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Indian Journal of Biotechnology
Vol 2, July 2003, pp 322-333
Microbial Secondary Metabolites Production and Strain Improvement
J Barrios-Gonzalez *, F J Fernandez and A Tomasini
Depto de Biotecnologfa, Universidad Aut6noma Metropolitana, Iztapalapa, Apdo Postal 55-535,
Mexico
0
F 09340, Mexico
Received 20 November 2002; accepted 20 February 2003
Microbial secondary metabolites are compounds produced mainly by actinomycetes and fungi, usually late in
the growth cycle (idiophase). Although antibiotics are the best known secondary metabolites (SM), there are others
with an enormous range of other biological activities mainly in fields like: pharmaceutical and cosmetics, food, agri-
culture and farming. These include compounds with anti-inflammatory, hypotensive, antitumour, anticholesterole-
mic activities, and also insecticides, plant growth regulators and enviromnental friendly herbicides and pesticides.
These compounds are usually produced by liquid submerged fermentation, but some of these metabolites could be
advantageously produced by solid-state fermentation. Today, strain improvement can be performed by two alterna-
tive strategies, each having distinct advantages, and in some cases all these approaches can be used in concert to in-
crease production such as classical genetic methods with mutation and random selection or rational selection (in-
cluding genetic recombination); and molecular genetic improvement methods. The latter can be applied by: amplifi-
cation of SM biosynthetic genes, inactivation of competing pathways, disruption or amplification of regulatory genes,
manipulation of secretory mechanisms and expression of a convenient heterologous protein. It is visualized that in
the near future, genomics will also be applied to industrial strain improvement.
Keywords: secondary metabolites, new activities, classical and molecular genetic improvement
Introduction
Secondary metabolites (SM) are compounds with
varied and sophisticated chemical structures, pro-
duced by strains of certain microbial species, and by
some plants. Although antibiotics are the best known
SM, there are other such metabolites with an enor-
mous range of biological activities, hence acquiring
actual or potential industrial importance.
These compounds do not play a physiological role
during exponential phase of growth. Moreover, they
have been described as SM in opposition to primary
metabolites (like amino acids, nucleotides, lipids and
carbohydrates), that are essential for growth.
A characteristic of secondary metabolism is that
the metabolites are usually not produced during the
phase of rapid growth (trophophase), but are synthe-
sized during a subsequent production stage (idio-
phase). Production of SM starts when growth is lim-
ited by the exhaustion of one key nutrient source: car-
bon, nitrogen or phosphate. For example, penicillin
biosynthesis by Penicillium chrysogenum starts when
glucose is exhausted from the culture medium and the
*
Author for correspondence:
Tel: 55-5804-6453; Fax: 55-5804-4712
E-mail: jbg@xanum.uam.mx
fungus starts consuming lactose, a less readily utilized
sugar.
Most SM of economic importance are produced by
actinomycetes, particularly of the genus Streptomyces,
and by fungi.
Biosynthetic Families
Microbial SM show an enormous diversity of
chemical structures. However, their biosynthetic
pathways link them to the more uniform network of
primary metabolism. It has been shown that SM are
formed by pathways which branch off from primary
metabolism at a relatively small number of points,
which define broad biosynthetic categories or fami-
lies:
(1) Metabolites derived from shikimic acid (aro-
matic amino acids).
Examples are ergot alkaloids and the antibiotics
candicidin and chloramphenicol.
(2) Metabolites derived from amino acids.
This family includes the ~-lactam antibiotics: peni-
cillin, cephalosporins and cephamycins, as well as
cyclic peptide antibiotics such as gramicidin or the
immunosupressive agent cyclosporine.
(3) Metabolites derived from Acetyl-CoA (and re-
lated compounds, including Kreb's cycle inter-
mediates).
BARRIOS-GONZALEZ
et al:
MICROBIAL SECONDARY METABOLITES
This family can be subdivided into polyketides and
terpenes. Examples of the former group include the
antibiotic erythromycin, the insecticidal-antiparasitic
compound avermectin and the anti tumour agent doxo-
rubicin. An example of the second group is the non
citotoxic antitumour agent taxol.
(4) Metabolites derived from sugars.
Examples of SM in this group are streptomycin and
kanamycin (Smith
&
Berry, 1976).
Since secondary biosynthetic routes are related to
the primary metabolic pathways and use the same in-
termediates, regulatory mechanisms i.e. induction,
carbon catabolite regulation and/or feedback regula-
tion, apparently operate in conjunction with an overall
control, which is linked to growth rate (Demaim &
Davis,1989; Doull
&
Vining, 1995).
New Bioactive Compounds
The last two decades have been a phase of rapid
discovery of new activities and development of major
compounds of use in different industrial fields,
mainly: pharmaceutical and cosmetics, food, agricul-
ture and farming (Table 1).
Microbial SM are now increasingly being used
against diseases previously treated only by synthetic
drugs, e.g. as anti-inflammatory, hypotensive, antitu-
mour, anticholesterolemic, uterocontractants, etc.
Moreover, new microbial metabolites are being used
in non medical fields such as agriculture, with major
herbicides, insecticides, plant growth regulators and
environmental friendly herbicides and pesticides as
well as antiparasitic agents.
This new era has been driven by modem strategies
to find microbial SM. Earlier, whole cell assay meth-
ods, like bioassays, are being replaced by new and
sophisticated, target-directed, mode-of-action screens.
In this way, culture broths of new isolates are tested in
key enzymatic reactions or as antagonistic or agonis-
tic of particular receptors. This new approach relies
on the knowledge of the biochemical and molecular
details of different diseases or physiological processes
(Barrios-Gonzalez et aI, 2003).
