Importance of microbial natural products and the need to revitalize their discovery

Abstract

Microbes are the leading producers of useful natural products. Natural products from microbes and plants make excellent drugs. Significant portions of the microbial genomes are devoted to production of these useful secondary metabolites. A single microbe can make a number of secondary metabolites, as high as 50 compounds. The most useful products include antibiotics, anticancer agents, immunosuppressants, but products for many other applications, e.g., antivirals, anthelmintics, enzyme inhibitors, nutraceuticals, polymers, surfactants, bioherbicides, and vaccines have been commercialized. Unfortunately, due to the decrease in natural product discovery efforts, drug discovery has decreased in the past 20 years. The reasons include excessive costs for clinical trials, too short a window before the products become generics, difficulty in discovery of antibiotics against resistant organisms, and short treatment times by patients for products such as antibiotics. Despite these difficulties, technology to discover new drugs has advanced, e.g., combinatorial chemistry of natural product scaffolds, discoveries in biodiversity, genome mining, and systems biology. Of great help would be government extension of the time before products become generic.

Full text

REVIEW
Importance of microbial natural products and the need
to revitalize their discovery
Arnold L. Demain
Received: 22 June 2013 / Accepted: 3 August 2013 / Published online: 30 August 2013
Ó Society for Industrial Microbiology and Biotechnology 2013
Abstract Microbes are the leading producers of useful
natural products. Natural products from microbes and
plants make excellent drugs. Significant portions of the
microbial genomes are devoted to production of these
useful secondary metabolites. A single microbe can make a
number of secondary metabolites, as high as 50 com-
pounds. The most useful products include antibiotics,
anticancer agents, immunosuppressants, but products for
many other applications, e.g., antivirals, anthelmintics,
enzyme inhibitors, nutraceuticals, polymers, surfactants,
bioherbicides, and vaccines have been commercialized.
Unfortunately, due to the decrease in natural product dis-
covery efforts, drug discovery has decreased in the past
20 years. The reasons include excessive costs for clinical
trials, too short a window before the products become
generics, difficulty in discovery of antibiotics against
resistant organisms, and short treatment times by patients
for products such as antibiotics. Despite these difficulties,
technology to discover new drugs has advanced, e.g.,
combinatorial chemistry of natural product scaffolds, dis-
coveries in biodiversity, genome mining, and systems
biology. Of great help would be government extension of
the time before products become generic.
Keywords Microbial natural products  Antibiotics 
Anticancer  Immunosuppressants 
Hypocholesterolemics  Drug discovery
Introduction
Secondary metabolites from natural sources (microbes,
plants, animals) have helped to double the human life span
during the 20th century, combated pain and suffering, and
revolutionized the practice of medicine [38]. Infectious
disease was the leading cause of human death in the world
in 1900 [125]. Recently, it has been the second-leading
cause of death worldwide and number three in developed
nations [75]. Microbes have provided compounds that have
cured or reduced the effect of human diseases for 70 years.
Microbial products include antibiotics against bacteria and
fungi, antitumor drugs, immunosuppressants, enzyme
inhibitors including hypocholesterolemics, antiparasitic
agents, bioherbicides, plant growth regulators, biopesti-
cides, and bioinsecticides. Other activities utilizing natural
products that are in use or being studied include treatments
for viral diseases, acne, malaria, prion diseases, diabetes,
hyperlipoproteinemia, obesity, gastric ulcers, iron-overload
disease (hemochromatosis) and alumina overload in kidney
dialysis patients.
The value and occurrence of natural products
Microbes have been very important in the production of
natural product drugs. Of 23,000 active compounds from
microorganisms, i.e., antimicrobials, antivirals, cytotoxic,
and immunosuppressive compounds, 42 % are made by
fungi and 32 % by filamentous bacteria, the actinomycetes.
Ever since the discovery of penicillin by Alexander
Fleming in 1928, its development in the early 1940s at
Oxford by Chain, Florey, Heatley and Abraham, and the
discovery of useful streptomycete products in the early
1940s at Rutgers University by Waksman, Woodruff,
A. L. Demain (&)
Research Institute for Scientists Emeriti (R.I.S.E.),
Drew University, Madison, NJ 07940, USA
e-mail: ademain@drew.edu
123
J Ind Microbiol Biotechnol (2014) 41:185–201
DOI 10.1007/s10295-013-1325-z

Schatz and Lechevalier, we have benefited from the
remarkable selective action of antibiotics on pathogenic
bacteria and fungi [39]. These include (but are not limited
to) penicillins, cephalosporins, tetracyclines, aminoglyco-
sides, chloramphenicol, macrolides, ansamycins, polyenes,
and glycopeptides. Over half of the antibiotics are pro-
duced by the actinomycetes, 10–15 % by non-filamentous
bacteria and about 20 % from filamentous fungi. Many
useful products are semi-synthetic derivatives of the above
compounds, which are produced by chemistry or by
bioconversion.
Out of about 1 million natural products, approximately
25 % are biologically active, i.e., show positive activities
or toxicity. About 60 % of these are from plants and most
of the rest from microbes; some are from animal sources.
We already know the structures of over 160,000 natural
products, about half from plants and half from microbes.
This figure grows at about 10,000 per year.
Although most known natural products have been
obtained from terrestrial environments, 129 bioactive
compounds were isolated from marine microbes from 2000
to 2003 [69]. It is clear that the marine environment has
yielded many new natural products [58]. About 20,000 are
known, despite the fact that \1 % of commensal micro-
biotic consortia of marine invertebrates are culturable.
Bacteria occupy up to 40 % of the biomass of sponges [88].
Sponges are one of the most important sources of biolog-
ically active products, and the producers are often their
bacterial symbionts. For example, the genes encoding the
biosynthesis of antitumor polyketides known as onnamides
and theopederins from the marine sponge Theonella swinhoei
are prokaryotic in sequence. The genes are similar to those
found in the terrestrial beetles which produce the antitumor
polyketide pederin; here the production is due to an uncultured
Pseudomonas symbiont. Thus, it appears that in both beetles
and sponges, the polyketides are made by symbiotic bacteria.
Marine cyanobacteria produce many compounds with
activities such as neurotoxic, antiproliferative, anticancer,
and anti-infective. These are Gram-negative photoautotro-
phic prokaryotes carrying out oxygenic photosynthesis.
They are known as blue-green bacteria due to their pro-
duction of C-phycocyanin, a blue green pigment used for
photosynthesis. Their products have anticancer, antibacte-
rial, antiviral, immunomodulatory and protease-inhibition
activities.
Approved marine products include Cytarabine (Cyto-
star
Ò
) for non-Hodgkin’s lymphoma, which was originally
isolated from a sponge, Vidarabine (Vira-A
Ò
), Ziconotide
(Prialt
Ò
) and Trabactedin (Yondelis
Ò
). Many of those in
clinical trials are for cancer.
The first genome sequenced from an obligate marine
actinobacterium was that of Salinispora tropica which
makes salinosporamides, sporolides and lymphostatin.
Genome sequencing revealed that 10 % of the genome was
devoted to secondary metabolism [109]. At least 19 bio-
synthetic loci were detected that produced siderophores,
polyketides, melanins, non-ribosomal and ribosomal pep-
tides, terpenoids and aminocyclitols. Genome sequencing
of another marine actinobacterium, Salinispora arenicola,
revealed 39 different biosynthetic loci of natural products.
Symbionts of marine invertebrate animals have also
revealed interesting natural products [43]. Cyanovirin, a
101-amino-acid protein isolate from Nostoc ellipsospori-
um, is a fusion inhibitor of HIV and also inhibits influenza
A and B viruses.
Natural products make excellent drugs. From 1981 to
2002, 60 % of new chemical entities (NCEs) for cancer
were natural, as were 75 % of the NCEs for infectious
disease. Natural products or compounds related to them
constitute over 60 % of approved and pre-new drug
application (NDAs) candidates, not including biologicals
such as monoclonal antibodies or vaccines [76]. These
include natural products (6 %), derivatives of natural
products (27 %), synthetic compounds with natural product
pharmacophores (5 %), and synthetic mimics of natural
products (23 %). About 78 % of antibacterials and 74 % of
antitumor agents are natural or related to natural products.
Approximately half of the leading pharmaceutical products
on the market are natural or related compounds. For
example, from 1981 to 2002, natural products were the
basis of 74 % of all new chemical entities for cancer, 48
out of 74 anti-hypertensive agents, and seven out of ten
anti-migraine agents. Almost 50 % of new drugs intro-
duced into the marketplace between 1985 and 2005 were
natural or related products. In 2003, the market for such
compounds amounted to $40 billion.
