin PKS Cardiovascular (HM) Penicillium brevicom- Fungus 662
pactum
Coumermycin Aminocoumarin Antibacterial (RT) Streptomyces rishiriensis Actinomycete 353
Cyclosporin A NRPS Immunomodulator Tolypocladium inflatum Fungus 50,544
(HM)
d-Cycloserine Other Antitubercular (HM) Streptomyces lavendulae Actinomycete 3045
Daptomycin NRPS Antibacterial (HM) Streptomyces roseospo- Actinomycete 2183
rus
Dalbavancin NRPS Antibacterial (HM) Nonomuraea sp. Actinomycete 235
Daunorubicin PKS II Antitumor (HM) Streptomyces peucetius Actinomycete 53,337
Echinocandin NRPS Antifungal (HM) Aspergillus nidulans Fungus 3079
Epothilone NRPS-PKS I Anti-tumor (HM) Sorangium cellulosum Myxobacteria 971
Ergometrine Ergolin alkaloid Cardiovascular (HM) Claviceps purpurea Fungus 2180
Erythromycin PKS I Antibacterial (HM) Saccharopolyspora Actinomycete 31,179
erythraea
Filipin PKS I Antifungal (RT) Streptomyces filipinensis Actinomycete 1303
Fusidic acid Terpine Antibacterial (HM) Fusidium coccineus Fungus 2167

13
J Ind Microbiol Biotechnol (2016) 43:155–176 163

Table 1  continued
Secondary metabolite Biosynthetic origin Major use Producing organism Type of organism Pubmed citationsa
Geldanamycin PKS I Antitumor (S) Streptomyces hygro- Actinomycete 1627
scopicus
Gentamicin Aminoglycoside Antibacterial (HM) Micromonospora pur- Actinomycete 27,233
purea
Gramicidin S NRPS Antibacterial (RT) Bacillus subtilis Bacillales 3841
Hygromycin Aminoglycoside Antibacterial (AH) Streptomyces hygro- Actinomycete 2092
scopicus
Josamycin PKS I Antibacterial (HM) Streptomyces narbon- Actinomycete 676
ensis
Kanamycin (amikacin) Aminoglycoside Antibacterial (HM) Streptomyces kanamy- Actinomycete 18,934 (8055)
ceticus
Kirromycin NRPS-PKS I Antibacterial (RT) Streptomyces collinus Actinomycete 219
Lasalocid PKS I Coccidiostat (AH) Streptomyces lasaliensis Actinomycete 875
Lincomycin (clindamy- Other Antibacterial (HM) Streptomyces lincoln- Actinomycete 7702 (9958)
cin) ensis
Lipiaramycin (fidax- PKS I Antibacterial (HM) Dactosporangium Actinomycete 155 (264)
omicin) aurantiacum
Lipstatin (Xenical) Fatty acyl-lactone Antiobesity (HM) Streptomyces toxitricini Actinomycete 25 (1559)
Lovastatin PKS Cardiovascular (HM) Asperigillus terreus Fungus 10,266
Milbemycin PKS I Anti-parasidic (AH) Streptomyces hygro- Actinomycete 736
scopicus
Mithramycin PKS II Antitumor (HM) Streptomyces plicatus Actinomycete 1342
Mitomycin C Quinone Antitumor (HM) Streptomyces lavendulae Actinomycete 18,112
Moenomycin Phosphoglycolipid Antibacterial (AH) Streptomyces ghanaensis Actinomycete 228
Monensin PKS I Coccidiostat (AH) Streptomyces cinnamon- Actinomycete 4675
ensis
Mycophenolic acid PKS Immunomodulator Penicillium brevicom- Fungus 7109
(HM) pactum
Narasin PKS I Coccidiostat (AH) Streptomyces aureofa- Actinomycete 221
ciens
Natamycin (pimaracin) PKS I Antifungal (HM) Streptomyces (multiple Actinomycete 817 (894)
sp.)
Neomycin Aminoglycoside Antibacterial (HM) Streptomyces fradiae Actinomycete 13,737
Netropsin NRPS Antitumor (RT) Streptomyces ambofa- Actinomycete 719
ciens
Nikkomycin Peptidyl-nucleoside Antifungal Streptomyces tendae Actinomycete 246
Nisin RiPP Food preservative Lactococcus lactis Lactobacillales 1720
Nystatin PKS I Antifungal (HM) Streptomyces noursei Actinomycete 4686
Oligomycin PKS I Toxin (RT) Streptomyces avermitilis Actinomycete 4784
Oxytetracycline PKS II Antibacterial (HM) Streptomyces rimosus Actinomycete 7379
Paclitaxel Isoprenoid Antitumor (HM) Several endophytic fungi Fungus 27,268
Penicillin NRPS Antibacterial (HM) Penicillium crysogenum Fungus 96,006
Phosphomycin Phosphone Antibacterial (HM) Streptomyces wedmo- Actinomycete 2410
rensis
Pleuromutalin Diterpine Antibacterial (HM) Clitopilus scyphoides Fungus 161
Pneumocandin NRPS Antifungal (HM) Glarea lozoyensis Fungus 77
Polymyxin (colistin) NRPS Antibacterial (HM) Paenibacillus polymyxa Bacillales 9611 (4084)
Polyoxin (D) Nucleoside Antifungal (CP) Streptomyces cacaoi Actinomycete 120
Pristinamycin IA NRPS Antibacterial (HM) Streptomyces pristi- Actinomycete 521
naespiralis
Pristinamycin IIA NRPS-PKS I Antibacterial (HM) Streptomyces pristi- Actinomycete 131
naespiralis

13

164 J Ind Microbiol Biotechnol (2016) 43:155–176

Table 1  continued
Secondary metabolite Biosynthetic origin Major use Producing organism Type of organism Pubmed citationsa
Ramoplanin NRPS Antibacterial (HM) Actinoplanes sp. ATCC Actinomycete 155
33076
Rapamycin NRPS-PKS I Immunomodulator Streptomyces hygro- Actinomycete 26,695
(HM) scopicus
Rebeccamycin Alkaloid Antitumor Lechevalieria aerocolo- Actinomycete 128
negenes
Rifamycin (rifampicin) PKS I Antibacterial (HM) Amycolatopsis mediter- Actinomycete 19,004 (24,279)
ranei
Ristocetin NRPS Antibacterial (RT) Amycolatopsis lurida Actinomycete 2953
Salinomycin PKS I Coccidiostat (AH) Streptomyces albus Actinomycete 624
Sinefungin Nucleoside Antifungal (RT) Streptomyces griseolus Actinomycete 255
Spectinomycin Aminoglycoside Antibacterial (HM) Streptomyces spectabilis Actinomycete 2408
Spinosyn (spinosad) PKS I Insecticidal (CP) Saccharopolyspora Actinomycete 92 (548)
spinosa
Spiramycin PKS I Antibacterial (HM) Streptomyces ambofa- Actinomycete 1367
ciens
Staurosporin (aglycone) Alkaloid Antitumor (S) Streptomyces stauro- Actinomycete 10,116
sporeus
Streptomycin Aminoglycoside Anti-tubercular (HM) Streptomyces griseus Actinomycete 29,437
Streptothricin Aminoglycoside Antibacterial Streptomyces (multiple Actinomycete 337
species)
Streptozotocin Glucosamine-nitros- Antitumor (HM) Streptomyces acromo- Actinomycete 24,436
ourea genes
Surfactin NRPS Surfactant Bacillus subtilis Bacillales 595
Tacrolimus (FK-506) NRPS-PKS I Immunomodulator Streptomyces tsukubae- Actinomycete 19,514 (19,841)
(HM) nsis
Teicoplanin NRPS Antibacterial (HM) Actinoplanes teichomy- Actinomycete 3358
ceticus
Tetracenomycin PKS II Antitumor Streptomyces glauce- Actinomycete 101
scens
Tetracycline PKS II Antibacterial (HM) Streptomyces rimosus Actinomycete 35,863
Thienamycin Other Antibacterial (HM) Streptomyces cattleya Actinomycete 324
Thiostrepton RiPP Antibacterial (AH, RT) Streptomyces azureus Actinomycete 486
Tobramycin Aminoglycoside Antibacterial (HM) Streptoalloteicus hindu- Actinomycete 6636
stanus
Tunicamycin Nucleoside Antibacterial (RT) Streptomyces chartreusis Actinomycete 4971
Tylosin PKS I Antibacterial (AH) Streptomyces fradiae Actinomycete 1557
Undecylprodigiosin Other Antibacterial (RT) Streptomyces coelicolor Actinomycete 161
Validamycin Glycoside Antifungal (CP) Streptomyces hygro- Actinomycete 99
scopicus
Viomycin NRPS Antibacterial (HM) Streptomyces sp. 11861 Actinomycete 831
Virginiamycin NRPS, NRPS-PKS I Antibacterial (AH) Streptomyces virginiae Actinomycete 1372

AH animal health, CP crop protection, HM human medicine, PKS polyketide synthase, NRPS nonribosomal peptide synthetase, S scaffold for
chemical semi-synthesis, RiPP ribosomally synthesized and post-translationally modified peptide, RT research tool
a
  PubMed citations on 8/31/2015
b
  Compounds of commercial products in parentheses correspond to citations in parentheses

soil sample from a golf course in Tokyo in the early 1970s
from which his research group discovered avermectin from
a new species of Streptomyces that he named S. avermiti-
lis. Other examples include the erythromycin producer
Saccharopolyspora erythraea, discovered in 1949 in the
garden of a local physician in the Phillipines employed by
Eli Lilly and Company [http://www.ipsnews.net/1994/11/
medicine-philippines-who-really-discovered-erythromycin-
1-an-inter-press-service-feature], the daptomycin producer
Streptomyces roseosporus, tracked to a soil sample from

13
J Ind Microbiol Biotechnol (2016) 43:155–176 165
Mount Ararat in Turkey [41], and the spinosad producer
Saccharopolyspora spinosa, tracked to a defunct sugar mill
rum still in the Virgin Islands [125]. The practice of collect-
ing soil from foreign sources and bringing samples back to
the home country went on unabated until countries signed
the treaty known as the Convention on Biological Diversity
(CBD) in the early 1990s. Until then, the countries from
which the soil samples originated did not share in the reve-
nues brought by the subsequent drugs that were developed.