Production
Liquid Fermentation
Secondary metabolites are generally produced in
industry by submerged fermentation (SmF) by batch
or fed-batch culture. An improved strain of the pro-
ducing microorganism is inoculated into a growth
medium in flasks and then transferred to a relatively
323
small fermenter or "seed culture". This culture, when
in rapid growth phase, is used to inoculate a fermenter
tank, in the range of 30,000 to 200,000 litres, with
production medium. Several parameters, like medium
composition, pH, temperature, agitation and aeration
rate, are controlled. The different regulatory mecha-
nisms mentioned previously are bypassed by envi-
ronmental manipulations. Hence, an inducer such as
methionine is added to cephalosporine fermentations,
phosphate is restricted in chlortetracycline fermenta-
tion, and glucose is avoided in penicillin or erythro-
mycin fermentation. The fermentation processes of
antibiotics regulated by carbon are now conduced
with slowly utilized sources of carbon, generally lac-
tose. When glucose is used, it is usually fed at a slow,
continuous rate to avoid catabolite regulation. Also
nitrogen sources like soybean meal are used to avoid
nitrogen (ammonium) regulation. In some cases, a
precursor is used to increase one specific desirable
metabolite, for example lysine is added as precursor
and cofactor to stimulate cephamycin production by
Streptomyces clavuligerus (Khetan et aI, 1999). Agi-
tation is provided by turbine impellers at a power in-
put of 1-4 W/litre and air has to be supplied at flow
rates of 0.5-1.0 v/v per min. Exit gas is generally
analyzed to monitor O2and CO2concentrations. This
can provide metabolic information to regulate the
feeding rates of precursors and nutrients.
Some natural antibiotics and other SM are chemi-
cally modified, in a subsequent stage to produce semi-
synthetic derivatives.
Solid-state Fermentation
Solid-state fermentation (SSF) holds an important
potential for the production of secondary metabolites
(Barrios-Gonzalez et aI, 1988; Tomasini et aI, 1997;
Robinson et aI, 2001). This fermentation system has
been used in several oriental countries since antiquity,
to prepare diverse fermented foods from grains like
soybeans or rice (Hesseltine, 1977a, b). However,
different SSF systems, that could be called non-
traditional have been developed in the last 15 years. A
modem SSF definition is the one proposed by Lon-
sane et al (1985)-a microbial culture that develops
on the surface and at the interior of a solid matrix and
in absence of free water. Today, two types of SSF can
be distinguished, depending on the nature of solid
phase used (Barrios-Gonzalez & Mejia, 1996).
(a) Solid culture of one support-substrate phase-
solid phase is constituted by a material that
324 INDIAN J BIOTECHNOL, JULY 2003
Activity Examples
Table l-Biological activities of some microbial secondary metabolites of industrial importance
Producing Micro-organism
Antibacterials Cephalosporin
Cephamycin
Chloramphenicol
Erythromycin
Kanamycin
Tetracyclin
Penicillin
Rifamycin
Spectinomycin
Streptomycin
Anticholesterolemics Lovastatin
Monacolin
Pravastatin
Antifungals Amphotericin
Aspergillic acid
Aureofacin
Candicidin
Griseofulvin
Nystatin
Oligomycin
Antitumourals Actinomycin D
Bleomycin
Doxorubicin
Mitomycin C
Taxol
Enzyme inhibitors Clavulanic acid
Plants Growth Regulators
Growth Promoters
Gibberellin
Monensin
Tylosin
Herbicidals Bialaphos
Inmunosuppresives Cyclosporin A
Rapamycin
Tacrolimus (FK-506)
Insecticides and Antiparasitics Avermectin
Milbemycin
Pigments Astaxanthin
Monascin
Phaffia rhodozyma
Monascus purpureus, M. ruber
Acremonium chrysogenum
Streptomyces clavuligerus
Streptomyces venezuelae
Saccharopolyspora erythraea
Streptomyces kanamyceticus
Streptomyces aureofaciens
Penicillium chrysogenum
Amycolatopsis mediterranei
Streptomyces spectablis
Streptomyces griseus
Aspergillus terreus
Monascus ruber
Penicillium citrinum, Streptomyces carbophilus
Streptomyces nodosus
Aspergillus flavus
Streptomyces aureofaciens
Streptomyces griseus
Penicillium griseofulvum
Streptomyces nourse,
S.
aureus
Streptomyces diastachromogenes
Streptomyces antibioticus,
S.
parvulus
Streptomyces verticillus
Streptomyces peucetius
Streptomyces lavendulae
Taxomyces andreanae, plants
Streptomyces clavuligerus
Gibberellafujikuroi
Streptomyces cinnamonensis
Streptomyces fradiae
Streptomyces hygroscopicus
Tolypoclaudium inflatum
Streptomyces hygroscopicus
several Streptomyces species
Streptomyces avermitilis
Streptomyces hygroscopicus
BARRIOS-GONZALEZ et al: MICROBIAL SECONDARY METABOLITES
assumes, simultaneously, the functions of sup-
port and of nutrients. source. Agricultural or even
animal goods or wastes are used as support-
substrate.
(b) Solid culture of two substrate-support phase-
solid phase is constituted by an inert support im-
pregnated with a liquid medium. Inert support
serves as a reservoir for the nutrients and water.
Materials as sugarcane bagasse pith or polyure-
thane can be used as inert support.
Fungi and actinomycetes, the main micro-
organisms producer of SM grow well is SSF, because
the conditions are similar to their natural habitats,
such as soil, and organic waste materials (Table 2).
The advantages of SSF in relation with SmF in-
clude: energy requirements of the process are rela-
tively low, since oxygen is transferred directly to the
microorganism. SM are often produced in much
higher yields, often in shorter times and often sterile
conditions are not required (Barrios-Gonzalez et ai,
1988; Ohno et
al,
1993; Balakrishna
&
Pandey, 1996;
Rosenblitt et al, 2000).