Certain structures are well represented among successful
natural products, e.g., isoprenoids, alkaloids and polyke-
tides. The number of known isoprenoids, including terpe-
noids and carotenoids, amount to about 50,000. The
alkaloids number over 16,000 and the polyketides around
10,000. Most of the polyketides are produced by bacteria
and fungi. Markets for such compounds included $12 bil-
lion for terpenoids [91] and $17 billion for polyketides.
One microbe often produces many secondary metabolite
compounds [13]. A gentamicin-producing strain of Mi-
cromonospora forms 50 isolatable secondary metabolites.
Further bacterial examples include 12 compounds made by
Streptomyces sp. Go40/14, 12 by sp. A1, 24 by sp Tn64, 30
by sp Go.40/10 and 32 by sp.Tn3634 [98]. A single strain
of Myxococcus xanthus produces 38 different epothilones
[59]. Aspergillus ochraceus produces 16 compounds.
Another fungus, Sphaeropsideles sp F-24
0
707 produces 19
compounds. These high numbers were obtained by varying
nutritional conditions, physical parameters or adding
inhibitors. Many of the detected compounds were
186 J Ind Microbiol Biotechnol (2014) 41:185–201
123

previously unknown, Biosynthetic genes of microorgan-
isms are present in clusters coding for large multidomain
and multimodular enzymes, e.g., polyketide synthases,
prenyltransferases, non-ribosomal peptide synthases and
terpene cyclases. By sequencing the Aspergillus nidulans
genome, it was determined that the organism could produce
27 polyketides, 14 non-ribosomal peptides, one terpene,
and two indole alkaloids [14]. Multiple gene clusters
encoding secondary metabolites are common in species of
Streptomyces, other filamentous actinomycetes and myco-
bacteria [23]. Streptomyces coelicolor and Streptomyces
avermitilis contain 20–30 of these clusters. On the other
hand, non-filamentous bacterial genomes seem to lack
them. Genes adjacent to the biosynthetic gene clusters
encode regulatory proteins, oxidases, hydroxylases and
transporters. Strategies to activate silent genes have been
recently reviewed [15].
Antibiotics
The selective action exerted on pathogenic bacteria and
fungi by microbial secondary metabolites ushered in the
antibiotic era and for over 50 years, we have benefited
from this remarkable property of ‘‘wonder drugs’’. Natural
products have served us well in combating infectious
bacteria and fungi. Microbial and plant secondary metab-
olites helped to double our life span during the 20th cen-
tury, reduced pain and suffering, and revolutionized
medicine. Most antibiotics are either (1) natural products of
microorganisms, (2) semi-synthetically produced from
natural products, or (3) chemically synthesized based on
the structure of the natural products. Already known are
15,000 antibiotics with 150 on the market [111].
Antibiotics have been crucial in the increase in average
life expectancy in the US from 47 years in 1900 to 74 for
males and 80 for women in 2000 [67]. They have been
virtually the only drugs utilized for chemotherapy against
pathogenic microorganisms. They are defined as low
molecular weight organic natural products (secondary
metabolites or idiolites) made by microorganisms that are
active at low concentration against other microorganisms.
The most important microbiological antibiotics include the
b-lactams (penicillins and cephalosporins), tetracyclines,
aminoglycosides, chloramphenicol, macrolides, and
glycopeptides.
The first medically useful antibiotic was discovered by
Alexander Fleming in 1928 as a product of the fungus
Penicillium notatum but its purification, isolation, and
structure were not known for another 15 years. This was
finally accomplished by the elegant work of Ernst Chain,
Howard Florey, Norman Heatley, and Edward Abraham at
Oxford University. In the early 1940s, the Selman
Waksman group at Rutgers University, which included H.
Boyd Woodruff, Albert Schatz and Hubert Lechevalier,
discovered useful antibiotics produced by the actinomy-
cetes, i.e., filamentous bacteria. These included actinomy-
cin, the aminoglycosides (aminocyclitols) including
streptomycin and neomycin, and many other antibiotics.
After these successes, new compounds continued to be
discovered. Benjamin Duggar’s group at Lederle Labora-
tories of American Cyanamid (now Pfizer) announced the
discovery of chlortetracycline (aureomycin, biomycin) in
1948 as produced by Streptomyces aureofaciens. It was
approved for use that year against both Gram-positive and
Gram-negative bacteria. This was soon followed by the
discovery at Pfizer of oxytetracycline (Terramycin). A
completely different compound, chloramphenicol, was
discovered by scientists at Parke-Davis (now Pfizer) in
cooperation with workers at Yale University and Univer-
sity of Illinois. chloramphenicol is produced by Strepto-
myces venezuelae but has been made by chemical synthesis
due to its relatively simple structure. Additional amino-
glycosides were discovered such as kanamycin by Hamao
Umezawa at the Institute for Microbial Chemistry in Tokyo
and gentamicin by Marvin Weinstein and coworkers at
Schering-Plough (now Merck) in New Jersey (USA).
Aminoglycosides are broad-spectrum in activity, stable
chemically and bactericidal.
For some unknown reason, the filamentous bacteria
(actinomycetes) are amazingly prolific in the number of
antibiotics they can produce [7]. About 75 % of the anti-
biotics are produced by actinomycetes and about 75 % of
these are made by a single genus, Streptomyces. Strains of
Streptomyces hygroscopicus make almost 200 antibiotics.
One Micromonospora strain can produce 48 aminoglyco-
side antibiotics. Streptomyces griseus strains produce over
40 different antibiotics. Other organisms making antibiot-
ics include strains of Bacillus subtilis producing over 60
such compounds. Twelve percent of the antibiotics are
produced by non-filamentous bacteria and about 20 % are
made by filamentous fungi. The antibiotics vary in size
from small molecules like cycloserine (102 Daltons) and
bacilysin (270 Daltons) to polypeptides, such as nisin
which contains 34 amino acid residues. Myxobacterium
xanthus devotes 9 % of its genome to production of sec-
ondary metabolites, which is twice that of S. coelicolor
[44]. Myxobacteria, as a group, produce more than 300
antibiotics.
More than 350 agents have reached the world market as
antimicrobials. They include (a) natural products, (b) semi-
synthetic antibiotics and (c) synthetic chemicals [19]. The
commercial antibiotics include the cephalosporins (45 %),
penicillins (15 %), quinolones (11 %), tetracyclines (6 %),
macrolides (5 %); the remainder include the aminoglyco-
sides, ansamycins, glycopeptides, lipopeptides and
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123

polyenes. The strictly synthetics include the sulfa drugs,
azoles, oxazolidinones (linezolid), quinolones, and
fluoroquinolones.
There have been about 40 b-lactam compounds used in
medicine. Despite the fact that b-lactamases are the major
cause of resistance development and there are over 450
such enzymes, b-lactams are still very useful due to the
discovery of b-lactamase inhibitors. These include clavu-
lanic acid, and the carbapenems. The latter include do-
ripenem (S-4661), which has broad spectrum activity
including Pseudomonas aeruginosa, as well as tomope-
nem, ceftobiprole, ceftaroline, faropenem and meropenem.
Tuberculosis is about the worst disease caused by bac-
terial infection with Mycobacterium tuberculosis killing
1.6–2 million people each year. A combination of the
carbapenem meropenem with clavulanic acid has been
found to inhibit M. tuberculosis, even when the organism is
in its ‘‘persistent’’ state, and also extensively drug-resistant
strains [62]. Since they are both FDA-approved drugs, this
combination is being used to treat patients with previously
untreatable TB.
Tetracyclines have had a significant contribution to the
antibiotic era. Chlortetracycline was discovered in 1948
followed by oxytetracycline in 1950. These were the first
broad-spectrum antibiotics known. Resistance eventually
developed via antibiotic efflux which was combated by the
second-generation tetracyclines, i.e., minocycline and
doxycycline, which were semi-synthetic, more lipophilic
and thus taken up to a greater degree by the resistant
strains, resulting in a decrease in net efflux. However,
resistance based on ribosomal protection soon developed.
These resistant strains were attacked by the third-genera-
tion tetracycline, the glycylcycline tigecycline, approved in
2005. Another third-generation tetracycline is PTIC-0796.
Doxycycline hyclate (Periostat
Ò
) is also used for peri-
odontal disease by inhibiting enzymes that breakdown gum
tissue.
Polyketide macrolides are another group of useful
antibiotics. The term macrolide is a shortened version of
macrolactone glycoside which was proposed by the famous
chemist R.B. Woodward in 1957. The term refers to a
macrolactone containing one or more deoxy sugars. They
are made by actinomycetes, e.g., Streptomyces, Micromo-
nospora, Saccharopolyspora, and Actinoplanes. In the
early 1950s, the polyketide macrolide erythromycin was
approved for oral use in outpatients. It was later accom-
panied in medical use by other macrolides, i.e., oleando-
mycin, pikromycin, amphotericin B, midecamycin,
josamycin, and carbomycin. Tylosin came along for use in
animals. Their major problems were acid instability, poor
bioavailability, and rapid elimination. Thus, they had to be
given three or four times per day. Semi-synthetic second-
generation macrolides followed, e.g., clarithromycin and
azithromycin. Midecamycin was replaced by miokamycin
and rokitamycin, while tylosin was improved upon by til-
micosin. Others included dirithromycin, florithromycin,
and roxithromycin. These second-generation compounds
were more acid-stable, eliminated from the body at a lower
rate, and only required dosing at one or two times per day.