CBD ensured that revenues would return to these countries.
During the first three decades of antibiotic discovery in
the pharmaceutical industry, many important NPs were dis-
covered from microbial sources, many of which, or their
second, third, or fourth generation semi-synthetic deriva-
tives, are still in use today. Many of the important NPs
listed in Table 1 were discovered in this timeframe. Thou-
sands of new chemical entities (NCEs) were discovered
from soil screening, but only a small number were devel-
oped as drugs. Nevertheless, the success achieved from the
commercial launch of a single compound was sufficient to
keep the process going unchanged for more than 30 years,
at least in the companies with which we are familiar. In
general, soil screening was a low-throughput process
which, as described below, was limited by the slow growth
of the actinomycetes and fungi examined, and importantly
by the amount of shaker space available to companies.
In the early years of screening, most companies focused
on isolating and identifying Streptomyces as the major bac-
terial genus to search for NCEs. As can be seen in Table 1,
Streptomyces species yielded many important compounds
in several antibiotic classes: macrolides (tylosin, spiramy-
cin); aminoglycosides (neomycin, kanamycin,); β-lactams
(cephamycin, carbapenems); tetracyclines (tetracycline,
chlortetracycline, oxytetracycline); polyenes (candicidin,
amphotericin B, nystatin); peptides (actinomycin); and
chloramphenicol. However, three important antibiotics
discovered by Eli Lilly and Company, erythromycin, van-
comycin and tobramycin [8], were originally thought to
be produced by Streptomyces species, but the producing
actinomycetes were later reclassified as Saccharopolyspora
erythraea, Amycolatopsis orientalis, and Streptoalloteicus
hindustanus, respectively (Table 1). In the late 1950s, the
US-based Schering Corp. collected a large group of soil
actinomycetes classified as Micromonospora and began
screening them for antibacterial activity [124, 127]. This
genus yielded the aminoglycosides gentamicin (a fam-
ily consisting of more than 5 congeners), sisomicin, the
fortimicins, and sagamicin, the macrolides rosaramicin,
juvenimicin, and the megalomicins, as well as the oligo-
saccharide everninomicin. Only gentamicin was developed
as an antibacterial drug. Lepetit (later Dow Lepetit then
Biosearch Italia) began to screen rare actinomycetes in the
1960s. The company discovered the macrolide lipiarmycin
from Dactosporangium (later developed as fidoxamicin
by Optimer and Cubist), and the glycopeptides teichopla-
nin and ramoplanin from different Actinoplanes species. It
is now apparent from the early discoveries in the pharma-
ceutical industry that in addition to the highly productive
Streptomyces, other less abundant actinomycetes have con-
tributed substantially to the wealth of important NPs dis-
covered in the first 30 years.
Detection, fermentation and isolation of secondary
metabolites
To determine if they produced active compounds, indi-
vidual colonies isolated from agar plates were grown in
small shake flask fermentations employing several media
compositions, and in some cases under a range of tempera-
tures and agitation speeds. Initial detection for antibacterial
or antifungal activity involved assay of solvent extracted
culture broths. Small amounts of extracted material were
deposited on filter paper discs which were then placed on
agar plates seeded with a susceptible test organism. Detec-
tion was as simple as visualization of growth inhibition of
test organisms after overnight incubation. Activities were
quantified by standard MIC analyses employing panels of
various pathogens. For more polar compounds that were
not solvent extractable (e.g., peptidic or glycosidic agents),
Schering used an agar plug assay. Small (2–3 cm diam-
eter) plugs were removed from agar plates seeded with a
test organism, allowing the administration of up to 5 mL
of whole culture broths of soil isolates (Micromonospora
strains). Detection of small zones of inhibition after over-
night incubation was used to identify antibiotic producers.
After the activities were identified, the active components
were often separated by paper chromatography and assayed
by bioautography: the chromatogram was placed on an agar
plate seeded with a susceptible test strain; after overnight
incubation, the plate was examined for zones of inhibition
corresponding to the migration of the active ingredient.
Bioautography was used to determine if there was more
than a single active compound in the culture. After initial
discovery, product titers were improved initially by fer-
mentation optimization to produce enough material for iso-
lation and evaluation. In some cases, the producing strain
was subjected to mutagenesis and selection for product titer
improvement at an early stage using chemical or physical
mutagenic agents. Promising strains were then scaled in
pilot plant fermenters to obtain greater quantities of mate-
rial for isolation and structural characterization, and further
activity evaluation. Because of the ease of collecting sam-
ples and obtaining different colonies, each colony (strain)
was typically given a single chance, as described above, to
produce an active product, although many companies kept
the isolated colonies as lyophilized spore suspensions in
13

166
their culture collections. Initial shake flask cultures were
generally discarded. Because of the sheer volume of shake
flasks and shaker capacity, companies could test only a
limited number of different microbes per year. At Eli Lilly
and Company, that number was about 35,000, translating
to about 1,000,000 strains screened over three decades.
[8, 11]. By the mid-1990s, when NP screening was halted,
Abbott Laboratories had screened about 400,000 isolates
(J. McAlpine, personal communication).
We estimate, for all companies, institutes, and research
laboratories worldwide engaged in screening during the
period from 1950 to 2000, that about 10–20 million isolates
were screened. Undoubtedly, many of the more common
actinomycete species were screened many times by many
companies. From these, thousands of NCEs were identified
and hundreds, either the NPs themselves or their deriva-
tives, were developed as drugs. Although, as we describe
briefly below, the methods to screen for pharmacological
activities have changed dramatically over the last 50 years,
the processes employed to isolate organisms from envi-
ronmental samples, to grow them in liquid culture, and to
extract and purify natural products have undergone only
minor evolution. High-speed shakers capable of providing
sufficient aeration to allow actinomycetes to grow in micro-
titer plates [80] replaced the need for shake flask cultivation
so that more isolates could be screened in less space, and
major improvements in NMR [29] and MS [49] analyses
have made structure determination of complex NPs easier
and quicker. Large-scale fermentation has remained basi-
cally unchanged.
The first 30 years of NP discovery followed the para-
digm: (1) phenotypic screening, (2) compound isola-
tion and structural characterization, (3) mode of action
studies in some cases, (4) preclinical development, and
if successful, (5) clinical development and commerciali-
zation. Efforts were directed primarily to discover anti-
bacterial and antifungal compounds. All of the NPs with
antibacterial or antifungal activities discovered during
this period, with a single exception, employed pheno-
typic screens that used cell killing (or growth inhibition)
as the read-out. Because Merck was interested in discov-
ering agents specifically targeted to the biosynthesis of
the cell, they developed a screen in which the read out
was the conversion of bacteria to spheroplasts. This led
to the discovery of fosfomycin [53, 110]. Moreover, with
a single exception, all the antibacterials and antifun-
gals discovered during this era that went on to be devel-
oped as drugs were all NPs. Naladixic acid is based on
the structure of the NP quinine, and was identified in a
search for synthetic derivatives of anti-malarial drugs,
but was found to have serendipitous antibacterial proper-
ties. In the 1980s, a number of natural quinolones with
13
J Ind Microbiol Biotechnol (2016) 43:155–176
antibacterial properties were discovered from the bacte-
rium Pseudonocardia spp. CL3849, as well as various
plant sources [50].
As early as the 1950s, companies became interested in
expanding their screening efforts to find compounds for
treatment of cancer. Anti-cancer agents were screened by
observing the reduction of transplanted or experimentally
induced tumors in rodents or by observing cytotoxic effects
of tumor cell lines grown in culture. Whole animal tests
were also used to distinguish antibacterial or antifungal
activity from cytotoxic activity and were used to inform
companies that antibacterial agents such as kirromycin or
oligomycin, or the antifungal agent rapamycin, for exam-
ple, were too toxic to be considered for drug development.
On the other hand, the same assays were used to initially
identify the anti-tumor activities of daunorubicin, mito-
mycin C, bleomycin, and camptothecin (Fig. 1) [39, 46,
63, 122]. In the 1960s and 1970s, companies also started
to become interested in finding NPs for treatment of heart
disease, neurologic disorders, and metabolic diseases, but
facile assays to screen for effective compounds in these
therapeutic areas were not readily available. Sandoz (now
Novartis) discovered cyclosporine A (Fig. 4) using an
assay developed to test both immunosuppressive and anti-
tumor activities in a single mouse. [55, 109]. Blood drawn
from mice 7 days after they were immunized with sheep
erythrocytes (antigen) agglutinated when the antigen was
added to the test tube. To test for immunosuppressive activ-
ity, the agent was given by intravenous administration to
the mice at the time of inoculation with the antigen, and for
a number of days thereafter. If the blood drawn at 7 days
did not agglutinate in the presence of the antigen, the com-
pound was said to have blocked the immune response–
immunosuppressive. Because the same mice carried trans-
planted tumors, antitumor activity could be measured by
suppression of tumor growth after the test compound was
administered. This expensive, low-throughput method was
replaced by the mixed lymphocyte reaction (MLR) test in
the 1980s, which is described below.
The second 30 years (1970s–2000s):
knowledge‑based approaches
As mentioned above, other than some miniaturization of
organism cultivation and improvements in NMR and MS
analyses for structure determination, used extensively
for dereplication purposes (to eliminate rediscovery of
known compounds), the processes used to produce NPs
for discovery during the second 30 years remained largely
unchanged. New sources for NPs were sought during this
period. The hallmark of the second 30 year period was the
J Ind Microbiol Biotechnol (2016) 43:155–176 167
great expansion in screening methodologies and strategies.