In SSF, parameters to control are similar to the
ones controlled for SmF. Particular parameters like
initial moisture content, particle size and medium
325
concentration have to be optimized in this culture
system. It has been shown that penicillin production
in a SSF impregnated bagasse system, is strongly
controlled by the proportions of bagasse, nutrients and
water. Combinations that supported very high peni-
cillin yields were identified in this work (Dominguez
et al, 2001). Interestingly, these conditions caused
growth phases with different characteristics, but all
allowed a slow but adequate supply of nutrients to the
fungus during the idiophase, supporting a characteris-
tic low but steady respiratory activity during produc-
tion phase (Dominguez et al, 2000).
Reports on enzymes production suggest that high
producing strains in SmF are generally poor producers
in SSF (Shankaranand et al, 1992). Barrios-Gonzalez
et al (1993) reported that high yielding strains for
SmF cannot be relied upon to perform well in SSF.
This situation dictates the need to develop high-
yielding strains particularly suited for SSF. These
special strains can be developed faster by using hiper-
producing strains developed for SmF as parental
strains (Barrios-Gonzalez et
al,
1993a).
Many comparative studies between SmF and SSF
claim higher yields for products made by SSF
(Pandey et ai, 1999, 2000), indicating that some of
Metabolite Substrate/Support
Table 2-Secondary metabolites produced by solid state fermentation system
Reference
Penicillin Sugarcane bagasse
Cephalosporin rice grains
Cyclosporin A wheat bran
Cephamycin C
Tetracycline
Pyrazines
Oxycetracycline
wheat straw
sweet potato residue
wheat and soybean
sweet potato residue
Iturine
Surfactin
Soybean curd
Soybean curd
Gibberellic acid wheat bran
cassava
polyurethane
Pigments rice grains
Ergot alkaloids Sugarcane bagasse Trejo et al, 1993
Microorganism Use
Penicillium chrysogenum Antibiotic
Streptomyces sp Antibiotic
Tolypocladium inflatum Antibiotic
Streptomyces clavuligerus Antibiotic
Streptomyces viridifaciens Antibiotic
Aspergillus oryzae Aroma
Streptomyces rimosus Antibiotic
Bacillus subtillis Antifungal
Bacillus subtillis Surfactant,
antibiotic
Gibberella fujikuroi Vegetal
hormone
Food and
pharmaceuti-
cals
Medical
Monascus purpureus
Claviceps fusiformis
Barrios-Gonzalez et al, 1988,
1993b
Wang et al, 1984; Jerami &
Demain, 1989
Sekar & Balaraman, 1998;
Ramana et al 1999
Kota & Sridhar, 1998
Yang & Ling, 1989
Serrano-Carre6n et al, 1992
Yang & Yuan, 1990; Yang
& Wang, 1996
Ohno et
al,
1993, 1996
Ohno et al, 1995
Kumar & Lonsane, 1987a,b;
Bandelier et
al,
1997; Agosin
et
al,
1997; Tomasini et
al,
1997
Lontong& Suwanarit, 1990;
Rosenblitt et al, 2000
326 INDIAN J BIOTECHNOL, JULY 2003
these metabolites could be commercially produced by
SSP. However, commercial production application of
secondary metabolites by SSF remains unexploited in
Western countries, mainly due to problems associated
with scale-up. Most of these problems have been
studied and some solutions proposed. Taking in ac-
count these points, some bioreactors have been de-
signed (Mitchell et al, 2000, 2002; Hardin et al, 2000;
Nagel et al, 2001, 2002; Suryanarayan et al, 2001;
Junter et al, 2002; Miranda et al, 2003).
In South-East Asia, where SSF is more common,
research attention was directed towards the industri-
alization of this culture method. In India, a fermenta-
tion industry, started industrial production of micro-
bial enzymes and secondary metabolites by SSF.
Suryanarayan (2002) designed a solid state bioreactor
in which the system is contained, and the fermentation
product can be extracted from the solid matrix with-
out opening the reactor. Finally this reactor is oper-
ated automatically. Since January 2001, this reactor is
being used to produce lovastatin, the first secondary
metabolite produced industrially by SSF.
Strain Improvement
The science and technology of manipulating and
improving microbial strains, in order to enhance their
metabolic capacities for biotechnological applications,
are referred to as strain improvement. The microbial
production strain can be regarded as the heart of a
fermentation industry, so improvement of the produc-
tion strain(s) offers the greatest opportunities for cost
reduction without significant capital outlay (Parekh et
al,
2000). Moreover, success in making and keeping a
fermentation industry competitive depends greatly on
continuous improvement of the production strain(s).
Improvement usually resides in increased yields of the
desired metabolite. However, other strain characteris-
tics can also be improved. Typical examples include
removal of unwanted cometabolites, improved utili-
zation of inexpensive carbon and nitrogen sources or
alteration of cellular morphology to a form better
suited for separation of the mycelium from the prod-
uct and/or for improved oxygen transfer in the fer-
menter.
Today, strain improvement can be performed by
two alternative strategies: 1) Classical genetic meth-
ods (including genetic recombination); and 2) Mo-
lecular genetic methods.
Each has distinct advantages, and in some cases all
these approaches can be used in concert to increase
production.
Classical Genetic Methods
Strain development by this strategy has typically
relied on mutation, followed by random screening.
After this, careful fermentation tests are performed
and new improved mutants are selected. Mutation can
be carried out with physical mutagens like UV-light
or chemical mutagens like N-methyl-N' -nitro-N-
nitrosoguanidine or ethyl methanesulphonate (Baltz,
1999). This empirical approach has a long history of
success, best exemplified by the improvement of
penicillin production, in which modem reported titles
are 50 g/l, an improvement of at least 4,000 fold over
the original parent (Peberdy, 1985). Other examples
include fungal or actinomycetal cultures capable of
producing metabolites in quantities as high as 80 g/l
(Rowlands, 1984; Vinci
&
Byng, 1999).