Third-generation ‘‘ketolides’’ appeared in the late 1980s,
mainly to overcome resistance to the earlier compounds.
These included telithromycin, introduced in 2001 in Eur-
ope and in 2004 in the USA. Lipiarmycin (dificidin, ti-
acamycin B, OPT-80) is particularly active against
Clostridium difficile infections and has been approved.
Additional macrolides include spiramycin. The major tar-
gets of the macrolides are respiratory pathogens, sexually
transmitted infections (Chlamidia trachomatis), Legion-
naires’ disease (Legionella pneumophila), Lyme disease
(Borrelia burgdorferi), peptic ulcers (Helicobacter pylori),
gonorrhea (Neisseria gonorrhoeae), and Mycobacterium
avium in AIDS patients. They are also effective against
skin and soft tissue pathogens (staphylococci, Propionib-
acter acne, and Streptococcus pyogenes). They are all
effective orally vs. Gram-positive bacteria and some Gram-
negative organisms such as Hemophilus influenzae and
Mannheimia spp. All macrolides inhibit protein synthesis.
Macrolides being used for other uses include immuno-
suppressants such as sirolimus (rapamycin), and tacrolimus
(FK506), antiparasitics such as avermectin, and antitumor
agents such as the epothilones. The biosynthesis of poly-
ketide macrolides has been subjected to genetic engineering
[83, 123], and combinatorial biosynthesis has become very
important [119] as it has for cyclic lipopeptide antibiotics.
Peptides are an important part of the antibiotic area [8,
52]. They include vancomycin, teicoplanin, the streptog-
ramins, and the bacteriocins. Streptogramins include pri-
stinamycin, and the virginiamycin M and virginiamycin S
pair, which act synergistically and are produced by Strep-
tomyces virginiae. The glycopeptide vancomycin was for
years the molecule of choice to treat infections caused by
antibiotic-resistant bacteria. However, over the years,
resistance to vancomycin developed, especially in the case
of infections by VRE. This problem has been combated by
the use of the related lipoglycopeptide teicoplanin (Tar-
gocid) as well as daptomycin, linezolid, synercid, and tel-
evancin. Teicoplanin also has fewer side-effects than
vancomycin and a longer half-life in the human body.
Lipophilic analogs of teicoplanin and ristocetin have
improved antibiotic activity. One has antiviral activity vs.
influenza [89]. Song [103] described five antimicrobials
which could replace vancomycin and new carbapenems
active against Gram-negative infections. They are the
glycopeptides dalbavancin, televancin and oritavancin, the
lipopeptide daptomycin, the cephalosporins ceftobiprole
and ceftaroline and the diaminopyrimidine iclaprim.
188 J Ind Microbiol Biotechnol (2014) 41:185–201
123

Dalbavancin is a semi-synthetic lipoglycopeptide derived
from teicoplanin. Televancin is a lipoglycopeptide that
inhibits cell wall formation and disrupts membrane barrier
function. Ceftobiprole is a cephalosporin that has both
Gram-positive and Gram-negative antibacterial activity.
Iclaprim is an inhibitor of dihydrofolate reductase that
inhibits Gram-positive and Gram-negative bacteria. Gram-
negative and Gram-positive activity is also shown by do-
ripenem, a carbapenem approved by FDA in 2007.
More than 1,000 antimicrobial peptides are known.
Bacteriocins are ribosomally synthesized antimicrobial
peptides and are divided into different groups. One group
(the cationic peptide type A1 antibiotics) is made up of the
lantibiotics, which contain unusual amino acids, one of
which is lanthionine. More than 50 lantibiotics produced by
Gram-positive bacteria are known. Type A(1) lantibiotics
are potent and broad- spectrum, have low toxicity, and are
active in vivo [6]. They include nisins A and Z, subtilin,
nukacin ISK-1, several lacticins, mersacidin, actagardine,
cinnamycin, SapB, sublancin, gallidermin, plantaricin W,
mutacin and epidermin. Some combinations work syner-
gistically. The most well known lantibiotic, nisin, was
discovered in the 1920s and has been used as a food pre-
servative for more than 40 years. It has no human toxicity
but is broken down in the gastrointestinal tract and has low
stability at physiological pH levels. Nisin inhibits bacterial
peptidoglycan synthesis and produces membrane pores by
reacting with the cell wall precursor lipid II. It is active
against bacterial mastitis, oral decay, enterococcal infec-
tion, peptic ulcers, and enterocolitis. It is also reported to
inhibit experimental vascular graft infection by methicillin-
resistant Staphylococcus epidermidis. Mersacidin and ac-
tagardine inhibit methicillin-resistant Staphylococcus aur-
eus (MRSA), bacterial mastitis, oral decay and acne.
Gallidermin and epidermin are effective against acne,
eczema, folliculitis, and impetigo. Lacticin 3147 works
against bacterial mastitis, MRSA, enterococci, and acne.
Cinnamycin acts against inflammation and viral infection.
Despite these important abilities, nisin and other lantibi-
otics have not been extensively used for therapy [101].
Apparently, their manufacture is both time-consuming and
expensive, and the high cost limits their use. Furthermore,
since they are administered by injection rather than orally,
their production is subject to intensive regulation con-
cerning sterility.
In the search for new antibiotics, many of the new
products are made by chemists by modification of natural
antibiotics; this process is called ‘‘semi-synthesis’’. As
early as 1974, over 20,000 semi-synthetic penicillins, 4,000
cephalosporins, 2,500 tetracyclines, 1,000 rifamycins, 500
kanamycins, and 500 chloramphenicols had been prepared.
Completely synthetic antimicrobials include the quino-
lone and fluoroquinolone groups, discovered as inhibitors
of DNA gyrase. They are related to the structure of a
natural product, the alkaloid quinine [81]. The quinolone
era started with the discovery of chloroquine (called Res-
oquine) for malaria at the Bayer company in the early
1930s. It was followed in 1962 at the Sterling-Winthrop
Research Institute in New York by Lesher and coworkers
who synthesized nalidixic acid (Negram
Ò
), a 4-quinolone
with strong Gram-negative antibacterial activity [108].
Additional quinolones were developed in 1968. These
included cinoxacin, rosoxacin, pipemidic acid, piromidic
acid, and oxolinic acid.
Work on the fluoroquinolones was first done at Riker
Laboratories in Minnesota in the mid-1970s. It was dis-
covered that fluorine at C6 markedly improved activity of
the quinolones and thus the commercial fluoroquinolones
were born. In 1981, the Merck company reported on their
6-fluoroquinolone, norflacin, which was licensed from
Kyorin Pharmaceuticals. This was followed by develop-
ment of ciprofloxacin (Cipro
Ò
), ofloxacin, enoxacin, per-
floxacin, moxifloxacin and levofloxacin. Ciprofloxacin,
developed at Bayer, became very successful due to its high
activity. It was approved by FDA as an oral drug in 1987
and as an IV drug in 1991.
Of the 25 top-selling drugs in 1997, 42 % were natural
products or derived from natural products [21]; of these,
antibiotics contributed 67 % of sales. The worldwide
market for antibiotics is $35 billion. If one includes anti-
viral agents, the figure reached $55 billion in 2000. Anti-
biotics from species of Streptomyces alone had a market of
$25 billion in 2001 [61]. The market for antifungal drugs in
2002 reached $4 billion [32]. At their peaks, individual
groups of antibiotics reached impressive sales figures.
b-Lactam antibiotics constituted a major part of the market:
cephalosporins sold for $11 billion, penicillins for $8 bil-
lion, and carbapenems and other b-lactams for $3 billion,
making a total of around $22 billion. Sales of macrolides
reached $7 billion, mainly involving tylosin, clarithromy-
cin, azithromycin and erythromycin. Aminoglycoside sales
reached $1.8 billion and tetracycline sales reached $1.4
billion. Combined sales of the glycopeptides vancomycin
and teicoplanin were $1 billion. The market for all quin-
olones amounted to $6.4 billion with fluoroquinolones
accounting for $3.2 billion, dominated by levofloxacin
(Levaquin
Ò
). Markets for the synthetic azoles reached $2
billion [53]. Global sales of oral antibiotics amounted to
$25 billion in 2005 [31]. The antiviral market was $16
billion.