Advancements in recombinant DNA and other technolo-
gies enabled researchers to rapidly determine mechanism
of action (MOA) of NPs, their semi-synthetic analogues,
and synthetic compounds, and rapidly led to the develop-
ment of biochemical or whole cell assays, collectively
known as target-based screening. In addition, the growing
understanding of cell cycle regulation, the role of recep-
tors in biologic responses, the genetic bases of cancer, etc.,
along with improvements in cell culture methodology, led
to the development of a large number of selective pheno-
typic screens for agents active against cancers, various neu-
rologic, metabolic, or cardiac disorders or abnormalities.
Both target-based and phenotypic screening are too vast
to be described comprehensively here, but are covered in
a number of reviews [74, 81, 102, 118]. We present here a
brief overview of some of the approaches taken, including
a number of examples that led to successful development of
NP-based drugs.
New sources of NPs
Phenotypic screening of soil samples for NPs continued
in the 1970s and 1980s, but was expanded, as described
below, beyond the search for antibacterial or antifungal
drugs. In addition, new sources beyond soils were sought
for NP discovery. This included marine environments, with
a heavy focus on the discovery of novel anti-cancer agents.
Samples collected by certified divers or sampling devices
included bacteria, algae, and marine invertebrates (tuni-
cates, corals, bryzoans, sea slugs, sponges, etc.), and the
NPs extracted from the samples were tested in new anti-
cancer screens described below.
Phenotypic screening
As described above, the early cancer drugs, mitomycin C,
daunoribucin, and actinomycin (Fig. 3) were discovered
using cancer cell lines transplanted into animals. Starting in
the 1960s, companies and academic laboratories interested
in anti-cancer agents began to produce stable cell lines that
were differentiated for specific types of cancer (e.g., lung,
breast, etc.) to screen fermentation broths, as well as their
compound collections consisting of purified NPs, semi-
synthetic and synthetic chemicals. In the 1980s, the US
National Cancer Institute began to develop their own col-
lection of stable, differentiated specific cancer cell lines,
culminating in the collection known as NCI-60, a panel of
60 cell lines differentiated for breast, lung, colon, prostate,
renal, ovarian, or CNS cancers, as well as those for leuke-
mia, and melanoma, and set up a service to test investiga-
tional compounds (including NPs) for efficacy (providing
dose response curves) and specificity (IC50 determinations
using the entire NCI-60 panel). For compounds discov-
ered before the panel was in use, for example paclitaxel
(Fig.  3), the results provided by NCI led to the develop-
ment of the drug for use in breast, lung and ovarian cancer.
The primary use of the NCI-60 panel today, however, is for
screening (efficacy, specificity, and MOA) of new NPs, and
other putative anti-cancer agents [104].
As of 2014, three compounds derived from NPs from
marine invertebrates have been approved for treatment
of various cancers. These include eribulin, a synthetic
derivative of halichondrin B (Fig. 3), trabectidin, a semi-
synthetic derivative of the NP safricin B, and PM-10450,
a synthetic compound derived from jorumycin from a sea
slug, and renieramycin J from a marine invertebrate. It is
not yet known if these compounds are produced from the
marine invertebrates themselves, of from yet to be identi-
fied bacterial symbionts. A number of other semi-synthetic
or synthetic derivatives, or antibody-conjugates of marine
invertebrate NPs are in various stages of clinical trials. The
compound salinosporamide A (Fig. 3), isolated from the
marine bacterium Salinispora tropica is the single example
to date of a potential anti-cancer NP from a marine bacte-
rium that made it into clinical trials [30].
A second example of a phenotypic screen developed in
the second period of NP discovery is the mixed lympho-
cyte reaction (MLR), used to screen for immunosuppres-
sive agents. In general when lymphocytes of two different
allotypes are mixed, an immunologic response causes them
to grow and divide. If one of the set of lymphocytes is uv-
inactivated, it can serve as a stimulator for growth and divi-
sion of the un-irradiated responder lymphocytes. Growth
can be measured by following 3H-thymidine incorporation
(into DNA) or by following OD570 in cells treated with the
tetrazolium dye MTT (3-[4,5-dimethylthiozol-2-yl]-2,5-di-
phenyltetrazoium bromide) [37, 82]. Immunosuppressive
activity is detected by inhibition of growth of the responder
cells. Separate cell-based assays are used to distinguish the
immunosuppression from cytoxicity. Many versions of the
MLR have been developed, from employing whole blood
from different donors, to using splenocytes drawn from dif-
ferent lines of mice [60]. Validation of the MLR employed
the use of cyclosporin A (Fig. 4) to demonstrate immu-
nosuppression. The MLR assay played a prominent role
in the discovery of two new NPs currently approved as
immunosuppressive agents, FK506 (tacrolimus) and rapa‑
mycin (Fig. 4). The immunosuppressive activity of FK506,
produced by Streptomyces tsukubaensis, was detected at
Fujisawa (now Astellas) in a primary screen using the MLR
assay. Rapamycin, isolated from a strain of Streptomyces
hygroscopicus collected on Easter Island, was discovered in
the early 1970s by microbiologists at Ayerst Research Lab-
oratories in an antifungal screen, but the compound proved
to be too toxic to develop as an antifungal drug [123]. Two
13

168
years after the initial report of the antifungal activity, phar-
macologists at Ayerst reported immunosuppressive activity
demonstrating the complete prevention of experimentally
induced immunogenic disorders in rats [75]. Work on the
mode of action of rapamycin, including demonstration that
at least one binding target of the drug differed from that of
FK506 continued during the 1980s, but serious develop-
ment of rapamycin as an immunosuppressive drug did not
begin until the late 1990s, after direct comparisons of activ-
ity between rapamycin and FK506 or cyclosporin A using
the MLR assay were published [52].
Target‑based approaches
In phenotype screens, the mode of action (i.e., molecular
target) of the compound tested is not known or assumed.
In contrast, target-based screening is based on the implica-
tion or the validated understanding that interaction of tested
compound with the designated target (usually an enzyme,
enzyme complex, or a receptor) will result in the desired
pharmacological effect. The screen is an assay that pro-
vides a measurable read-out if the tested compound inter-
acts specifically with the target. Target-based assays can be
as simple as in vitro enzyme assays. Target-based assays
can also employ genetically engineered yeast or bacteria in
whole-cell survival read outs, similar to that used in pheno-
typic screening.
Only a very small number of NPs were identified by
using a specific enzyme assay and later developed as drugs.
Two are described here: lovastatin and clavulanic acid. As
early as the middle 1960s, the biochemical pathway of cho-
lesterol biosynthesis was fairly well understood and the
enzyme hydroxymethylglutaryl-CoA reductase (HMGR)
was known to convert HMG-CoA to the intermediate
mevalonate. Akira Endo at Sankyo developed a three step
assay (two phenotypic screens and a target-based assay)
using liver tissues extracted from rats to screen fermenta-
tion broths to find NPs that specifically inhibited HMGR
[43]. In the first screen, the broths were screened examin-
ing the conversion of 14C-acetate to 14C-non-saponifiable
lipids (e.g., cholesterol). Broths that exhibited inhibition
were then screened in an assay that measured conversion
of 3H-mevalonate to 3H-lipids. Broths that did not inhibit
this conversion were then assayed for the conversion of
14
C-HMG-CoA to 14C-mevalonate. Using this approach,
Endo discovered the compound ML-236B, later named
compactin (Fig. 4), from the broth of a strain of Penicil-
lium citrinum that was isolated from a rice sample collected
from a vendor in Kyoto. Compactin was not developed
as a drug, however. Using screening strategies analogous
to that used to discover compactin, Endo and a group at
Merck independently discovered lovastatin, a methylated
analog of compactin, from two different fungi, Monascus
13
J Ind Microbiol Biotechnol (2016) 43:155–176
ruber and Aspergillus terreus, respectively [2, 42]. Merck
developed lovastatin to become the first in class of the
statins successfully used to treat hypercholesteremia. Sec-
ond-generation synthetic derivatives of compactin and lov-
astatin, known under the trade names Pravachol®, Zocor®,
and Lipitor® are among the world’s largest selling drugs.
In retrospect, the contributions that Endo brought to human
health with the discovery of compactin may not have been
adequately recognized.
Clavulanic acid was discovered by Beecham [98] in a
culture broth of a cephamycin producing strain of Strep-
tomyces clavuligerus first described by Lilly scientists
in 1971 [8, 83]. Although clavulanic acid was not active
enough to be considered for development as an antibacte-
rial agent, it was later shown to be an effective competi-
tive inhibitor of a number of β-lactamases which, by as
early as the 1950s, were known to be mediators of clinical
resistance of Gram positive and Gram negative pathogens
to penicillin and other β-lactam antibiotics [95]. Clavu-
lanic acid has been combined with amoxicillin, a semi-syn-
thetic derivative of penicillin, in the drug Augmentin® to
widen the spectrum of pathogens susceptible to antibiotic
treatment.
During the second 30 year period, discovery of new NPs
from screening continued. We cannot accurately state the
number of NCEs discovered during this period, but the
number of patents filed world-wide exceeded 1300 [70].
Yet less than two dozen novel NPs, their-semi-synthetic or
completely synthetic derivatives that were discovered dur-
ing this period were developed as drugs [114]. In contrast
to the first 30 year period, which gave rise to the introduc-
tion of dozens of antibacterials NPs into clinical practice,
the only novel anti-bacterial NP discovered during the sec-
ond 30 year period that went through clinical development
was daptomycin; the echinocandins represent the only class
of novel antifungal NPs discovered during this period that
were later approved as drugs. On the other hand, numerous
2nd, 3rd, and in the case of the cephalosporins, 4th genera-
tion semi- or completely synthetic derivatives of antibac-
terial NPs, representing many chemical classes, were suc-
cessfully developed during this period.