The advantage of mutation/selection is simplicity,
since it requires little knowledge of the genetics, bio-
chemistry and physiology of the product biosynthetic
pathway. Moreover, it does not need sophisticated
equipment and requires minimal specialized technical
manipulation. Another important advantage is effec-
tiveness, since it leads to rapid titer increases.
A drawback of this strategy is that it is labour in-
tensive. In the last 10-15 years, these random screen-
ing methods have been replaced by less empirical,
directed selection techniques or rational selection.
Rational selection. Rational screening allows for
significant improvement in the efficiency of the se-
lection stage. In this process, a selection is made for a
particular characteristic of the desired genotype, dif-
ferent from the one of final interest, but easier to de-
tect. In its more effective form, a rational screen will
eliminate all undesirable genotypes, allowing very
high numbers of isolates to be tested easily.
The design of these methods requires some basic
understanding of the product metabolism and pathway
regulation. This knowledge can be used to propose
environmental conditions, or the addition of a chemi-
cal that could be a chromogenic or selective reagent, a
dye or an indicator organism. For example, a toxic
precursor of penicillin (phenylacetic acid) was added
to the agar medium, where the sensitive parent strains
were prevented from growing, while only resistant
mutants propagated. In this case, 16.7% of the resis-
tant mutants produced more antibiotic than the pa-
rental strain (Barrios-Gonzalez et al, 1993b).
In another example, carotenoids have been shown
to protect the yeast, Phaffia rodozyma from singlet
oxygen damage (oxidative stress). Combination of
BARRIOS-GONZALEZ et al: MICROBIAL SECONDARY METABOLITES
Rose Bengal and thymol (oxidation/reduction reaction
detection) in visible light has been used to select ca-
rotenoid over-producing strains (Schroeder & John-
son, 1995a, b).
Vinci and Byng (1999) have given some examples
of selection by rational screening. These include re-
sistance to chloroacetete, fluoroacetate or chloroacet-
amide for overproduction of polyketides; resistance to
2-deoxyglucose to overcome glucose repression; re-
sistance to methylammonium chloride to overcome
repression by ammonium ion, and arsenate resistance
to overcome phosphate repression.
Micro-organisms possess regulatory mechanisms
which control production of their metabolites, thus
preventing overproduction. For primary metabolite
production, microbiologists have found that elimi-
nating or decreasing the particular mechanism (de-
regulating) in the microbe causes overproduction of
the desired product. However, factors that turn on
secondary product formation are complex (induction,
feedback regulation, nutritional regulation by source
of carbon, nitrogen and/or phosphorus as well as a
global physiological control), most of which are by-
passed by nutritional manipulations of the culture.
Some success has been achieved by applying concepts
derived from mutation of regulatory controls of pri-
mary metabolism. For example, a way to produce
feed-back resistant mutants of primary metabolism is
to select for analogue-resistant mutants. The analogue
technique has been successfully applied to secondary
metabolism. The fungi, Penicillium chrysogenum and
Acremonium chrysogenum are producers of the ~-
lactamic antibiotics penicillin and cephalosporin, re-
spectively, which are derived from amino acid precur-
sors. Mutants resistant to analogs of lysine and
methionine yielded a much higher frequency of supe-
rior strains (Elander
&
Lowe, 1992). In a similar
maimer, Pospisil et al (1998) evaluated analog resis-
tant mutants of monensine over-producing strains of
Streptomyces cinnamonensis. When a secondary me-
tabolite like an antibiotic is itself a growth inhibitor,
the antibiotic can be used to select resistant cultures,
some of which are superior producers (Elander &
Vournakis, 1986).
Genetic recombination methods are represented by
sexual or parasexual crosses in fungi and conjugation
in actinomycetes. However, it is very often performed
by protoplast fusion in both organisms (Elander
&
Lowe 1992). This strategy becomes an important
complement to mutagenesis, once several independent
327
lineages of mutants have been established. It repre-
sents a means to construct strains with many different
combinations of mutations that influence production.
A situation where recombination (by protoplast fu-
sion) of related species of actinomycetes or related
species of fungi seems particularly attractive is when
one strain has been subjected to years of genetic de-
velopment and produces high levels of a SM, and the
other is a new isolate that produces low levels of a
new SM. The productivity of the newly identified SM
may be increased by generating recombinants from
the two strains.
Molecular Genetic Methods
To carry out these strategies, some biochemical and
molecular genetic tools, including identification of the
biosynthetic pathway, adequate vectors and effective
transformation protocols for the particular species
have to be developed or made available. After this,
the biosynthetic gene or genes have to be cloned and
analyzed. Molecular biology of actinomycetes and
fungi has been successfully developed to a degree that
its application to industrial strain improvement is now
a reality.
Genetic engineering methods have also provided
the tools to know in detail the nature of the modifica-
tions that have occurred during the decades of genetic
improvement of industrial strains (mainly by random
mutagenesis).
Characterization of high producing strains. The
genes responsible for antibiotic biosynthesis are
grouped together in clusters in most fungi and acti-
nomycetes. It has been found that in industrial peni-
cillin production strains, like P. chrysogenum AS-P- .
78 or P2, the cluster of penicillin biosynthetic genes is
amplified in a tandem array. In these strains, a DNA
region of -106.5 kb (containing these genes) has been
amplified between 5 and 7 times, while only one copy
is found in the original isolate (NRRL 1951). The se-
quence TTT ACA has been found flanking the ampli-
fied region, as well as linking the different copies. In
the much higher-producing industrial strain, P.
chrysogenum El, there are 12 to 14 copies of the bio-
synthetic cluster, being the size of the amplified re-
gion of only -57.9 kb, in this case (Fierro et al, 1995).
Penicillin production correlates well with the number
of copies of the biosynthetic genes present in them. It
indicates that this cluster amplification has been an
important factor in achieving the great production
increases during the long process of development (by
328 INDIAN J BIOTECHNOL, JULY 2003
mutation and selection) of these strains. But not the
only one, since the intermediate level producer, Wis-
consin 54-1255, displays a 15-20 fold higher produc-
tion than the wild type, but they both have just one
copy of the biosynthetic genes.