Individual antimicrobials with annual markets over $1
billion dollars include augmentin, a combination of a semi-
synthetic penicillin and the b-lactamase inhibitor, clavu-
lanic acid ($2.1 billion), the quinolones ciprofloxacin ($1.8
billion) and levofloxacin/ofloxacin ($1.1 billion), the semi-
synthetic macrolides azithromycin (Zithromax
Ò
;$2
J Ind Microbiol Biotechnol (2014) 41:185–201 189
123

billion) and clarithromycin (Biaxin
Ò
; $1.6 billion) and the
semi-synthetic cephalosporin ceftriazone (Rocephin
Ò
; $1.1
billion) [107, 119].
Worldwide antibiotic production amounts to about
100,000 tons. Included are 60,000 tons of penicillins, 5,500
tons of tetracyclines, 2,500 tons of cephalosporins. Anti-
biotics that are natural or derived from natural products
include b-lactam antibiotics such as ampicillin (5,000 tons
per year), cephalexin (4,000 tons), amoxicillin (16,000
tons), and cefadroxil (1,000 tons). Macrolides at high
tonnage include azithromycin (1,500 tons) and clarithro-
mycin (1,500 tons). Glycopeptides such as vancomycin and
teicoplanin are produced at a total of 9,000 tons.
Anticancer agents
An extremely important concept for the further develop-
ment of natural products is that compounds which possess
antibiotic activity also possess other activities. Some of
these activities had been quietly exploited in the past, and it
became clear in the 1980s that such broadening of scope
should be expanded. Thus, a broad screening of antibioti-
cally active molecules for antagonistic activity against
organisms other than microorganisms, as well as for
activities useful for pharmacological or agricultural appli-
cations, was pursued in order to yield new and useful lives
for ‘‘failed antibiotics.’’ This resulted in the development of
a large number of simple in vitro laboratory tests, e.g.,
enzyme inhibition screens to detect, isolate and purify
useful compounds. Fortunately, we entered into a new era
in which microbial metabolites were applied to diseases
heretofore only treated with synthetic compounds, i.e.,
diseases not caused by bacteria and fungi and huge suc-
cesses were achieved. An area that experienced great
success was that of antitumor agents. Of the 140 anticancer
agents approved since 1940 and available for use, over
60 % can be traced to a natural product. Of the 126 small
molecules among them, 67 % are natural in origin [77]. In
2000, 57 % of all drugs in clinical trials for cancer were
either natural products or their derivatives [33].
In their review on the use of microbes to prescreen potential
antitumor compounds, Newman and Shapiro [78] concluded
that microorganisms have played an important role in identi-
fying compounds with therapeutic benefit against cancer.
Most of the important compounds used for chemotherapy of
tumors are microbially produced antibiotics. Approved anti-
tumor agents from microorganisms include actinomycin D
(dactinomycin), anthracyclines, including daunorubicin,
doxorubicin (adriamycin), epirubicin, pirarubicin, idarubicin,
valrubicin and amrubicin, glycopeptides (bleomycin, phleo-
mycin), the mitosane mitomycin C, and the anthracenones
(mithramycin, streptozotocin, pentostatin).
A modified anthracycline, 11-hydroxyaclacinomycin A,
was produced by cloning the doxorubicin resistance gene
and the aklavinone 11-hydroxylase gene dnrF from the
doxorubicin producer, Streptomyces peucetius subsp. cae-
sius, into the aclacinomycin A producer. The hybrid mol-
ecule showed greater activity against leukemia and
melanoma than aclacinomycin A. Another hybrid molecule
produced was 2
0
-amino-11-hydroxyaclacinomycin Y,
which was highly active against tumors. Additional new
anthracyclines have been made by introducing DNA from
Streptomyces purpurascens into Streptomyces galilaeus,
both of which normally produce known anthracyclines.
Novel anthracyclines were produced by cloning DNA
from the nogalomycin producer, Streptomyces nogalater,
into Streptomyces lividans and into an aclacinomycin-
negative mutant of S. galilaeus. Cloning of the actI, actIV,
and actVII genes from S. coelicolor into the 2-hydrox-
yaklavinone producer, S. galilaeus 31,671 yielded novel
hybrid metabolites, desoxyerythrolaccin and 1-O-methyl-
desoxyerythrolaccin. Similar studies yielded the novel
metabolite aloesaponarin II. Epirubicin (4
0
-epidoxorubicin)
is a semi-synthetic anthracycline with less cardiotoxicity
than doxorubicin. Genetic engineering of a blocked S.
peucetius strain provided a new method to produce it. The
gene introduced was avrE of the avermectin-producing S.
avermitilis or the eryBIV genes of the erythromycin pro-
ducer, Saccharopolyspora erythraea. These genes and the
blocked gene in the recipient are involved in deoxysugar
biosynthesis.
An unusual source of secondary metabolites is the my-
xobacteria, relatively large Gram-negative rods that move
by gliding or creeping. They form fruiting bodies and have
a very diverse morphology. Over 400 compounds had been
isolated from these organisms by 2005 but the first in
clinical trials were the epothilones, potential antitumor
agents that act like taxol but are active vs. taxol-resistant
tumors. They are 16-member ring polyketide macrolide
lactones produced by the myxobacterium Sorangium cell-
ulosum, which were originally developed as antifungal
agents against rust fungi [54], but have found their use as
antitumor compounds [56]. They contain a methylthiazole
group attached by an olefinic bond. They are active against
breast cancers, including those that are resistant to taxol
and other forms of chemotherapy. They bind to and sta-
bilize microtubules essential for DNA replication and cell
division, even more so than taxol. One epothilone, ixab-
epilone, produced chemically at Bristol Myers-Squibb
from epothilone B, was approved by FDA. By preventing
the disassembly of microtubules, epothilones cause arrest
of the tumor cell cycle at the GM2/M phase and induce
apoptosis (programmed cell death). The mechanism is
similar to that of taxol but epothilones bind to tubulin
at different binding sites and induce microtubule
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123

polymerization. Production of epothilone B by Sorangium
cellulosum is accompanied by the undesirable epothilone
A. Production of B over A was favored by adding sodium
propionate to the medium. Epothilone polyketides are more
water-soluble than taxol. The producing microbe is a very
slow grower (16-h doubling time) and low producer
(20 lg/ml).
Plants have been a useful source of anticancer agents.
Etoposide and teniposide were derived as semi-synthetic
derivatives of podophyllotoxin, an antimitotic metabolite
of mayapple roots [41]. The mayapple plant is an old
herbal remedy. Etoposide is a topoisomerase II inhibitor.
This essential enzyme is involved in eukaryotic cell growth
by regulating levels of DNA supercoiling [9]. Vinca alka-
loids, such as vinblastine and vincristine, originate from the
Madagascar periwinkle plant. The naphthoquinone pig-
ment shikonin is produced by cell culture of the plant
Lithospermum erythrorhizon, a herbal medicine remedy.
Shikonin and two derivatives inhibit tumor growth in mice
bearing Lewis lung carcinoma [68]. Other promising plant
products include curcumin, resveratrol, gingrerole, capsa-
icin, epogallocatechin gallate, genistein, flapopiridol, and
silymarin.
Taxol (paclitaxel) has been a very successful antitumor
molecule. It was originally discovered in plants but has
also been found to be a fungal metabolite [104]. This
diterpene alkaloid was approved for breast and ovarian
cancer and acts by blocking depolymerization of microtu-
bules. In addition, taxol promotes tubulin polymerization
and inhibits rapidly dividing mammalian cancer cells.
Taxol was originally isolated from the bark of the Pacific
yew tree (Taxus brevifolia) but it took six trees of
100 years of age to treat one cancer patient. It is now
produced by plant cell culture or by semi-synthesis from
taxoids made by Taxus species. These species make more
than 350 known taxoid compounds. Early genetic engi-
neering of S. cerevisiae yielded no taxadiene (the taxol
precursor) because too little of the intermediate, geranyl-
geranyl diphosphate, was formed. When the Taxus
canadensis geranylgeranyl diphosphate synthase gene was
introduced, 1 mg/l of taxadiene was obtained [36]. More
recent metabolic engineering studies [50] yielded a Sac-
charomyces cerevisiae strain producing over 8 mg/l tax-
adiene and 33 mg/l geranyl geraniol. The use of cells of the
plant Taxus chinensis to produce taxol became the indus-
trial means to make the compound. The addition of methyl
jasmonate, a plant signal transducer, increased production
from 28 to 110 mg/l. The optimum temperature for growth
of T. chinensis is 24 °C and that for taxol synthesis is
29 °C. Shifting from 24 to 29 °C at 14 days gave 137 mg/l
at 21 days [30]. There is a 6 week process yielding
153 mg/l with Taxus sp [18]. Taxol has sales of $1.6 billion
per year. Fungi such as Taxomyces adreanae,
Pestalotiopsis microspora, Tubercularia sp., and Phyl-
losticta citricarpa produce taxol [104, 106, 116] but the
production level is low, e.g., 265 lg/l produced by P. ci-
tricarpa [66]. Taxol has antifungal activity by the same
microtubule mechanism, especially against oomycetes
[105]. Oomycetes are water molds exemplified by plant
pathogens such as Phytophthora, Pythium, and
Aphanomyces.