During the 1990s, combinatorial chemistry was devel-
oped that could give rise to thousands of unique com-
pounds from hundreds of chemical scaffolds. Most phar-
maceutical companies either purchased or developed their
own ‘combi-chem’ libraries that numbered in the 5 × 105
to 4 × 106 range. In addition, advances in robotics together
with the development of receptor-binding or enzyme-based
assays with facile read-outs enabled companies to run these
libraries through multiple high-throughput screens (HTS)
simultaneously. To handle the large numbers, mixtures
were made containing known compounds and screened;
mixtures yielding “hits” were deconvoluted in subsequent
J Ind Microbiol Biotechnol (2016) 43:155–176 169
screening rounds. In contrast, NP preparation of broths
could not be scaled up practically and, hence, could not
keep pace with the generation of known synthetic chemi-
cals. The prospect of screening endless numbers of known
compounds rapidly versus the low-throughput phenotypic
screens of small numbers of broths that may or may not
have contained NPs was too enticing for management in
pharmaceutical companies to pass up. By the mid-1990s,
most pharmaceutical companies in the US and Europe
stopped screening natural products. The exception was
Novartis, which continues NP screening [102].
During the period between the mid-1990s to the mid-
2000s, millions of compounds were screened against hun-
dreds of targets in areas relating to metabolic diseases,
cancer, neurobiology, cardiovascular, immunology, and
infectious disease. More compounds were screened in this
period than in the preceding 60 years of drug discovery,
yet this effort returned very few quality compounds, and to
date has produced no approved drugs. GSK ran a library
of upwards of 500,000 synthetic compounds through 70
antibacterial screens on targets from Streptococcus pneu-
monia. All the targets were validated as essential proteins
involved in macromolecular biosynthesis, including targets
for which there were known NP antibiotics: MurA, fosfo-
mycin; FabI/FabH, cerulenin, thiolactomycin; PBP2, peni-
cillins, cepahlosporins. All of the screens either returned
no hits, or hits that were not turned into leads. The same
500,000 compound library was also run through whole
cell antibacterial screens using E. coli and Staphyloccocus
aureus, again resulting in no useful leads [96].
Mutasynthesis and precursor‑directed biosynthesis
Mutational biosynthesis, commonly referred to as muta-
synthesis, is the production of novel NPs through the
incorporation of analogs of NP intermediates into biosyn-
thetic pathways. This approach, coined by Kenneth Rine-
hart, was started in the late 1960s in his laboratory, and
used extensively throughout the 1970s and 1980s. Feed-
ing streptamine or epistreptamine, analogs of the normal
precursor 2-deoxystreptamine, to mutants of Strepomyces
fradiae blocked in neomycin biosynthesis, caused produc-
tion of neomycin analogs [103]. Similar mutasynthesis
programs were used to produce analogs of novobiocin,
the nucleoside antibiotic nikkomycin, as well as other
NPs [129]. The early studies were undertaken before full
knowledge of the biochemical pathways became available.
Understanding the steps in secondary metabolite biosynthe-
sis, coupled with mutagenesis to block specific steps has
enabled a related approach termed product-directed bio-
conversion [10]. An example is the approach was taken at
Eli Lilly and Company in the 1980s to generate macrolide
antibiotics related to tylosin. Macrocin, the immediate
precursor to tylosin, was produced by a mutant blocked in
terminal O-methylation generated by N-methyl-N’-nitro-
N-nitrosoguinadine (MNNG) mutagenesis, affording a fer-
mentation process to overproduce macrocin [22]. Purified
macrocin was then fed to Streptomyces thermotolerans, a
strain that expresses 4″-mycarosyl isovaleryl-CoA trans-
ferase, leading to the biosynthesis of 4″-isovalerylmacro-
sin, a highly active antibiotic that was evaluated as an ani-
mal health product by Lilly Elanco [121]. Although Lilly
Elanco chose not to develop this compound, other compa-
nies currently manufacture a related compound, 3-acetyl-
4″-isovaleryltylosin (AIV or Tylvalosin), for treatment of
Mycoplasma gallisepticum air sac infections in chickens,
by feeding tylosin to Streptomyces thermotolerans for bio-
conversion [135].
Other mutants blocked in tylosin biosynthesis produced
high levels of other precursors or shunt metabolites which
served as starting materials for semi-synthesis. One mutant
produced desmycosin, which lacked a single sugar resi-
due. Desmycosin was used as a scaffold for semi-synthesis
culminating in the discovery of tilmicosin [35], marketed
by Lilly Elanco as Micotil®. Semi-synthesis has also been
used to generate a second generation derivative of spino-
sad (Fig. 2), a glycosylated type I polyketide insecticide
marketed by Dow AgroSciences for plant crop protec-
tion. MNNG-induced mutants of Saccharopolyspora spi-
nosa blocked in spinosad biosynthesis were isolated and
evaluated for insecticidal activities [69, 125]. One of the
mutants was defective in the O-methylation of the 3′-OH
of the ramnosyl moiety and accumulated high levels of fac-
tors J and L, the starting materials for the semi-synthesis of
spinetoram, which is marketed as Radiant® by Dow Agro-
Sciences [45, 108].
Precursor-directed biosynthesis also employs the addi-
tion of specific compounds to the growth medium that ulti-
mately get incorporated into the pathway resulting in the
biosynthesis of a novel NP. Unlike mutasynthesis, however,
the compound supplied does not replace an intermediate in
the pathway, but a precursor, hence mutants employed are
not in the biochemical pathway per se. The best example
of precursor-directed biosynthesis is the production of the
anthelminthic compound doramectin. By the early 1980s,
it was well understood that the avemectins employed the
precursors isobutyryl-CoA (e.g., avemectin 1B; Fig. 2)
or 2-methylbutyryl-CoA to initiate their biosyntheses in
S. avermitilis. In the 1990s, the Pfizer group identified
the set of bkd genes in S. avermitilis that were respon-
sible for the breakdown of branched-chain amino acids
valine, isoleucine, and leucine that resulted in the produc-
tion of isobutyryl-CoA and 2-methylbutyryl-CoA, as well
as isovaleryl-CoA. After disrupting this pathway, they
found that numerous carboxylic acids could be introduced
into the “starting” position of avermectin, giving rise to a
13

170
variety of novel analogs [38]. Among them was the com-
pound named doramectin, which carries cyclohexane in
place of the isobutyate or 2-methylbutyrate side chain. Ser-
endipitously, the S. avermitilis avermectin producing strain
contains one or more acyl-CoA synthetases that have broad
substrate specificities which, upon direct feeding of free
acids, enable them to generate a variety of acyl-CoAs that
can initiate avermectin biosynthesis. A similar approach to
changing the structure of the starter employed in rapamy-
cin biosynthesis was attempted. The dihydroxycyclohexyl
starter unit of rapamycin was replaced with a hydroxycy-
clohexyl- or hydroxycyclohepatanyl-unit when the corre-
sponding carboxylic acids were fed to a rapK mutant of the
rapamycin producer, Streptomyces hygroscopicus, blocked
in the production of the natural starter [48]. Similarly, feed-
ing of sulfur-containing analogs of the precursor pipecolic
acid to a rapL mutant blocked in pipecolic acid biosynthe-
sis generated a number of sulfide- or sulfoxide-containing
rapamycin analogs [65]. None of these naturally produced
rapalogs were advanced to clinical trials, however.
Two important scientific and technological develop-
ments, briefly reviewed here, took place during this 30 year
period that had significant impact on NP discovery: the
development of genetic tools for streptomycetes, and the
detailed understanding of NP biosynthesis.
Genetic tools for streptomycetes
The development of recombinant DNA (rDNA) technol-
ogy in Escherichia coli took place in the early 1970s; NPs
played an important role in its development as all vectors
used for cloning depended on the use of genes that con-
ferred resistance to antibiotics (penicillin, kanamycin,
chloramphenicol, etc.) [33]. Development of rDNA tech-
nologies in Streptomyces species lagged for about a dec-
ade. Some of the initial advancements included the devel-
opment of plasmid vectors and protoplast transformation
and regeneration techniques for a variety of both academic
and industrial strains [e.g., see 27, 76]. Later, bifunctional
vectors were constructed that facilitated engineering of
secondary metabolite genes in E. coli followed by conju-
gation into streptomycetes and site-specific integration
into bacteriophage attachment (attB) sites for stable main-
tenance of the engineered genes or gene clusters [18, 28].
These advancements were followed by improved genetic
engineering methods facilitated by the use of the λ RED
recombination system of cloned genes and gene clusters
in bacterial artificial chromosomes (BACs) in E. coli, fol-
lowed by conjugation and site-specific integration into any
of a number of attB sites to facilitate combinatorial bio-
synthesis [3, 20, 87, 88]. In addition, all analytical meth-
ods developed for determining gene expression (transcrip-
tomics, RNA-Seq), enzyme levels (proteomics), precursor
13
J Ind Microbiol Biotechnol (2016) 43:155–176
supply (metabolomics) in E. coli were directly applicable
to streptomycetes.
Biochemistry and genetics of NP biosynthesis
Biochemical pathways for the production of penicillins
and cephalosporins were determined by the middle 1960s,
employing feeding experiments and biochemical assays on
isolated enzymes [97]. When DNA cloning in actinomy-
cetes and DNA sequencing became available, biochemical
pathways of NPs were inferred largely from the sequences
of the genes that determined the corresponding enzymes,
although enzymology was always prominent in academic
labs interested in NP biosynthesis. Involvement of genes
in biochemical pathways was often determined by gene
disruption, followed in some instances by complementa-
tion analysis. As more biosynthetic genes were sequenced,
informatics was used to construct the biochemical steps
determined by the predicted enzymes. This paradigm has
been used for more than 100 NPs where pathways have
been verified genetically, as well as for hundreds more
of their structural relatives for which genetic verifica-
tion is lacking. All of these data established that, overall,
biochemical pathways of NPs appear to contain discrete,
single function enzymes that act on and release diffusible
compounds, and large multifunctional enzyme complexes
that carry out many processive enzymatic steps in a linear
order where all compounds are tethered to the complex
in the form of thioester linkages. A brief overview is pro-
vided here. Detailed descriptions of biochemical pathways,
genetics of NP biosynthesis, and structural information of
multifunctional enzymes can be found in other reviews [54,
56, 71, 111, 128]. Aminoglycosides and other NPs consist-
ing exclusively of sugars, deoxysugars, or acylated sugars,
are produced by discrete single-function enzymes. The
tripeptide backbones of penicillins and cephalosporins, the
tetrapeptide subtilisin, and the cyclic tridecapeptide dap-
tomycin are each produced by enzyme complexes collec-
tively named non-ribosomal peptide synthetases (NRPS).