These and other findings have influenced the
strategies that are being used to apply genetic engi-
neering to strain improvement of antibiotics and other
SM producing strains.
Targeted duplication or amplification of SM pro-
duction genes. Although this method has not yet been
harnessed as a general method to improve product
yields, there are some encouraging reports, both in
actinomycetes and in fungi. This strategy can be di-
vided into two different approaches: targeted gene
duplication (or amplification) and whole pathway
amplification.
A prerequisite for the former is to identify the rate
limiting step in the biosynthetic pathway and to clone
the gene. Ideally, the first step would be to identify a
neutral site in the chromosome where genes can be
inserted without altering the fermentation properties
of the strain. Then the neutral site is cloned and incor-
porated into the vector with the antibiotic gene. In this
way, after transformation, the gene is inserted into the
chromosomal neutral site by homologous recombina-
tion (Baltz, 1998).
An example of the neutral site cloning was the tar-
geted duplication of the tylF gene that encodes the
rate limiting O-methylation of macrocin in the tylosin
biosynthesis in an industrial production strain of
Streptomyces fradiae. Transformants that contained
two copies of the tylF gene produced 60% more ty-
losin than the parental strain (Solenberg et al, 1996;
Baltz et al, 1997).
It is important to note that in many organisms, par-
ticularly industrial antibiotic producing fungi, ho-
mologous recombination is not a frequent event (or
not easy to achieve). In these cases the plasmid inte-
grates in different sites in the different transformants
obtained. However, a very simple screening for high
producers among them will indicate the cases where
the gene integrated in an adequate site of the chromo-
some.
With the development of genetic tools for fungi,
including more efficient transformation techniques,
first in Aspergillus nidulans (Yelton et al, 1984) and
later in Acremonium chrysogenum (Pefialva et al,
1985; Queener et al, 1985; Skatrud, 1987) and P.
chrysogenum (Beri
&
Turner, 1987; Cantoral et al,
1987; Sanchez et al, 1987), the gene amplification
effect was studied in these organisms.
Skatrud and coworkers (1989) successfully ampli-
fied the gene cejEF of the cephalosporin pathway in
A. chrysogenum. This caused a decrease in the inter-
mediate penicillin N and a 30% increase in cephalo-
sporin C production. Even better results (3 fold
cephalosporin C production increase) were obtained
when gene cejG (last step in the pathway) was ampli-
fied in A. chrysogenum ClO (Gutierrez et al, 1991).
Kennedy
&
Turner (1996), working with A. nidu-
lans, performed a variation of this strategy: promoter
replacement. That is, exchanging the gene's promoter
for a stronger and/or less regulated one, hence ob-
taining the same effect as with gene amplification.
They performed a promoter fusion to the first gene of
the pathway pcbAB resulting in a 30 fold increase in
penicillin yields. It is important to note, however, that,
penicillin production in this model organism is very
small compared with the strains of P. chrysogenum.
Integration of additional copies of the second or the
third gene of this three steps pathway, has not had an
important effect on penicillin yields (Barredo, 1990;
Fernandez, 1997). However, introduction of addi-
tional copies of these two genes together in the origi-
nal fragment caused a 40% increase in the penicillin
low producing strain P. chrysogenum Wis. 54-1255
(Veenstra et al, 1991). Recently, the introduction of
the complete penicillin cluster in the same strain was
studied. Transformants were isolated with production
increases of 124 to 176% (Theilgaard et al, 2001).
There are two reports of gene cluster amplifications
in actinomycetes leading to yield enhancements
(Gravius et al, 1994; Peschke et al, 1995).
Inactivation of competing pathways. Molecular ge-
netics also provides the means to block a pathway that
competes for a common intermediate, key precursors
such as cofactors, reducing power and energy supply.
Such strains could be able to channel the precursors to
the SM biosynthesis. This can be done by transposon
mutagenesis in actinomycetes, gene disruption or by
inserting an antisense synthetic gene.
o-aminoadipic acid, is one of the 3 amino acid pre-
cursors of penicillin biosynthesis, and it is also a
branching point, leading to the synthesis of lysine.
Disruption of gene lys2 of P. chrysogenum, which
connects n-aminoadipic towards lysine, has generated
auxotrophs of the amino acid that show 100% in-
crease in penicillin yields (Casqueiro et al, 1999). In
microorganisms where homologous recombination is
BARRIOS-GONZALEZ et al: MICROBIAL SECONDARY METABOLITES
not easy to achieve, such as P. chrysogenum, inacti-
vation of a gene could probably be done easier by
transforming with an antisense gene or an antisense
oligonucleotide.
Regulatory genes. A task much more complicated
than identifying the biosynthetic pathway and cloning
the corresponding genes is investigating its regulation
at a molecular level. However, the same molecular
genetics tools are allowing important advances in this
complicated field.
It is encouraging that the amplification of a regu-
latory gene (ccaR), required for cephamycin and cla-
vulanic acid production in Streptomyces clavuligerus,
results in a 3 fold overproduction of both industrial ~-
lactam compounds. (Perez-Llarena et al, 1997).
Moreover, disruption of negatively acting regulatory
gene mmy of methylenoomycin biosynthesis increased
production 17 fold, whereas introduction of a single
copy of the positively acting gene actII raised the
synthesis of actinorhodine 35-fold in Streptomyces
coelicolor (Hobbs et
al,
1992; Gramajo et aI, 1993;
Bibb, 1996). Research with actinomycetes is more
advanced in this area, where transposon mutagenesis
appears to be a useful procedure to identify (disrupt)
and clone regulatory genes (Solenberg
&
Baltz, 1994;
Baltz, 2001).
Basic knowledge on regulatory mechanisms will
also present the opportunity to delete negatively cis
acting regulatory elements in the promoter region, as
well as insertion of activating sequences.