Camptothecin is a modified monoterpene indole alkaloid
produced by certain plants (angiosperms) [115]. It also is
produced by an endophytic fungus (Entrophospora inf-
requens) from the plant Nathapodytes foetida. It is used for
recurrent colon cancer and has unusual activity against
lung, ovarian, and uterine cancers [5]. Colon cancer is the
second-leading cause of cancer fatalities in the USA and
the third most common cancer among US citizens. Cam-
ptothecin is known commercially as Camptosar (Pharma-
cia) and Campto (Aventis and Yakult) and achieved sales
of $1 billion in 2003 [70]. Its water-soluble derivatives
irinotecan and topotecan are used clinically. In view of the
low concentration of camptothecin in tree roots and poor
yield from chemical synthesis, the fungal fermentation is
very promising for industrial production of camptothecin.
Its cellular target is type I DNA topoisomerase. When
patients become resistant to irenotecan, its use can be
prolonged by combining it with the monoclonal antibody
Erbitux (Cetuximab
Ò
) from ImClone/BMS. Erbitux blocks
a protein that stimulates tumor growth and the combination
helps metastatic colorectal cancer patients expressing epi-
dermal growth factor receptor (EGFR). This protein is
expressed in 80 % of advanced metastatic colorectal can-
cers. The drug combination reduces invasion of normal
tissues by tumor cells and the spread of tumors to new
areas.
Metastatic testicular cancer, although rather uncommon
(1 % of male malignancies in the USA; 80,000 in the year
2000 as compared to 190,000 cases of prostate cancer), is
the most common carcinoma in men aged 15–35. It is quite
interesting that the cure rate for disseminated testicular
cancer was 5 % in 1974; later, it rose to 90 % mainly due
to combination chemotherapy with the natural products
bleomycin and etoposide and the synthetic cisplatin [46].
Angiogenesis (recruitment of new blood vessels) is
necessary for tumors to obtain oxygen and nutrients.
Tumors actively secrete growth factors that trigger angio-
genesis. The concept of angiogenesis was established by
Prof. Judah Folkman [25]. He proposed that tumor growth
depends on angiogenesis and proposed the use of angio-
genesis inhibitors as antitumor agents, i.e., to target acti-
vated endothelial cells. He further proposed that the
vascular endothelial growth factor (VEGF) is involved in
angiogenesis and that it could be a target for anti-angio-
genic drugs. Fumagillin, produced by Aspergillus
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123

fumigatus, was one of the first agents found to act as an
anti-angiogenesis compound. Next to come along for
angiogenesis inhibition were its oxidation product ovalacin
and the fumagillin analogue TNP470 (=AGM-1470).
TNP470 binds to and inhibits type 2 methionine amino-
peptidase (MetAP2). This interferes with amino-terminal
processing of methionine, which may lead to inactivation
of enzymes essential for proliferation and in vitro migra-
tion of endothelial cells. In animal models, TNP470
effectively treated many types of tumor and metastases.
Fumagillin also has immunosuppressive activity that is
correlated with its anti-angiogenic activity. MetAP2 is also
the fumagillin target in yeast, which makes MetAP2 and
another similar enzyme MetAP1. Wild-type yeast
expressing both map1 and map2 genes are insensitive
whereas map1 mutants are sensitive to fumagillin and
ovalicin; map2 mutants are insensitive. The primary
function of these two enzymes is to remove the initiator
methionine at the amino terminus of proteins. This post-
translational processing step is necessary for the myris-
toylation, which is required for protein targeting and
stability of certain proteins. N-myristoylation is essential
for viability of S. cerevisiae and Candida neoformans.
Differences between substrate specificity of the C. neo-
formans enzyme and the mammalian enzyme exists which
suggests the possibility of developing novel antifungal
agents [27]. Inhibition of MetAP2 might be expected to
make short-lived enzymes more stable to desirable degra-
dation and this might inhibit angiogenesis. Fumagillin is
active against Nosia sp, a fungus causing disease of hon-
eybees. Also, TNP470 is potent against microsporidia,
obligate intracellular parasites causing diarrhea and wast-
ing syndrome in immunocompromised patients including
those with AIDS.
The first anti-angiogenesis drug on the market for cancer
was Avastin (bevacizumab), a monoclonal antibody from
Genentech/Roche. It acts against the vascular endothelial
growth factor (VEGF), an angiogenic factor, and is used
for metastatic colorectal cancer. Additional FDA-approved
angiogenesis inhibitors are pegaptanib (Macugen
Ò
) and
ranibizumab (Lucentis
Ò
). Macugen is an aptamer of VEGF
and Lucentis is an anti-VEGF antibody. By 2008, ten anti-
angiogenesis drugs had been approved. Eight are used
against cancer and two are employed for treatment of age-
related macular degeneration. Anti-angiogenesis therapy is
now known as one of four cancer treatments. The other
three are surgery, radiotherapy, and chemotherapy. By the
end of 2007, 23 anti-angiogenic drugs were in Phase III
clinical trials and more than 30 were in Phase II.
Genome mining is useful for identifying genetic units
with potential for synthesizing new drugs [97, 118]. As a
result of such an effort with Micromonospora sp., a new
antitumor drug was discovered [127]. The compound,
ECO-04601, is a farnesylated dibenzodiazapene that
induces apoptosis.
An analog of the immunosuppressive agent rapamycin
with antitumor activity is temsirolimus. It inhibits mTOR,
which is a kinase. The drug, produced by Wyeth, was
approved for renal cell carcinoma [93].
The marine environment offers new opportunities for
the discovery of antitumor agents [63]. The genus Sali-
nospora and its 2 species, S. tropica and S. areniola, have
been isolated around the world, S. tropica makes a bicyclic
beta-lactone gamma-lactam called salinosporamide A
which is a proteasome inhibitor and has antitumor activity.
Also the genus Marinophilus contains species that produce
novel polyenes with potent antitumor activity. The sym-
bionts of marine invertebrate animals continue to reveal
interesting natural products [43]. Variants of the toxic
dolastin from the sea hare Dolabella auricalaria seem
promising against cancer. These include soblidotin (T2F
1027) which completed Phase II against soft tissue sar-
coma, and synthadotin (=tasidotin = 1LX 651), which is at
the same clinical stage against melanoma, prostate and
non-small cell lung cancers. These are thought to be pro-
duced by cyanobacteria sequestered by the marine inver-
tebrates in their diet.
A promising drug for clinically reversing multidrug
resistance in tumor cells is the non-immunosuppressive
cyclosporin A derivative valspodar (PSC-833) of Novartis
[94].
Immunosuppressants
Cyclosporin A was originally discovered as a narrow
spectrum antifungal peptide produced by the mold, Toly-
pocladium nivenum (previously Tolypocladium inflatum).
Discovery of its immunosuppressive activity led to its use
in heart, liver, and kidney transplants and to the over-
whelming success of the organ transplant field. Sales of
cyclosporin A reached $1 billion in 1994. Although
cyclosporin A had been the only product on the market for
many years, two other products, produced by actinomy-
cetes, provided new opportunities. These are rapamycin
(=sirolimus) [113] and the independently discovered ta-
crolimus (=FK506, Fujimycin). They are both narrow
spectrum polyketide antifungal agents, which are 100-fold
more potent that cyclosporin as immunosuppressants and
less toxic. All three immunosuppressants were originally
described as antifungal agents. Non-immunosuppressive
derivatives have been developed with good antifungal
activity [34]. Tacrolimus and rapamycin have both been
used clinically for many years. Tacrolimus was almost
abandoned by the Fujisawa Pharmaceutical Co. after initial
animal studies showed dose-associated toxicity. However,
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Dr. Thomas Starzl of the University of Pittsburgh, realizing
that the immunosuppressant was 30 to 100-fold more
active than cyclosporin tried lower doses which were very
effective and non-toxic, thus saving the drug and many
patients after that, especially those that were not respond-
ing to cyclosporin [4]. Since its introduction (1993 in
Japan; 1994 in USA), tacrolimus has been used for trans-
plants of liver, kidney, heart, pancreas, lung, intestines and
for prevention of graft-versus-host disease. Recently, a
topical preparation has been shown to be very active
against atopic dermatitis, a widespread skin disease. Ta-
crolimus had a market of $2 billion in 2007.
Rapamycin does not exhibit the nephrotoxicity of cyclo-
sporin A and tacrolimus and is synergistic with both com-
pounds in immunosuppressive action [95]. By combining it
with either, kidney toxicity is markedly reduced. Rapamycin
has been the basis of chemical modification to yield important
products such as everolimus, temsirolimus (CCI-779) and
deforolimus (A23573). Rapamycin is not only an immuno-
suppressant but also has antifungal, antitumor, neuroprotec-
tive, autoimmune and anti-aging properties. Its biosynthesis,
regulation and mutagenic improvement of production have
been reviewed by Park et al. [84].