NRPSs consist of modules, which themselves consist of
domains that are responsible for the incorporation of one
selected amino acid into the nacent polypeptide chain. The
peptide backbone of the glycopeptide vancomycin consist-
ing of seven amino acids is biosynthesized by three NRPS
proteins containing a total of seven modules. The sugar,
vancosamine, is generated from glucose by seven discrete
enzymes. Interestingly, the NRPS that produces the cyclic
decapeptide cyclosporin A consists of a single protein con-
taining ten modules. Similarly, the large macrocyclic back-
bones of the polyketides erythromycin, tylosin, spinosyn A,
avermectin, amphotericin B, etc., are produced by specific
multifunctional enzymes designated polyketide synthases
(PKSs) which incorporate, in a processive fashion, specific
J Ind Microbiol Biotechnol (2016) 43:155–176 171
acyl-CoA precursors, into a growing acyl (polyketide)
chain which is tethered to the multienzyme as a thioester
until released at the termination of synthesis. These type
I PKSs are also composed of modules which themselves
consist of enzymatic domains responsible for a single step
in the biosynthesis of the polyketide backbone. The sugar
components of erythromycin, tylosin, and others are pro-
duced by discrete enzymes.
Early cloning experiments, followed by sequencing of
biosynthesis genes initially, and entire genomes afterward
consistently revealed that the genes for entire pathways of
NP biosynthesis were clustered, even in eukaryotes (e.g.,
fungi) whose genomes are composed of multiple chromo-
somes. The erythromycin biosynthesis cluster consists of
three large PKS genes (35 kb) flanked by 17 genes involved
in the biosynthesis of sugars, hydroxylation of the pol-
yketide backbone, methylatation of one of the sugars, and
a thioesterase, as well as a gene conferring resistance to
erythromycin [113]. The entire cluster is greater than 50 kb.
Some NP biosynthesis clusters are much larger. Some clus-
ters (e.g., tylosin [34]), contain a number of positive and
negative regulatory genes. Clustering facilitates the clon-
ing of the entire NP biosynthesis pathway into vectors, as
described above, for production of NPs in heterologous
hosts, or for combinatorial biosynthesis.
Combinatorial biosynthesis
At Eli Lilly and Company, advances in genetic engineering
methodologies facilitated the generation of novel polyke-
tides and glycopeptides. Building on the precursor-directed
biosynthesis studies described earlier [7], scientists at Lilly
cloned the 4″-mycarosyl isovaleryl-CoA transferase gene
(carE) from Streptomyces thermotolerans and expressed
it in Streptomyces ambofaciens, the spiramycin producer
[44]. Expression of carE in S. ambofaciens caused the pro-
duction of isovalerylspiramycin. This served as an early
validation of what would later be termed combinatorial
biosynthesis [10]. A second example was the reprogram-
ming of the polyketide synthase involved in spiramycin
biosynthesis by exchanging a module with a different acyl-
transferase specificity to produce a novel hybrid polyketide
[72]. A novel glycopeptide was generated by cloning the
glucosyltransferase gene (gtfE) from the vancomycin pro-
ducer, A. orientalis, into the A47934 producer, Streptomy-
ces toyocaensis [107]. This was the first novel glycopeptide
produced by genetic engineering, and it further validated
the concept of combinatorial biosynthesis in Streptomyces
species.
At Cubist Pharmaceuticals, the use of λ RED recom-
bineering of lipopeptides cloned in BAC vectors facilitated
combinatorial biosynthesis around the core cyclic peptide
structures of daptomycin and A54145 to generate about
120 novel derivatives for evaluation as potent antibacterial
agents active against Gram-positive pathogens [3, 4, 20, 87,
88]. The work at Cubist helped define the “rules” for suc-
cessful engineering of NRPS pathways, and demonstrated
that complex pharmacological properties can be modulated
by changing the primary amino acid sequences of the tride-
capeptides of daptomycin and A54145 in ways not address-
able by synthetic chemistry.
Combinatorial biosynthesis of modular PKSs has had
limited successes at Lilly, Abbott, Kosan, and Biotica.
Changes to the structure of erythromycin, tylosin, pikromy-
cin, epothilone and other polyketides were initially accom-
plished by domain swaps employing cumbersome and time
consuming double reciprocal, two step recombination in
the producing organism [93, 100, 116, 117]. At Kosan, the
erythromycin PKS was re-synthesized employing E. coli
codon usage and unique restriction sites so that domain
swaps could be done combinatorially using restriction site
cloning leading to multiple changes in the structure of the
erythromycin polyketide backbone [77]. In addition, scien-
tists at Kosan showed that the terminal two modules of the
erythromycin PKS, carried on a single PKS polypeptide,
could be replaced by the last two modules of the pikro-
mycin or oleandomycin PKSs to yield hybrid polyketides.
This work was carried out by combining plasmids carrying
separate PKS modules from the ery, pik, or ole PKSs in the
host Streptomyces lividans [116]. At Kosan, hybrid PKS
composed from the tylosin and chalcomycin PKSs or the
tylosin and spiramycin PKSs produced the expected hybrid
macrolactone backbones. When these hybrid PKSs were
placed in a strain of Streptomyces fradiae deleted for the
native PKS genes, the hybrid macrolactones produced were
then acted upon by the tylosin tailoring enzymes (glycosyl
transferases, hydroxylases, methytransferases) to produce
novel, fully elaborated NPs [99]. More progress in PKS
engineering has been made in recent years as better under-
standing of PKS structure has emerged, but the promise of
new polyketide-based drugs has not yet been realized.
Genomics‑based approaches (2000s and beyond)
In the early 2000s, the first two Streptomyces genome
sequences revealed the striking observation that many
more secondary metabolite gene clusters (SMGCs) are
encoded in these large genomes than predicted from their
expressed secondary metabolomes [24, 31, 58, 59]. These
observations have since been generalized to other actino-
mycetes with large genomes [6, 14, 17, 23, 57, 62, 84, 90].
It is now estimated that <10 % of SMGCs are expressed in
sufficient quantities to be observed under routine fermenta-
tion analyses, and the others require special conditions or
genetic manipulations to reveal their products [21, 89, 133,
13

172
134]. Among the actinomycetes, the number of SMGCs
varies widely [14, 84], and actinomycetes with very large
genomes tend to be the most “gifted” [17, 19]. An extreme
example is Streptomyces rapamycinicus (a rapamycin pro-
ducer) which has a 12.7 Mb genome with 3.0 Mb (24 %)
devoted to the biosynthesis of 48 SMGCs [23]. Sequence
information also revealed the high frequencies of biosyn-
thetic clusters of the NPs discovered in the early discovery
period, as well as the presence of antibiotic “resistomes”
(multiple genes for antibiotic resistance) among soil iso-
lates [131]. Indeed, current knowledge on mechanisms of
antibiotic resistance can be exploited to select directly for
producers of vancomycin and related glycopeptides from
soil actinomycetes [119]. This concept can be extended to
other antibiotic classes where the mechanism of antibacte-
rial resistance is known and easily selectable.
With inexpensive genome sequencing, advances in
understanding secondary metabolite biosynthesis, and
advances in analytical and bioinformatics methods, it is
now possible to predict at least partial structures of new
SMs related to known SMs, and truly novel SMs by scan-
ning DNA sequences [78, 120, 126]. Although gene clus-
ter sequences are not yet available for all known SMs, this
process continues to improve as more SMGC sequences
become available. Predicting new and novel pathways from
genome sequence data bypasses the tedious task of derep-
lication of known compounds, which has been the bane of
natural products discovery in recent years [9, 11, 13]. Fur-
thermore, improved technology for DNA synthesis, clon-
ing, and heterologous expression allows the rapid trans-
plantation of novel NP clusters from the sequenced host
into well characterized, genetically tractable engineered
hosts that have high levels of needed precursors, and con-
stitutively expressed strong promoters, together with new
high-throughput MS detection methodology [105, 132], to
enable high probability production and detection of novel
NPs [21].
Genome mining and activation of cryptic pathways
As described above, it is now apparent that actinomy-
cetes with large genomes encode multiple (mostly cryptic)
SMGCs [1, 6, 23, 24, 31, 58, 59, 90] ranging from ~20 to
50 per genome. It is now feasible to sequence 10,000 [62]
to over 100,000 actinomycete genomes in pools (Warp
Drive Bio, unpublished data) to identify new and novel
SMGCs. There are a number of genetic and fermentation
methods to activate the expression of cryptic or poorly
expressed SMGCs. Genetic methods include: (1) chemical
and transposon mutagenesis; (2) gene cluster duplication/
amplification; (3) overexpression of positive regulators;
(4) disruption of negative regulators; (5) modulation of the
transcription apparatus; (6) modulation of the translation
13
J Ind Microbiol Biotechnol (2016) 43:155–176
apparatus; (7) modulation of post translation of carrier
proteins; (8) heterologous expression; (9) synthetic biol-
ogy refactoring of promoters and ribosome binding sites;
and (10) combinations of (1) through (9). These and other
methods to activate or improve the production of SMs are
reviewed in detail elsewhere [15, 16, 21].
Aside from exploring multiple fermentation media, there
are a number of elicitors or stimulators of SM production,
including certain types of stress or chemicals that can be
added to fermentations (e.g., heat, ethanol, rare earth ele-
ments, glucosamine, and others) [89, 133, 134] or antibiot-
ics added at sub-inhibitory concentrations [5, 115]. This is
an area ripe for future investigations.