Secretion mechanisms. This is another point now
under study with an important potential for molecular
strain improvement. In fact several protein-
hiperproducing yeast strains have been constructed by
increasing specific genes of the secretion path (like
genes kar2 and pdi1) or by disruption of genes like
pmr1.
Enhanced bipA (kar2 analogue in filamentous
fungi) mRNA levels have been observed in various
Aspergillus strains expressing recombinant extracel-
lular proteins. (Punt et aI, 1998; Sagt et al, 1998).
However, the correlation between BiP induction and
secretion efficiency remains unclear. pdiA genes, en-
coding protein disulphide isomerase, also are potential
targets for secretion pathway manipulation. Notice-
able differences in the Trichoderma reesei pdiA ex-
pression levels were observed under conditions sup-
porting high levels of protein secretion compared to
those supporting low levels of protein secretion (Sa-
loheimo et aI, 1999).
329
Unfortunately, the amplification of these genes in
Aspergillus niger has not succeeded in increasing het-
erologous proteins production in the fungus (Conesa
et aI, 2001).
Expression of heterologous enzyme activities. An
alternative strategy for strain improvement is to in-
corporate a new enzymatic activity in the strain (het-
erologous gene) that will lead to the formation of a
new related product of industrial interest. This could
only be obtained through a difficult and expensive
process of chemical synthesis. Transformation of
A. chrysogenum with a D-aminoacid oxidase of Fu-
sarium solani and a cephalosporin acylase from Pseu-
domonas diminuta, caused the direct synthesis of 7-
ACA, the substrate for the production of semi-
synthetic cephalosporins (Isogai et aI, 1991).
When an oxygen transporter bacterial protein,
similar to hemoglobin, was introduced in A. chryso-
genum, transformants were isolated with increased
cephalosporin C production yields (De Modena et
al,
1993).
Another example is the disruption of gene cejEF in
an industrial strain of A. chrysogenum, and the inte-
gration of the gene cefE from Streptomyces clavu-
ligerus. The transformants obtained could produce
great amounts of desacetoxicephalosporin C, product
that can easily be transformed into the other precursor
of semi-synthetic cephalosporins, 7-ADCA (Velasco
et aI, 2000).
Combinatorial biosynthesis. Another interesting
strategy is the development of novel antibiotics, pro-
duced by using non conventional compounds as sub-
strates of the biosynthetic enzymes of the micro-
organism. These enzymes can be modified or mutated
in such a way as to increase their affinity for those
unnatural substrates.
Generation of new antibiotics can also be per-
formed by the so called combinatorial biosynthesis. In
this case, different activity modules of enzymes like
polyketide synthases can be rearranged by genetic
engineering to obtain a microbial strain that synthe-
sizes an antibiotic with novel characteristics. An Eli
Lilly research group engineered Streptomyces toyo-
caensis, the producer of the non-glycosylated hepta-
peptide (similar to teicoplanin core) to produce hybrid
glycopeptides. They expressed the glycosyltransferase
genes from vancomycin- and chloroeremomycin-
producing strains of A. orientalis in this organism,
generating a novel monoglycosilated derivative (So-
lenberg et aI, 1997).
330 INDIAN J BIOTECHNOL, JULY 2003
Perspectives
It is visualized that discoveries of new antibiotics
and other SM, useful in the medical field as well as in
other productive activities, will continue at a fast rate,
driven by the new target-directed strategies to find
microbial SM. Generation of new SM will also be
performed by the so called combinatorial biosynthe-
sis. Economic production of these compounds will
depend on the fermentation production process and on
the application of adequate strain improvement meth-
ods.
Even though molecular genetic improvement is just
starting to become a practical reality, the next impor-
tant scientific and technological advance is already
appearing on the horizon, challenging researchers
imagination and creativity.
The end of the human genome project has liberated
a great technical potential for DNA sequencing. Part
of this capacity is now being directed to sequencing
the genomes of model microorganisms. The complete
genomes of E. coli, the yeasts Saccharomyces cere-
visiae and Schizosaccharomyces pombe, and of other
50 microorganisms, have already been sequenced;
while ,the sequencing of the fungi Aspergillus nidu-
lans and Neurospora crassa are in progress. After this
group, the turn is of microorganisms of industrial im-
portance. Moreover, the entire genomic sequences of
Streptomyces coelicolor and Streptomyces avermitilis
have very recently been published (Omura et
al,
2001;
Bentley et al, 2002).
Hence, the challenge is to apply this huge amount
of information to genetic improvement strategies and
methods (genomics).The knowledge (availability) of
the complete nucleotide sequence of a species, sup-
ported by the genomic sequence information and
functional annotations of many other microbial ge-
nomes, will enable us to identify all the genes present
in SM producing microorganism. This information
will facilitate metabolic reconstruction; that is the
prediction of the pathways (genes) associated with the
particular SM biosynthesis, like the synthetic pathway
itself, precursor biosynthesis, cofactors biosynthesis,
reducing power, regulatory circuits, etc. This infor-
mation could be useful in designing rational screens.
In a very modem approach to molecular genetics
strain improvement, this information will facilitate
rapid testing of the metabolic reconstruction predic-
tions by gene disruption analysis. Genes whose dis-
ruption causes a decrease in product yield should be
amplified. The inactivation of genes encoding for a
competing function or a negative regulatory element
should cause an increase in product titers. In this way,
genes that should be amplified and genes that should
be inactivated can be identified. On the other hand,
multiple transcript analysis by DNA micro arrays, of
different strains and environmental and physiological
conditions, will provide additional and complemen-
tary information about. the relevance of many genes.
In a near future, a number of genetic and molecular
genetics methods will be available to improve fer-
mentation product yields and other strain characteris-
tics. Some are effective and simple (like mutation and
selection), others are more expensive and sophisti-
cated and have been applied successfully in a few in-
dustrial cases, but with high theoretical potential. The
choice of approaches which should be taken will be
driven by the economics of the biotechnological proc-
ess, and the genetic tools available for the strain of
interest.