Studies on the mode of action of these immunosuppressive
agents have markedly expanded current knowledge of T cell
activation and proliferation. Rapamycin, tacrolimus and
cyclosporin A all act by interacting with an intracellular pro-
tein (an immunophilin) thus forming a novel complex which
selectively disrupts signal transduction events of lymphocyte
activation. By binding to its immunophilin (FKBP), rapamy-
cin inhibits a unique growth regulation path utilized by lym-
phocytes in responding to several cytokines. The targets of
cyclosporin A, tacrolimus and sirolimus are inhibitors of
signal transduction cascades in microorganisms and humans.
In humans, the signal transduction pathway is required for
activation of T cells. The targets are highly conserved from
microbial eukaryotes to humans. When these compounds
enter cells, they form complexes with their immunophilins
and inhibit the latter’s prolyl isomerase activity, usually
involved in protein folding, but this is not the crucial step. The
combination of cyclosporin or tacrolimus and their specific
immunophilin inhibits calcineurin, a serine-threonine-specific
protein phosphatase that is normally activated by calmodulin
in response to increases in intracellular Ca
2?
. A previously
unknown protein called mTOR (a member of the family of
lipid/protein kinases) is part of the sirolimus-sensitive signal
transduction pathway. Rapamycin combined with its immu-
nophilin inhibits TOR kinase which normally transduces
growth promoting signals that are sent in response to nutrients
(e.g., amino acids) and growth factors.
TOR has phosphatidylinositol lipid kinase activity,
which is involved in cell cycle regulation. TOR proteins of
yeast and mammals share sequence similarity to protein
and lipid kinases although their predominant activity is
thought to be that of phosphatidylinositol lipid kinase.
Many homologues exist in yeast and mammalian cells
(called RAFT, FRAP, mTOR, SEP, RAPT1). TORs
respond to nitrogen sources and other growth factors to
regulate translation, transcription and cell cycle progres-
sion. The sensitivity of yeast to cyclosporin A and tacrol-
imus is due to the need for calcineurin to promote yeast
survival during cation stress. In Cryptococcus neoformans,
calcineurin is needed for virulence. Other fungi inhibitable
by cyclosporin and tacrolimus are Coccidiodes immitis,
Aspergillus niger, A. fumigatus and Neurospora crassa,
suggesting that calcineurin may be necessary for viability
in these species. Two non-immunosuppressive analogs of
cyclosporin are active against Cryptococcus neoformans
[34]. Like the immunosuppressive cyclosporin, they act by
binding to cyclophilin A and inhibiting the action of the
fungal calcineurin. A non-immunosuppressive tacrolimus
derivative, a C18 hydroxy C21 ethyl analogue called
L-685,818, inhibits C. neoformans by inhibition of calci-
neurin. It is non-immunosuppressive because its combina-
tion with human immunophilin (human FKBP12) does not
inhibit vertebrate calcineurin but when in combination with
fungal FKBP12, it does. Thus, it is possible to exploit
subtle differences in the structures of human and fungal
FKBP12. Since TOR proteins are involved negatively in
nutrient repression, addition of rapamycin induces certain
yeast genes whose transcription is repressed by nitrogen
abundance, glucose abundance and also genes involved in
the diauxic shift [27]. Rapamycin and its less immuno-
suppressive analogs are effective against Candida albicans
and C. neoformans via FKBP12-dependent inhibition of
TOR kinases [35]. These enzymes are necessary for sta-
tionary phase entry, expression of ribosomal protein genes,
nitrogen catabolite repression and translation.
Rapamycin also has antitumor activity. Whereas cyclo-
sporin A promotes tumor growth and many transplant patients
are killed by tumors, rapamycin inhibits tumor growth by
interfering with angiogenesis and also is an inducer of apop-
tosis [57, 90]. Rapamycin is also able to reverse multidrug
resistance to antitumor agents in mammalian cells. Cyclo-
sporin and tacrolimus also have this ability. In addition to their
actions as immunosuppressants, antitumor agents and anti-
fungal agents, the ascomycins, structurally related to rapa-
mycin, have anti-inflammatory action and are being used for
topical treatment of skin diseases such as atopic dermatitis,
allergic contact dermatitis and psoriasis [28]. Tacrolimus is
also being studied for skin diseases.
Cyclosporin A has activity against the malaria parasite
Plasmodium falciparum in agreement with its genome
containing sequences encoding cyclophilin and calcineurin
[42]. Another activity of tacrolimus and rapamycin is
stimulation of nerve cells [55]. They thus might find use for
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combating neurological disorders. Certain ascomycin
derivatives made by combinatorial biosynthesis are being
studied for nerve regeneration [92]. Cyclosporin A analogs
are being clinically tested against the inflammatory disease
asthma and have shown promising results [45]. They
exhibit decreased nephrotoxicity and have different phar-
macology and metabolism.
A very old broad-spectrum antibiotic compound, my-
cophenolic acid, has an amazing history. Bartolomeo Gosio
(1863–1944), was an Italian physician who discovered the
compound in 1893 [10]. Gosio isolated a fungus from
spoiled corn which he named Penicillium glaucum, which
was later reclassified as Penicillium brevicompactum.He
isolated crystals of the compound from culture filtrates in
1886 and found them to inhibit growth of Bacillus an-
thracis. This was the first time an antibiotic had been
crystallized and the first time that a pure compound had
ever been shown to have antibiotic activity. The work was
forgotten but fortunately the compound was rediscovered
by Alsberg and Black [3] and given the name mycophen-
olic acid. They used a strain originally isolated from
spoiled corn in Italy called Penicillium stoloniferum,a
synonym of P. brevicompactum. The chemical structure
was elucidated many years later by workers in England
[12]. Mycophenolic acid has antibacterial, antifungal,
antiviral, antitumor, antipsoriasis and immunosuppressive
activities. It was never commercialized as an antibiotic
because of its toxicity, but its 2-morpholinoethylester was
approved as a new immunosuppressant for kidney trans-
plantation in 1995 and for heart transplants in 1998. The
ester is called mycophenolate mofetil (CellCept
Ò
) and is a
prodrug that is hydrolyzed to mycophenolic acid in the
body.
Immunosuppressants, such as cyclosporin A and ta-
crolimus, convert the normally fungistatic activity of az-
oles (i.e., fluconazole) against C. albicans, Candida
glabrata and Candida krusei into fungicidal activity [80].
They do this by inhibiting the protein phosphatase calci-
neurin. Even non-immunosuppressive analogs of tacroli-
mus have this ability. Non-azole drugs that inhibit other
steps of ergosterol biosynthesis (terbinafine, fen-
propimorph) are also improved in activity by immuno-
suppressants and their non-immunosuppressive analogs.
Rapamycin extends life span in mice and is being con-
sidered for possible use against progeria (Hutchinson–
Gilford progeria syndrome) in children. This is a rare dis-
ease that resembles accelerated aging and kills children in
their teens. Rapamycin, when tested on cells from children
suffering with progeria, promoted cleavage of progerin, the
mutant protein that accumulates in cells of affected chil-
dren, and it extended survival of the cells [26].
Prodigiosins, red microbial pigments produced by Ser-
ratia marcescens, have immunosuppressive and anticancer
activities [82]. Pigments produced by Monascus have
antibiotic, immunosuppressive, and hypotensive activities.
Hypocholesterolemic agents
Only 30 % of the cholesterol in the human body comes
from the diet. The remaining 70 % is synthesized by the
body, mainly in the liver. Many people cannot control their
cholesterol at a healthy level by diet alone but must depend
on hypocholesterolemic drugs. The statins inhibit de novo
production of cholesterol in the liver, the major source of
blood cholesterol. High blood cholesterol leads to athero-
sclerosis, which is a causal factor in many types of coro-
nary heart disease, a leading cause of human death. Statins
were a success because they reduced total plasma choles-
terol by 20–40 % whereas the previously used fibrates only
reduced it by 10–15 % [65]. The statins are microbially
produced enzyme inhibitors, inhibiting 3-hydroxy-3-
methylglutaryl-coenzyme A reductase, the regulatory and
rate-limiting enzyme of cholesterol biosynthesis in liver.
The discovery and developments of the statins present a
fascinating story. Their history has been described by Akira
Endo, the discoverer of the first statin, compactin [48].