Advances in combinatorial biosynthesis
Early attempts at combinatorial biosynthesis as a means
to produce novel NP structures were limited by a lack of
understanding of the fundamental structural requirements
of these megasynthases for activity. Much progress has
been made in understanding the important protein–protein
interactions critical to the functioning of PKS and NRPS
megaenzymes [12, 20, 40, 66, 73, 128, 130]. In addition,
much is now known about effective approaches for com-
binatorial biosynthesis of post-PKS and other modifica-
tions of SMs [12, 51, 91, 92]. Combinatorial biosynthesis
can also be coupled with medicinal chemistry to further
expand the numbers and variety of compounds available
for evaluation [67]. With the rapid accumulation of new
DNA sequences for novel PKS, NRPS and other SMGCs
afforded by inexpensive microbial genome sequencing,
which can serve as important new sources of “parts and
devices” for combinatorial biosynthesis, it is time to rein-
vigorate this important approach as an adjunct to microbial
genome mining for drug discovery.
The future of natural product discovery
We believe that three key elements will drive natural prod-
ucts discovery from microbes in the coming years. 1. We
have already learned that microbes (particularly actino-
mycetes) with large genomes encode multiple SMGCs,
most of which are cryptic, and many of which appear to
encode novel SMs. Many more cryptic pathways will be
uncovered by inexpensive genome sequencing. Though
structural details of truly novel SMs cannot be predicted
accurately by bioinformatics alone, the current programs
are rapidly improving their predictive power and can read-
ily identify truly novel SMGCs [61, 78, 105, 120, 126].
These novel gene clusters can be expressed by genetic and
physiological manipulations of the producing microbes or
by expression in a heterologous hosts. 2. Scientists have
J Ind Microbiol Biotechnol (2016) 43:155–176 173
sampled a miniscule fraction of soil environments [9, 13]
and metagenomic and other studies indicate that there
exists a wealth of novel SMGCs yet to be discovered [32,
64, 79, 94, 106]. Vastly expanded sampling of soil will
provide an inexhaustible source of new microbes (cultiva-
ble or not) for genome mining. 3. Advances in structural
biology of complex PKS megaenzymes [40, 128, 130] and
in functional analysis of engineered NRPS megaenzymes
[20] bode well for future applications of combinatorial
biosynthesis to exploit novel PKS and NRPS modules and
domains identified by genome mining. These three cor-
nerstones will be supported by continuing advancements
in enzymology, structural biology, bioinformatics, tran-
scriptomics, proteomics, metabolomics, and advances in
mass spectral analysis. In addition, as understanding of
cellular function and the molecular basis of human disease
increases, more “smart-screens” will be developed to find
NPs with desired activities. Indeed, we predict a second
“Golden Age for Antibiotics” and natural product discov-
ery. Stay tuned!
References
1. Aigle B, Lautru S, Spiteller D, Dickschat JS, Challis GL, Leb-
lond P, Pernodet JL (2014) Genome mining of Streptomyces
ambofaciens. J Ind Microbiol Biotechnol 41:251–264
2. Alberts AW, Chen J, Kuron G et al (1980) Mevinolin: a highly
potent competitive inhibitor of hydroxymethylglutaryl-CoA
reductase and a cholesterol-lowering agent. Proc Natl Acad Sci
USA 77:3957–3961
3. Alexander D, Rock J, He X, Miao V, Brian P, Baltz RH (2010)
Development of a genetic system for lipopeptide combinatorial
biosynthesis in Streptomyces fradiae and heterologous expres-
sion of the A54145 biosynthetic gene cluster. Appl Environ
Microbiol 76:6877–6887
4. Alexander D, Rock J, Gu JQ, Mascio C, Chu M, Brian P, Baltz
RH (2011) Production of novel lipopeptide antibiotics related
to A54145 by Streptomyces fradiae mutants blocked in biosyn-
thesis of modified amino acids and assignment of lptJ, lptK and
lptL gene functions. J Antibiot 64:79–87
5. Amando SI, Sakurai T, Endo K et al (2011) A cryptic antibiotic
triggered by monensin. J Antibiot 64:703
6. Bachmann BO, Van Lanen SG, Baltz RH (2014) Microbial
genome mining for accelerated natural products discovery:
is a renaissance in the making? J Ind Microbiol Biotechnol
41:175–184
7. Baltz RH (1982) Genetics and biochemistry of tylosin produc-
tion: a model for genetic engineering in antibiotic-producing
Streptomyces. Basic Life Sci 19:431–444
8. Baltz RH (2005) Natural product discovery and development at
Eli Lilly and Company: one scientists view. SIM News 55:5–16
9. Baltz RH (2005) Antibiotic discovery from actinomycetes:
will a renaissance follow the decline and fall? SIM News
55:186–196
10. Baltz RH (2006) Combinatorial biosynthesis of novel antibi-
otics and other secondary metabolites in actinomycetes. SIM
News 56:148–160
11. Baltz RH (2006) Marcel Faber Roundtable: is our antibiotic
pipeline unproductive because of starvation, constipation or
lack of inspiration? J Ind Microbiol Biotechnol 33:507–513
12. Baltz RH (2006) Molecular engineering approaches to peptide,
polyketide and other antibiotics. Nat Biotechnol 24:1533–1540
13. Baltz RH (2007) Antimicrobials from actinomycetes: back to
the future. Microbe 2:125–131
14. Baltz RH (2008) Renaissance in antibacterial discovery from
actinomycetes. Curr Opin Pharmacol 8:557–563
15. Baltz RH (2010) Streptomyces and Saccharopolyspora hosts for
heterologous expression of secondary metabolite gene clusters.
J Ind Microbiol Biotechnol 37:759–772
16. Baltz RH (2011) Strain improvement in actinomycetes in the
postgenomic era. J Ind Microbiol Biotechnol 38:657–666
17. Baltz RH (2011) Function of MbtH homologs in nonribosomal
peptide biosynthesis and applications in secondary metabolite
discovery. J Ind Microbiol Biotechnol 38:1747–1760
18. Baltz RH (2012) Streptomyces temperate bacteriophage integra-
tion systems for stable genetic engineering of actinomycetes
(and other organisms). J Ind Microbiol Biotechnol 39:661–672
19. Baltz RH (2014) MbtH homology codes to identify gifted
microbes for genome mining. J Ind Microbiol Biotechnol
41:357–369
20. Baltz RH (2014) Combinatorial biosynthesis of cyclic lipopep-
tide antibiotics: a model for synthetic biology to accelerate the
evolution of secondary metabolite biosynthetic pathways. ACS
Synth Biol 3:748–759
21. Baltz RH (2015) Genetic manipulation of secondary metabo-
lite biosynthesis for improved production in Streptomyces and
other actinomycetes. J Ind Microbiol Biotechnol. doi:10.1007/
s10295-015-1682-x
22. Baltz RH, Seno ET (1981) Properties of Streptomyces fradiae
mutants blocked in biosynthesis of the macrolide antibiotic
tylosin. Antimicrob Agents Chemother 20:214–225
23. Baranasic D, Gacesa R, Starcevic A et al (2013) Draft genome
sequence of Streptomyces rapamycinicus strain NRRL 5491,
the producer of the immunosuppressant rapamycin. Genome
Announc 1:e00581–e00613
24. Bentley SD, Chater KF, Cerdeño-Tárraga AM et al (2002)
Complete genome sequence of the model actinomycete Strepto-
myces coelicolor A3(2). Nature 417:141–147
25. Bérdy J (2012) Thoughts and facts about antibiotics: where we
are now and where we are heading. J Antibiot 65:385–395
26. Bhanot A, Sharma R, Noolvi MN (2011) Natural sources as
potential anti-cancer agents: a review. Int J Phytomed 3:9–26
27. Bibb MJ, Ward JM, Hopwood DA (1978) Transformation of
plasmid DNA into Streptomyces at high frequency. Nature
274:398–400
28. Bierman M, Logan R, O’Brien K, Seno ET, Rao RN, Schoner
BE (1992) Plasmid cloning vectors for the conjugal trans-
fer of DNA from Escherichia coli to Streptomyces spp. Gene
116:43–49
29. Breton RC, Reynolds WF (2013) Using NMR to identify and
characterize natural products. Nat Prod Rep 30:501–534
30. Butler MS, Robertson AA, Cooper MA (2014) Natural product
and natural product derived drugs in clinical trials. Nat Prod
Rep 31:1612–1661
31. Challis GL (2014) Exploitation of the Streptomyces coelicolor
A3(2) genome sequence for discovery of new natural prod-
ucts and biosynthetic pathways. J Ind Microbiol Biotechnol
41:219–232
32. Charlop-Powers Z, Owen JG, Reddy BVB, Ternei MA, Guima-
raes DO, de Frias UA, Pupo MT, Seepe P, Feng Z, Brady SF
(2015) Global biogeographic sampling of bacterial secondary
metabolism. eLife 4:e05048
13

174
33. Cohen SN, Chang AC, Boyer HW, Helling RB (1973) Con-
struction of biologically functional bacterial plasmids in vitro.
Proc Natl Acad Sci USA 70:3240–3244
34. Cundliffe E (2008) Control of tylosin biosynthesis in Strepto-
myces fradiae. J Microbiol Biotechnol 18:1485–1491
35. Debono M, Willard KE, Kirst HA et al (1989) Synthesis and
antimicrobial evaluation of 20-deoxo-20-(3,5-dimethylpiperi-
din-1-yl)desmycosin and related cyclic amino derivatives. J
Antibiot 42:1253–1267
36. Demain AL (2014) Importance of microbial natural products
and the need to revitalize their discovery. J Ind Microbiol Bio-
technol 41:185–201
37. Denizot F, Lang R (1986) Rapid colorimetric assay for cell
growth and survival. Modifications to the tetrazolium dye pro-
cedure giving improved sensitivity and reliability. J Immunol
Methods 89:271–277
38. Denoya CD, Fedechko RW, Hafner EW et al (1995) A second
branched-chain α-keto acid dehydrogenase gene cluster (bkd-
FGH) from Streptomyces avermitilis: its relationship to aver-
mectin biosynthesis and the construction of a bkdF mutant
suitable for the production of novel antiparasitic avermectins. J
Bacteriol 177:3504–3511
39. DiMarco A, Gaetani M, Dorigotti L, Soldati M, Bellini O
(1964) Daunomycin: a new antibiotic with anti-tumor activity.