References
Agosin E et al, 1997. Production of gibberellins by solid substrate
cultivation of Gibberellafujikuroi. in Advances in Solid state
fermentation, edited by S Roussos, B K Lonsane, M Rairn-
bault
&
G Viniegra-Gonzalez. K1uwer Academic Publish-
ers,The Netherlands. Pp 355-366.
Balakrishnan K
&
Pandey A, 1996. Production of biologically
active secondary metabolites in solid state fermentation. JSci
Ind Res, 55, 365-372.
Baltz R H, 1997. Molecular genetic approaches to yield improve-
ment in actimomycetes. Drug Pharm Sci, 82, 49-62.
Baltz R H, 1998. New genetics methods to improve secondary
metabolite production in Streptomyces. JInd Microbiol Bio-
technol, 20, 360-363.
Baltz R H, 1999. Mutagenesis. in Encyclopedia of Bioprocess
technology: Fermentation, biocatalysis and separation, edited
by M C Flickinger
&
S W Drew. Wiley, New York. Pp 307-
311.
Baltz R H, 2001. Genetics methods and strategies for secondary
metabolite yield improvement in actinomycetes. Avan
Leeuwenhoek, 79, 251-259.
Bandelier S et al, 1997. Production of gibberellic acid by fed
batch solid state fermentation in an aseptic pilot scale reactor.
Proc Biochem, 32,141-145.
Barredo, 1990. Analisis de una regi6n del genoma de Penicillium
chrysogenum que contiene los genes pcbC ypenDE. Ph D
Thesis. University of Le6n, Spain.
Barrios-Gonzalez J
&
Mejia A, 1996. Production of secondary
metabolites by solid-state fermentation. Biotechnoi Annu
Rev, 2, 85-121.
Barrios-Gonzalez J et
al,
1988. Penicillin production by solid state
fermentation. Biotechnoi Leu, 10,793-798.
Barrios-Gonzalez J et
al,
1993a. Development of high penicillin
producing strains for solid-state fermentation. Biotechnoi
Adv, 10, 793-798.
BARRIOS-GONZALEZ et al: MICROBIAL SECONDARY METABOLITES
Barrios-Gonzalez, J et al, 1993b. Penicillin production by mutants
resistant to phenylacetic acid. JFerm Bioeng, 76, 455-458.
Barrios-Gonzalez J et al,2oo3. Production of Antibiotics. in Con-
cise Encyclopedia of Bioresource Technology, edited by
Ashok Pandey. The Haworth Press Inc., Binghamton, New
York (in press).
Bentley S D et al, 2002. Complete genome sequence of the model
actinomycete Streptomyces coelicolor A3(2). Nature(Lond),
417, 141-147.
Beri R K
&
Turner G, 1987. Transformation of Penicillium
chrysogenum using the Aspergillus nidulans amdS gene as a
dominant selective marker. Curr Genet, 11,6390-6411.
Bibb M, 1996. Colworth Prize Lecture. The regulation of antibi-
otic production in Streptomyces coelicolor A(3)2. Microbiol-
ogy, 142, 1335-1344.
Cantoral J Met al, 1987. High frequency transformation of Peni-
cillium chrysogenum. Bio/Technology, 5,494-497.
Casqueiro J et al, 1999. Gene targeting in Penicillium chryso-
genum: Disruption of the lys2 gene leads to penicillin over-
production. JBacteriol, 181, 1181-1188.
Conesa A et al, 2001. The secretion pathway in filamentous fungi:
A biotechnological view. Fungal Genet Bioi, 33,155-171.
DeModena J A et
al,
1993. The production of cephalosporin C by
Acremonium chrysogenum is improved by the intracellular
expression of a bacterial hemoglobin. Bio/Technology, 11,
926-929.
Dominguez M et
al,
2000. Respiration studies of penicillin solid-
state fermentation. JBiosci Bioeng, 89,409-413.
Dominguez M et
al,
2001. Optimization of bagasse, nutrients and
initial moisture ratios on the yields of penicillin in solid-state
fermentation. World JMicrobiol Biotechnol, 17, 751-756.
Elander R P
&
Vournakis J N, 1986. Enetic aspects of overpro-
duction of antibiotics and other secondary metabolites. in
Overproduction of Microbial metabolites, edited by Z Va-
neck
&
Z Hostalek. Butterworth, Boston. Pp 63-79.
Elander R P
&
Lowe D A, 1992. Fungal Biotechnology: An over-
view. in Handbook of Applied Mycology, Vol. 4, edited by
D K Arora, R P Elander
&
K G Mukerji. Marcel Dekker,
New York. Pp 1-34.
Fernandez F J, 1997. Caracterizacion de la expresion de los genes
implicados en la biosfntesis de penicilina: Reaccion en-
zimatica catalizada por el producto del ultimo gen (penDE)
de la ruta biosintetica de penicilina. Ph D Thesis. University
of Leon, Spain.
Fierro F et al, 1995. The penicillin gene cluster is amplified in
tandem repeats linked by conserved hexanuc\eotide se-
quences. Proc Natl Acad Sci USA, 92, 6200-6204.
Gramajo H C, et
al,
1993. Stationary-phase production of the
antibiotic actinorhodin in Streptomyces coelicolor A3(2) is
transcriptionally regulated Mol Microbiol,7, 837-845.
Gravius B et al, 1994. The 387 kb linear plasmid of Streptomyces
rimosus and the interactions with the chromosome. Microbi-
ology, 140, 2271-2277.
Gutierrez S et al, 1991. Expression of the penDE gene of Penicil-
lium chrysogenum encoding isopenicillin N acyltransferase
in Cephalosporium acremonium: production of benzylpeni-
cillin by the transformants. Mol Gen Genet, 225, 56-64.