They were first discovered in fungi in England and Japan in
1975–6. Compactin had been reported as an antifungal
agent by Brown et al. [20] of Glaxo from P. brevicom-
pactum. Earlier, in Japan, the Sankyo pharmaceutical
company had supported Endo for a sabbatical leave at
Albert Einstein Medical College (NYC) to study
phospholipid metabolism under Bernard Horecker. Upon
returning in 1968 to Sankyo, Endo screened 6,000 fungal
extracts for inhibition of cholesterol biosynthesis by rat
liver membranes and found two actives, i.e., ML-236A and
B, produced by Penicillium citrinum. ML-236B was
compactin (=mevastatin). It was found to inhibit
3-hydroxy-3-methylglutaryl coenzyme A reductase [49]. In
1976, Sankyo prepared a patent application on compactin.
However, Sankyo did not commercialize compactin and it
never became a commercial drug. In 1976, H. Boyd
Woodruff, who was Merck’s representative in Japan, heard
of the work of Endo, and requested a sample of compactin.
The two companies signed a confidentiality agreement and
the compactin sample was given to Merck. Merck did tests
in cultured mammalian cells, rats and dogs, and, by 1978,
had obtained promising results. At that time, the group of
Alberts at Merck started to screen for new inhibitors and
discovered lovastatin produced by Aspergillus terreus [2],
which they called Mevacor
Ò
. It had a structure similar to
compactin but contained a methyl group. At about the same
time, Endo [47] reported the discovery of lovastatin from
Monascus ruber and named it monocolin K (=mevinolin).
It was patented in Japan but without structural elucidation.
194 J Ind Microbiol Biotechnol (2014) 41:185–201
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Merck filed for a patent containing their findings and the
structure of lovastatin. The company received a US patent
in 1980 and lovastatin became the first statin on the market.
Sankyo, in their tests with compactin on dogs, apparently
noted intestinal tumors and in 1980, stopped further testing.
Merck also stopped testing at that time and this inactivity
existed for 2–3 years. However, since Merck had seen no
such tumors with lovastatin, they decided to resume their
efforts. Further clinical tests on lovastatin went into full
speed in 1982, and the drug was finally approved by FDA
in 1987, after clinical tests in humans had shown a low-
ering of total blood cholesterol of 18–34 %, a 19–39 %
decrease in low-density lipoprotein cholesterol (‘‘bad
cholesterol’’) and a slight increase in high-density lipo-
protein cholesterol (‘‘good cholesterol’’).
Merck later produced simvastatin (Zocor
Ò
) in which the
2-methylbutanoate side-chain of lovastatin was chemically
modified to 2,2-dimethylbutanoate; it was launched in
1988. Although compactin was never used medically,
Sankyo devised a bioconversion of it by hydroxylation
yielding pravastatin which became commercial in 1989 and
was then licensed to Bristol-Myers Squibb. The biocon-
version was carried out using actinomycetes [87, 100].
In 1985, a 28-year-old postdoctoral associate in the Uni-
versity of Rochester’s Chemistry Department, Bruce D. Roth,
was able to chemically synthesize one of the statins that Endo
had isolated from his fungus in the 1970s. Two years later, he
headed an 18-person group at the Parke-Davis company (now
Pfizer) working on synthesis of a synthetic statin called ator-
vastatin (Lipitor
Ò
). They compared atorvastatin in a further
clinical trial vs. fluvastatin, lovastatin, pravastatin, and sim-
vastatin; atorvastatin showed the best results. The FDA
approved it in January of 1997. Parke-Davis decided to co-
market it with Pfizer in 1996. By mid-1998, atorvastatin had
18 % of the statin market as compared to simvastatin’s 37 %.
Pfizer, in 2000, purchased Warner-Lambert, the parent of
Parke-Davis and became the sole owner of atorvastatin, which
became the leading drug in the world.
The largest segment of the pharmaceutical business is
for cholesterol-lowering drugs, amounting to about 30 %
of the market. Simvastatin reached a market of over $7
billion. Pravastatin attained sales of $5 billion. Atorvastatin
became the leading drug in the world at $12 billion per
year. A useful review on the statins was published by
Manzoni and Rollini [71].
Natural statins are produced by many fungi: A. terreus
and species of Monascus, Penicillium, Doratomyces, Eu-
penicillium, Gymnoascus, Hypomyces, Paecilomyces,
Phoma, Trichoderma, and Pleurotis. Although pravastatin
is commercially made by bioconversion of compactin,
certain strains of Aspergillus and Monascus can produce
pravastatin directly [72]. Basidiomycetes, such as Pleuro-
tus ostreatus, also produce lovastatin but at low levels [1].
Simvastatin has traditionally been made by synthetic
multistep processes starting with lovastatin. This can now
be avoided by use of an Escherichia coli strain over-
expressing lovD in the presence of a cell-membrane per-
meable thioester, i.e., dimethylbutyryl-5-methylmercapto-
propionate [121, 122]. The whole-cell procedure converts
monocolin J acid to simvastatin acid in high yields, i.e.,
over 99 %.
Statins reduce cardiovascular events including myocar-
dial infarction, stroke, and death [112]. Not only are they
active against arthrosclerosis, the most common cause of
death in Western countries, but also improve endothelial
function, and have anti-inflammatory, anti-atherothrombo-
sis, immunomodulation, and anti-migration activities. The
inflammatory effect is over and above their action in
lowering cholesterol [126]. Statins reduce total and LDL-
cholesterol and increase HDL-cholesterol. They also
reduce the occurrence of Alzheimer’s disease. Statins also
lower elevated C-reactive protein (CRP) levels indepen-
dent of their effect on cholesterol [29]. This is important
since half of all myocardial infarctions occur in patients
with normal LDL levels. High CRP is associated with
inflammatory response in atherosclerosis and is a predictor
of future cardiovascular mortality. Statins can also prevent
stroke, reduce development of peripheral vascular disease.
They are also showing beneficial effects for multiple
sclerosis and cancer. Experiments with oral statins showed
efficacy in a mouse model of multiple sclerosis. The effect
appears to be independent of cholesterol lowering. Other
activities being studied are stimulation of bone formation
and antioxidation [120].
Red yeast has been used as a traditional Chinese food
and medicine since 800
A.D. It is also known as red koji or
Hongqu. The fermentation organism is Monascus purpu-
reus whose pigments give the food its characteristic color.
The organism is used for rice, red wine, red soy bean
cheese, meat, and fish, and is authorized for food use in
China and Japan. The orange pigments, monascorubin and
rubropunctatin, have both antibacterial and antifungal
activities [73]. Li Shizhen, the noted pharmacologist of the
Ming Dynasty (1368–1644), reported the favorable effects
of red rice on blood circulation. More recent work has
shown that red rice lowers blood-lipid levels due to its
content of statins and clinical trials demonstrated the
lowering of cholesterol in humans.
Additional applications of natural products
Inhibitors of angiotensin-converting enzyme (ACE), which
are widely used for hypertension and congestive heart
failure, are chemicals based on peptides isolated from
snake venom [85].
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123

The predecessor of aspirin has been known as far back
as the fifth century
B.C., at which time it was extracted from
willow tree bark by Hippocrates. It probably was used even
earlier in Egypt and Babylonia for fever, pain, and child-
birth. Such salicylic acid derivatives have been found in
plants such as white willow, wintergreen and meadow-
sweet. Synthetic salicylates were produced on a large scale
in 1874 by the Bayer company in Germany.
Natural products have been useful in our battles against
viruses.
D-nucleoside analogs, usually D-ribose derivatives,
such as
D-ribavirin (Virazole
Ò
), are used as enzyme
inhibitors against hepatitis C virus [111]. They also include
vidarabin (Ara-A), idoxuridine and acyclovir (Zovirax
Ò
),
acting against DNA synthesis by herpes simplex virus.
Acyclovir was isolated from a sponge.
L-nucleoside ana-
logs, usually less toxic than the
D-forms, include levorine
(
L-ribavirin), lamivudine (L-b-1,3-oxathiolanylcytosine)
and
L-2
0
,3
0
-dideoxycytidine (L-ddc) against HIV and hep-
atitis C. Also important are the HIV inhibitors such as
azidothymidine (AZT) against reverse transcriptase, and
inhibitors of aspartyl protease, HIV entry, cell fusion and
integrase. The reverse transcriptase and protease inhibitors
that made it to the market were derived from natural
product leads screened at the National Cancer Institute
[124]. Then, natural product inhibitors of influenza virus
neuraminidase (sialidase) came upon the scene. Combina-
tions of the above small molecules are commonly used for
antiviral therapy. Recently, larger molecules such as
interferon alpha, monoclonal antibodies, polyclonal anti-
bodies, and nucleic acids are becoming important, all of
which are related to natural products.
Microbial toxins have also found use. The toxin from
Clostridium botulinum, botulinum A (Oculinum
Ò
), is used
for neck, eye and mouth spasms and as Botox
Ò
for
reduction of wrinkles.