Cancer Chemother Rep 38:31–38
40. Dutta S, Whicher JR, Hansen DA et al (2014) Structure of a
modular polyketide synthase. Nature 510:512–517
41. Eisenstein BI, Oleson FB Jr, Baltz RH (2010) Daptomycin:
from the mountain to the clinic with the essential help from
Francis Tally, MD. Clin Inf Dis 50:S10–S15
42. Endo AMK (1979) A new hypo-cholesteremic agent produced
by a Monascus species. J Antibiot 32:852–854
43. Endo A (2010) A historical perspective on the discovery of the
statins. Proc Jpn Acad Ser B 86:484–493
44. Epp JK, Huber ML, Turner JR, Goodson T, Schoner BE (1989)
Production of a hybrid macrolide antibiotic in Streptomyces
ambofaciens and Streptomyces lividans by introduction of a
cloned carbomycin biosynthetic gene from Streptomyces ther-
motolerans. Gene 85:293–301
45. Galm U, Sparks TC (2015) Natural product derived insecti-
cides: discovery and development of spinetoram. J Ind Micro-
biol Biotechnol. doi:10.1007/s10295-015-1710-x
46. Gallo RC, Whang-Peng J, Adamson RH (1971) Studies on the
antitumor activity, mechanism of action, and cell cycle effects
of camptothecin. J Nat Cancer Inst 46:789–795
47. Giddings LA, Newman DJH (2013) Microbial natural products:
molecular blueprints for antitumor drugs. J Ind Microbiol Bio-
technol 40:1181–1210
48. Gregory MA, Petkovic H, Lill RE, Moss SJ, Wilkinson B, Gais-
ser S, Leadlay PF, Sheridan RE (2005) Mutasynthesis of rapa-
mycin analogues through the manipulation of a gene governing
starter unit biosynthesis. Angew Chem Int Ed 44:4757–4760
49. Havlicek V, Lemr K, Schug KA (2013) Current trends in
microbial diagnostics based on mass spectrometry. Anal Chem
85:790–797
50. Heeb S, Fletcher MP, Chhabra SR, Diggle SP, Williams P,
Cámara M (2011) Quinolones: from antibiotics to autoinducers.
FEMS Microbiol Rev 35:247–274
51. Heide L (2014) New aminocoumarin antibiotics as gyrase
inhibitors. Int J Med Microbiol 304:31–36
52. Henderson DJ, Naya I, Bundick RV, Smith GM, Schmidt JA
(1991) Comparison of the effects of FK-506, cyclosporine A
and rapamycin on IL-2 production. Immunology 73:316–321
53. Hendlin D, Stapley EO, Jackson M et al (1969) Phosphonomy-
cin, a new antibiotic produced by strains of Streptomyces. Sci-
ence 166:122–123
13
J Ind Microbiol Biotechnol (2016) 43:155–176
54. Hertweck C (2015) Decoding and reprogramming complex pol-
yketide assembly lines: prospects for synthetic biology. Trends
Biochem Sci 40:189–199
55. Heusler K, Pletscher A (2001) The controversial early history of
cyclosporine. Swiss Med Wkly 131:299–302
56. Hur GH, Vickery CR, Burkart MD (2012) Explorations of cata-
lytic domains in non-ribosomal peptide synthetase enzymology.
Nat Prod Rep 29:1074–1098
57. Iftime D, Kylik A, Härtner T et al (2015) Identification and acti-
vation of novel biosynthetic gene clusters by genome mining in
the kirromycin producer Tü 365. J Ind Microbiol Biotechnol.
doi:10.1007/s100295-015-1685-7
Shiba T, Sakaki Y, Hattori M, Ōmura S (2003) Complete
58. Ikeda H, Ishikawas J, Hanamoto A, Shinose M, Kikuchi H,
genome sequence of and comparative analysis of the indus-
trial microorganism Streptomyces avermitilis. Nat Biotechnol
21:526–531
59. Ikeda H, Shin-ya K, Omura S (2014) Genome mining of the
Streptomyces avermitilis genome and development of genome-
minimized hosts for heterologous expression of biosynthetic
gene clusters. J Ind Microbiol Biotechnol 41:233–250
60. Itoh T, Ishii K, Irikura T, Ueno Y, Kojima A, Horie Y (1993) A
modified method of mixed lymphocyte reaction: establishment
of the assay system and its application to extracts of fungal cul-
tures. J Antibiot 46:1575–1581
61. Johnston CW, Connaty AD, Skinnider MA, Li Y, Grunwald
A, Wyatt MA, Kerr RG, Magarvey NA (2015) Informatic
search strategies to discover analogues and variants of natural
product archetypes. J Ind Microbiol Biotechnol. doi:10.1007/
s10295-015-1675-9
62. Ju KS, Gao J, Doroghazi JR et al (2015) Discovery of
phosphonic acid natural products by mining the genomes
of 10,000 actinomycetes. Proc Natl Acad Sci USA
112:12175–12180
63. Kanomori H, Shima T, Morita C, Hata T (1957) Studies on the
antitumor activity of mitomycin. J Antibiot 10:120–127
64. Katz M, Hover BM, Brady SF (2015) Culture-independent dis-
covery of natural products from soil metagenomes. J Ind Micro-
biol Biotechnol. doi:10.1007/s10295-015-1706-6
65. Khaw LE, Böhm GA, Metcalf S, Staunton J, Leadlay PF (1998)
Mutational biosynthesis of rapamycins by a strain of Streptomy-
ces hygroscopicus NRRL 5491 disrupted in rapL, encoding a
putative lysine cyclodeaminase. J Bacteriol 180:89–814
66. Khosla C, Hershlag D, Cane DE, Walsh CT (2014) Assembly
line polyketide synthases: mechanistic insights and unsolved
problems. Biochemistry 53:2875–2883
67. Kim E, Moore BS, Yoon YJ (2015) Reinvigorating natural
product combinatorial biosynthesis with synthetic biology. Nat
Chem Biol 11:649–659
68. Kinch MS, Haynesworth A, Kinch SL, Hoyer D (2014) An
overview of FDA-approved new molecular entities: 1827–2013.
Drug Discov Today 19:1033–1039
69. Kirst HA (2010) The spinosyn family of insecticides: real-
izing the potential of natural products research. J Antibiot
63:101–111
70. Koehn FE, Carter GT (2005) The evolving role of natural prod-
ucts in drug discovery. Nat Rev Drug Discov 4:206–220
71. Kudo F, Eguchi T (2009) Biosynthesis genes for aminoglyco-
side antibiotics. J Antibiot 62:471–481
72. Kustoss S, Huber M, Turner JR, Paschal JW, Rao RN (1996)
Production of a novel polyketide through the construction of a
hybrid polyketide synthase. Gene 183:231–236
73. Ladner CC, Williams GJ (2015) Harnessing natural product
assembly lines: structure, promiscuity, and engineering. J Ind
Microbiol Biotechnol. doi:10.1007/s10295-015-1704-8
J Ind Microbiol Biotechnol (2016) 43:155–176 175
74. Lay M, Ang T, Murima P, Pethe K (2015) Next-generation anti-
microbials: from chemical biology to first-in-class drugs. Arch
Pharm Res 38:1702–1707
75. Martel RR, Klicius J, Galet S (1977) Inhibition of the immune
response by rapamycin, a new antifungal antibiotic. Can J Phys-
iol Pharmacol 55:48–51
76. Matsushima P, Baltz RH (1985) Efficient plasmid transforma-
tion of Streptomyces ambofaciens and Streptomyces fradiae
protoplasts. J Bacteriol 163:180–185
77. McDaniel R, Thamchaipenet A, Gustafsson C, Fu H, Bet-
lach M, Ashley G (1999) Multiple genetic modifications of
the erythromycin polyketide synthase to produce a library of
novel “unnatural” natural products. Proc Natl Acad Sci USA
96:1846–1851
78. Medema MH, Fischbach MA (2015) Computational approaches
to natural product discovery. Nat Chem Biol 11:639–648
79. Milshteyn A, Schneider JS, Brady SF (2014) Mining the meta-
biome: identifying novel natural products from microbial com-
munities. Chem Biol 21:1211–1223
80. Minas W, Bailey JE, Duetz W (2000) Streptomycetes in micro-
cultures: growth production of secondary metabolites, and stor-
age and retrieval in the 96 well format. Antonie Van Leeuwen-
hoek 78:297–305
81. Moffat JG, Rudolph J, Bailey D (2014) Phenotypic screening in
cancer drug discovery—past, present and future. Nat Rev Drug
Discov 13:588–602
82. Mossman T (1983) Rapid colorimetric assay for cellular growth
and survival: application to proliferation and cytotoxic assays. J
Immunol Methods 65:55–63
83. Nagarajan R, Boeck LD, Gorman M, Hamill RL, Higgens CE,
Hoehn MM, Stark WM, Whitney JG (1971) β-Lactam antibiot-
ics from Streptomyces. J Am Chem Soc 93:2308–2310
84. Nett M, Ikeda H, Moore B (2009) Genomic basis for natural
product biosynthetic diversity in the actinomycetes. Nat Prod
Rep 26:1362–1384
85. Newman DJ, Cragg GM, Snader KM (2000) The influ-
ence of natural products upon drug discovery. Nat Prod Rep
17:214–234
86. Newman DJ, Cragg GM (2012) Natural products as sources
of new drugs over the 30 years from 1981 to 2010. J Nat Prod
75:311–335
87. Nguyen K, Ritz D, Gu JQ, Alexander D, Chu M, Miao V, Brian
P, Baltz RH (2006) Combinatorial biosynthesis of lipopeptide
antibiotics related to daptomycin. Proc Natl Acad Sci USA
103:17462–17467
88. Nguyen KT, He X, Alexander D, Li C, Gu JQ, Mascio C, Van
Praagh A, Mortin L, Chu M, Silverman JA, Brian P, Baltz RH
(2010) Engineered hybrid lipopeptide antibiotics related to
A54145 and daptomycin with improved properties. Antimicrob
Agents Chemother 54:1404–1413
89. Ochi K, Tanaka Y, Tojo S (2014) Activating the expression
of bacterial cryptic genes by rpoB mutations in RNA poly-
merase or by rare earth elements. J Ind Microbiol Biotechnol
41:403–414
90. Ohnishi Y, Ishikawa J, Hara H, Suzuki H, Ikenoya M, Ikeda
H, Yamashita A, Hattori M, Horinouchi S (2008) Genome
sequence of the streptomycin-producing microorganism Strep-
tomyces griseus IFO 13350. J Bacteriol 190:4050–4060
91. Olano C, Méndez C, Salas JA (2010) Post-PKS tailoring steps
in natural product-producing actinomycetes from the perspec-
tive of combinatorial biosynthesis. Nat Prod Rep 27:571–616
92. Olano C, Méndez C, Salas JA (2011) Molecular insights on
the biosynthesis of antitumor compounds by actinomycetes.