Natural products are not only of interest as pharma-
ceuticals, but also include compounds of use in nutrition
and as polymers. The annual tonnage of amino acids
includes 2.2 million for
L-glutamic acid, 1.5 million for L-
lysine, 77,000 for
L-threonine, and 16,000 for L-phenylal-
anine. Vitamin C is made at 100,000 tons and vitamin B
12
at 12 tons per year. Industrial biotechnology has penetrated
the chemical industry in the areas of fine and bulk chem-
icals, polymers and energy. In certain ways, industrial
biotechnology often outperforms conventional chemical
technology, e.g., higher reaction rates, increased conver-
sion efficiency, improved product purity, lower energy
consumption, and decreased generation of chemical waste
[102]. The natural polymer polylactic acid, which is made
at 140,000 tons per year, has properties similar to synthetic
polyethylene and polypropylene but is much more biode-
gradable [11]. Microbes are the workhorses of industrial
biotechnology because of high synthetic versatility, ease of
using renewable raw materials, high reaction rates, rapid
growth, and easy modification by genetic means.
Natural products have solved many other problems of
our world because of their unique activities. They include
anthelmintic agents such as avermectins [24, 79], bioin-
secticides (BT toxin, spinosyns, tetranactin, nikkomycins),
enzyme inhibitors (lipstain, desferal, acarbose, validam-
ycins, ancovenine, fibrostatin B, phthoxazoline, bialophos,
nikkomycin), terpenoids (sterols, gibberellins, carotenoids,
lycopene, drimane sesquiterpenes, b-carotene, ubiquinone,
artemisinin, amorphadiene, astaxanthin, farnesine, farne-
sol, isoprene), nutraceuticals (polyunsaturated fatty acids
such as docosahexaenoic acid [DHA] and arachidonic acid,
prebiotics, coenzyme Q10, omega-3-fatty acids), surfac-
tants (sophorolipids), polysaccharides (pullulan, xanthan,
succinoglycan, alginates), bioherbicides, 1,3-propanediol,
1,2-propanediol (propylene glycol), polymeric plastics
(poly[3-hydroxybutyrate], polyhydroxyalkanoates, poly-
acrylamide, polylactic acid), and the very important area of
vaccines.
Decrease of new drug discovery by the pharmaceutical
industry
The golden age of antibiotic discovery was from 1940 to
1980. New bioactive products from microbes were dis-
covered at an amazing pace, i.e., 200–300 per year in the
late 1970s increasing to 500 per year later. Genetics played
a great part in the development of useful industrial products
from microbes [110]. Production of antibiotics began with
penicillin in the late 1940s and proceeded with great suc-
cess until the 1980s when it became harder and harder to
discover new and useful products. The rate of discovery
dropped then and slowed down drastically in the 21st
century. The major problem has been the loss of interest by
the major pharmaceutical companies in these compounds
due to the years of preclinical and clinical development
required and the short period given to these organizations
by governments for sales before patent expiration. Also,
the short period of a patient’s use of such products limits
their sales as compared to drugs that are taken by patients
daily for many years.
The departure of much of the pharmaceutical industry
from the drug discovery effort has had its greatest negative
effect in the area of antibiotics. New antibiotics are sorely
needed because of (a) the development of antibiotic-resis-
tant pathogens, (b) the emergence of over 30 new diseases
since 1980 such as AIDS, Ebola virus, Hanta virus,
Cryptosporidium, Legionnaires’ disease, Lyme disease,
E. coli 0157:H7; (c) the existence of naturally resistant
bacteria such as P. aeruginosa causing fatal wound infec-
tions, burn infections, and chronic and fatal infections in
196 J Ind Microbiol Biotechnol (2014) 41:185–201
123

lungs of cystic fibrosis patients, Stenotrophomonas malto-
philia, Enterococcus faecium, Burkholderia cepacia, and
Acinetobacter baumanni and (d) the toxicity of some of the
current compounds.
In the 1990–2005 period, a number of large pharma-
ceutical companies emphasized combinatorial chemistry,
and departed from the natural product area. Unfortunately,
this attempt to replace natural products with synthetic
molecules failed because the chemistry did not create
sufficiently diverse or pharmacologically active molecules.
As a result, the numbers of (a) FDA drug applications,
(b) new drug approvals, (c) new and approved active
substances, (d) orphan drug applications, and (e) new
chemical entities of the pharmaceutical industry markedly
decreased since the late 1990s. The exit of many of the
major pharmaceutical companies has left much of the
discovery efforts to small companies, and the biotechnol-
ogy industry.
Despite the above problems, development of new anti-
biotics has continued, albeit at a much slower pace than in
the last century. We have seen the (1) appearance of newly
discovered antibiotics (e.g., candins), (2) development of
old but unutilized antibiotics (e.g., daptomycin, lipiarmy-
cin), (3) production of new semisynthetic versions of old
antibiotics (e.g., glycylcyclines, streptogramins), as well as
the (4) very useful application of old but underutilized
antibiotics (e.g., teicoplanin).
Possible solutions
Research on natural products must continue due to unmet
needs. It is obvious that we cannot ignore the opportunities
presented to us by natural products. They include their
novelty, complexity, remarkable diversity of structures and
activities, utility as biochemical probes, existence of novel
and sensitive assay methods, improvements in isolation,
purification and characterization, and new production
methods. The number of chirality centers, bridges, rings,
and functional groups in natural products make them much
more complex than synthetic compounds. Although com-
binatorial chemistry has not been of significant use, com-
binatorial biosynthesis has been helpful in the hunt for new
products. This has involved the genetic modification of a
producing organism, and/or exchange of genes between
organisms to create useful hybrid molecules [8, 60, 74,
119] including new erythromycins, spiramycins, tetrace-
nomycins, anthracyclines and nonribosomal peptides.
However, there still is an urgent need for more secondary
metabolites to be discovered for use in medicine.
What is needed are the further applications of (1) high-
throughput screening to natural product libraries and (2)
combinatorial chemistry using natural product scaffolds
[37, 64, 114]. There is a higher hit rate in high-throughput
screening of natural product collections than of combina-
torial libraries of synthetic compounds [16, 17]. Further-
more, the number of compounds in a chemical library is
not as important as the biological relevance, design and
diversity of the library, and a scaffold from nature provides
viable, biologically validated starting points for the design
of chemical libraries. It is clear that the role of combina-
torial chemistry, like those of structure–function drug
design and recombinant DNA technology decades ago, is
that of complementing and assisting natural product dis-
covery and development, not replacing them [86]. The
combining of natural product screening with high-
throughput screening, combinatorial chemistry, genomics,
proteomics, metabolomics, and discoveries in biodiversity
provide real opportunities for the future [40]. Application
of systems biology [96] to antibiotic production is another
potential solution.
Opportunity also exists in the deeper examination of the
microbial world. Bacteria have existed on earth for over
3 billion years, and eukaryotes have been around for
1 billion years. Since 95–99.9 % of organisms existing in
nature have not yet been cultured in the laboratory, only a
minor proportion of bacteria and fungi have thus far been
examined for secondary metabolite production. It has been
estimated that 30 g of soil contains 20,000 common bac-
terial species and perhaps 500,000 rare species. Another
estimate is that that 1 g of soil contains 1,000–10,000
species of undiscovered prokaryotes. About 65,000–70,000
fungal species have been recognized but it has been esti-
mated that from 0.25 to 9.9 million exist [99]. Their total
weight is thought to be higher than that of humans. Of the
fungal species that have been described, only about 16 %
have been cultured. The use of fungal ecology in the search
for new drugs is extremely important. The estimated
number of fungal species is more than five times the pre-
dicted number of plant species and 50 times the estimated
number of bacterial species. The well-known concept
involving the need for isolation of microbial strains from
different geographical and climatic locations around the
world, in order to insure microbial diversity in collections,
still gathers support. It is obvious that we need to improve
our methodology of finding new natural products.
What the government could do
The opportunities described above of combining screening
with modern techniques should be seriously considered by
the pharmaceutical industry. However, if we ever are to
solve the problem of fewer and fewer drugs being com-
mercialized, the government must also contribute. Con-
sider what the pharmaceutical industry has faced in the 21st
J Ind Microbiol Biotechnol (2014) 41:185–201 197
123

century [22, 51, 117]: 2–10 years for discovery, 4 years for
pre-clinical development, 1 year for clinical development
in Phase I, 1.5 years for Phase II, 3.5 years for Phase III,
1 year of FDA review and approval, and 1 year of post-
marketing testing. Thus, the total time is 14 years but in
certain cases, it has reached 22 years. From 1999 to 2003,
the total cost rose from $500–600 million to $900 million.
The US government has made an important step in the right
direction, i.e., the establishment of its translational center
for new drug discovery. However, what is clearly needed is
the granting of more time for the drug industry to market
their discovered products before they become generic
drugs. The time now is 22 years from the time of patent
approval and this is clearly too short when one considers
the length of time and the funds needed for discovery, pre-
clinical and clinical development, etc.
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