Microb Biotechnol 4:144–164
93. Olynyk M, Brown MJ, Cortés J, Staunton J, Leadlay PF (1996)
A hybrid modular polyketide synthase obtained by domain
swapping. Chem Biol 3:833–839
94. Owen JG, Charlop-Powers Z, Smith AG, Ternei MA, Calle PY,
Reddy BJB, Montiel D, Brady SF (2015) Multiplexed metagen-
omic mining using short DNA sequence tags facilitates targeted
discovery of epoxyketone proteasome inhibitors. Proc Natl
Acad Sci USA 112:4221–4226
95. Paradkar A (2013) Clavulanic acid production by Streptomyces
clavuligerus: biogenesis, regulation and strain improvement. J
Antibiot 66:411–420
96. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL (2007) Bad
drugs for bad bugs: confronting the challenges of antibacterial
discovery. Nat Rev Drug Discov 6:29–40
97. Perlman D, Bodanszky D (1971) Biosynthesis of peptide antibi-
otics. Annu Rev Biochem 40:449–464
98. Reading C, Cole M (1977) Clavulanic acid: a beta-lactamase-
inhibiting beta-lactam from Streptomyces clavuligerus. Antimi-
crob Agents Chemother 11:852–857
99. Reeves CD, Ward SL, Revill WP et al (2004) Production of
hybrid 16-membered macrolides by expressing combinations of
polyketide synthase genes in Streptomyces fradiae hosts. Chem
Biol 11:1466–1472
100. Ruan X, Pereda A, Stassi DL et al (1997) Acyltransferase
domain substitutions in erythromycin polyketide synthase yield
novel erythromycin derivatives. J Bacteriol 179:6416–6425
101. Rudolf JD, Yan X, Shen B (2015) Genome neighborhood net-
work reveals insights into enediyne biosynthesis and facilitates
prediction and prioritization for discovery. J Ind Microbiol Bio-
technol. doi:10.1007/s10295-015-1671-0
102. Schmitt EK, Hoepfner D, Krastel P (2015) Natural products as
probes in pharmaceutical research. J Ind Microbiol Biotechnol.
doi:10.1007/s10295-015-1691-9
103. Shier WT, Rinehart KL, Gottlieb D (1969) Preparation of four
new antibiotics from a mutant of Streptomyces fradiae. Proc
Natl Acad Sci USA 63:198–204
104. Shoemaker R (2006) The NCI60 human tumour cell line anti-
cancer drug screen. Nat Rev Cancer 6:813–823
105. Skinnider MA, Dejong CA, Rees PN, Johnston CW, Li H, Web-
ster AL, Wyatt MA, Magarvey NA (2015) Genomes to natural
products PRediction Informatics for Secondary Metabolomes
(PRISM). Nucleic Acids Res 43:9645–9662
106. Smanski MJ, Schlatter DC, Kinkel LL (2015) Leveraging eco-
logical theory to guide natural product discovery. J Ind Micro-
biol Biotechnol. doi:10.1007/s10295-015-1683-9
107. Solenberg PJ, Matsushima P, Stack DR, Wilkie SC, Thompson
RC, Baltz RH (1997) Production of hybrid glycopeptide antibiotics
in vitro and in Streptomyces toyocaensis. Chem Biol 4:195–202
108. Sparks TC, Crouse GD, Dripps JE, Anzeveno P, Martynow J,
Deamichis CV, Gifford J (2008) Neural network-based QSAR
and insecticide discovery: spinetoram. J Comput Aided Mol
Des 22:393–4001
109. Stähelin HF (1996) The history of cyclosporine A (Sandim-
mun®) revisited: another point of view. Experientia 52:5–13
110. Stapley EO, Hendlin D, Mata JM, Jackson M, Wallick H, Her-
nandez S, Mochales S, Currie SA, Miler RN (1969) Phosphon-
omycin. I. Discovery and in vitro biological characterization.
Antimicrob Agents Chemother 9:284–290
111. Strieker M, Tanović A, Marahiel MA (2010) Nonribosomal
peptide synthetases: structure and dynamics. Curr Opin Struct
Biol 7:77–84
112. Strohl WR, Woodruff HB, Monaghan RL et al (2001) The his-
tory of natural products research at Merck & Co. SIM News
51:5–19
13

176
113. Summers RG, Donadio S, Staver MJ, Wendt-Pienkowski E,
Hutchinson CR, Katz L (1997) Sequencing and mutagenesis
of genes from the erythromycin biosynthetic gene cluster from
Saccharopolyspora erythraea that are involved in l-mycarose
and d-desosamine production. Microbiology 143:3251–3261
114. Swinney DC, Anthony J (2011) How were new medicines dis-
covered? Nat Rev Drug Discov 10:507–519
115. Tanaka H, Nishiyama T, Amano S, Beppu T, Kobayashi M,
Ueda K (2015) Streptomyces metabolites in divergent micro-
bial interactions. J Ind Microbiol Biotechnol. doi:10.1007/
s10295-015-1680-z
116. Tang L, Fu H, McDaniel R (2000) Formation of functional
heterologous complexes using subunits from the pikromycin,
erythromycin and oleandomycin synthases. Chem Biol 7:77–84
117. Tang L, Chung L, Carney JR, Starks CM, Licari P, Katz L
(2005) Generation of new epothilones by genetic engineering
of a polyketide synthase in Myxococcus xanthus. J Antibiot
58:178–184
118. Terstappen G, Schlüpen C, Raggiaschi R, Gaviraghi G (2007)
Target deconvolution strategies in drug discovery. Nat Rev Drug
Discov 6:891–903
119. Thaker MN, Waglechner N, Wright GD (2014) Antibiotic
resistance-mediated isolation of scaffold-specific natural prod-
uct producers. Nat Protoc 9:1469–1479
120. Tietz JI, Mitchell DA (2015) Using genomics for natural prod-
uct structure elucidation. Curr Top Med Chem (Epub ahead of
print)
121. Turner J, Krupinski VM, Fukuda DS, Baltz RH (1985) Process
for preparing macrocin derivatives. US patent 4,559,301
122. Umezawa H (1965) Bleomycin and other antibiotics of high
molecular weight. Antimicrob Agents Chemother 5:1079–1085
123. Vezina C, Kudelski A, Sehgal SN (1975) Rapamycin (AY-
22,989), a new antifungal antibiotic. I. Taxonomy of the pro-
ducing streptomycete and isolation of the active principle. J
Antibiot 28:721–726
13
View publication stats
J Ind Microbiol Biotechnol (2016) 43:155–176
124. Wagman GH, Weinstein MJ (1980) Antibiotic from Micromon-
ospora. Ann Rev Microbiol 34:537–557
125. Waldron C, Matsushima P, Rosteck PR, Broughton MC, Turner
J, Madduri K, Crawford KP, Merlo DJ, Baltz RH (2001) Clon-
ing and analysis of the spinosad biosynthetic gene cluster of
Saccharopolyspora spinosa. Chem Biol 8:487–499
126. Weber T, Blin K, Duddela S et al (2015) antiSMASH 3.0—a
comprehensive resource for the genome mining of biosynthetic
gene clusters. Nucleic Acids Res 43:w237–w243
127. Weinstein MJ (2004) Micromonospora antibiotic discovery at
Schering/Schering Plough (1961–1973). SIM News 54:56–66
128. Weissman KJ (2015) The structural biology of biosynthetic
megaenzymes. Nat Chem Biol 11:660–670
129. Weist S, Süssmuth RD (2005) Mutational biosynthesis—a tool
for the generation of structural diversity in the biosynthesis of
antibiotics. Appl Microbiol Biotechnol 68:141–150
130. Whicher JR, Dutta S, Hansen DA et al (2014) Structural rear-
rangements of a polyketide synthase module during its catalytic
cycle. Nature 510:560–564
131. Wright GD (2007) The antibiotic resistome: the nexus of chem-
ical and genetic diversity. Nat Rev Microbiol 5:175–186
132. Wu C, Choi YH, van Wezel G (2015) Metabolic profiling as a
tool for prioritizing antimicrobial compounds. J Ind Microbiol
Biotechnol. doi:10.1007/s10295-015-1666-x
133. Yoon V, Nodwell JR (2014) Activating secondary metabo-
lism with stress and chemicals. J Ind Microbiol Biotechnol
41:415–424
134. Zhu H, Sandiford SK, van Wezel GP (2014) Triggers and cues
that activate antibiotic production by actinomycetes. J Ind
Microbiol Biotechnol 41:371–386
135. Zhu H, Wang W, Liu J, Caijin Q, Qiao J (2015) Immobilization
of Streptomyces thermotolerans 11432 on polyurethane foam to
improve production of Acetylisovaleryltylosin. J Ind Microbiol
Biotechnol 42:105–111