331
Hardin M T et al, 2000. Approach to designing rotating drum
bioreactors for solid-state fermentation on the basis of di-
mensionless design factors. Biotechnol Bioeng, 67, 274-282.
Hesseltine C W, 1977a. Solid-state fermentation Part 1. Proc Bio-
chem, 12, 24-27.
Hesseltine C W, 1977b, Solid-state fermentation Part 2. Proc
Biochem, 12, 29-32
Hobbs G et
al,
1992. An integrated approach to studying regula-
tion of production of the antibiotic methylenomycin by
Streptomyces coelicolor A3(2). JBacteriol, 174, 1487-1494.
Isogai T et al, 1991. Construction of a 7-aminocephalosporanic
acid (7ACA) biosynthetic operon and direct production of
7ACA in Acremonium chrysogenum. Bio/Technology, 9,
188-191.
Jermini M F G
&
Demain A L, 1989. Solid state fermentation for
cephalosporin production by Streptomyces clavuligerus and
Cephalosporium acremonium. Experientia, 45,1061-1065.
Junter B et
al,
2002. Use of frozen bagged seed inoculum for sec-
ondary metabolites and biocoversion processes at the pilot
scale. Biotechnol Bioeng, 79, 628-640.
Kennedy J
&
Turner G, 1996. 8(L-a-aminoadipyl)-L-cysteinyl-D-
valine synthetase is a rate limiting enzyme for penicillin pro-
duction in Aspergillus nidulans. Mol Gen Genet, 253, 189-
197.
Khetan A et
al,
1999. Precursor and cofactor as a check valve for
cephamycin biosynthesis in Streptomyces clavuligerus. Bio-
technol Prog, 15, 1020-1027.
Kota K P
&
Sridhar P, 1998. Solid state cultivation of Streptomy-
ces clavuligerus for producing cephamycin C. JSci Ind Res,
57,587-590.
Kumar P K R
&
Lonsane B K, 1987a. Gibberellic acid by SSF:
consistent and improvement yields. Biotechnol Bioeng, 30,
267-271.
Kumar P K R
&
Lonsane B K, 1987b. Extraction of gibberellic
acid from dry mouldy bran produced under solid-state fer-
mentation. Proc Biochem Biotechnol, 33, 139-143.
Lonsane B K et al, 1985. Engineering aspects of solid-state fer-
mentation. Enzyme Microbiol Technol, 7, 258-265.
Lotong N
&
Suwanarit P, 1990. Fermentation of ang-kak in plas-
tic bags and regulation of pigmentation by initial moisture
content. JAppl Bacteriol, 68, 565-570.
Miranda L
0
et al,2oo3. Efecto del mezc\ado, la adicion de agua y
lactosa en la produccion de penicilina por Penicillium
chrysogenum en fermentacion en estado solido usando un
biorreactor dinamico. Informacion Tecnological, 14(l)(in
press).
Mitchell D A et al, 2000. New developments in solid-state fer-
mentation. II Rational approaches to the design, operating
and scale-up of bioreactors. Proc Biochem, 35, 1211-1225.
Mitchell D A et al, 2002. The potential for establishment of axial
temperature profiles during solid-state fermentation in rotat-
ing drum bioreactors. Biotechnol Bioeng, 80, 114-122.
Nagel F J et al, 2001 Temperature control in a continuously mixed
bioreactor. Biotechnol Bioeng, 72, 219-230.
Nagel F J et al, 2002 Simultaneous control of temperature and
moisture content in a mist bioreactor for solid-state fermen-
tation. Biotechnol Bioeng (in press)
332 INDIAN J BIOTECHNOL, JULY 2003
Ohno A et
al,
1993. Production of the antifungal peptide antibi-
otic, iturin, by Bacillus subtilis N B22. Biotechnol Lett, 14,
1165-1168.
Ohno A et
al,
1995. Production of a lipopeptide antibiotic, surfac-
tin, by recombinant Bacillus subtilis in solid state fermenta-
tion. Biotechnol Bioeng, 47, 209-213.
Ohno A et
al,
1996. Use of soybean curd residue, okra, for solid
state substrate in the production of a lipopeptide antibiotic,
iturin A, by Bacillus subtilis NB22. Proc Biochem, 16, 483-
489.
Omura S et al, 2001. Genome sequence of an industrial microor-
ganism Streptomyces avermitilis: deducing the ability of pro-
ducing secondary metabolites. Proc Natl Acad Sci USA, 98,
12215-12220.
Pandey A P et
al,
1999. Solid state fermentation for the produc-
tion of industrial enzymes. Curr Sci, 77, 149-162.
Pandey A P et al, 2000. New developments in solid state fermen-
tation: l-Bioprocesses and products. Proc Biochem, 35, 1153-
1169.
Parekh S et
al,
2000. Improvement of microbial strains and fer-
mentation processes. Appl Microbiol Biotechnol, 54, 287-
301.
Peberdy J F, 1985. Biology of Penicillins. in Biology of Industrial
Microorganisms, edited by A Demain
&
N Solomon. Benja-
min Cummings, Menlo Park. Pp 407-431.
Pefialva M A et
al,
1985. Studies on transformation of Cephalo-
sporium acremonium. in Molecular Genetics of Filamentous
Fungi, edited by W E Timberlake.
&
Alan R. Liss, New
York. P 59.
Perez-Llarena F Jet al, 1997. A regulatory gene (ccaR) required
for cephamycin and clavulanic acid production in Streptomy-
ces clavuligerus: amplification results in overproduction of
both beta-Iactarn compounds. JBacteriol, 179,2053-2059.
Peschke U et al, 1995. Molecular characterization of the lincomy-
cin-production gene cluster of Streptomyces lincolnensis 78-
11. Mol Microbiol, 16, 1137-1158.
Pospisil S et al, 1998. Depression and altered feedback regulation
of valine biosynthesis pathway in analogue-resistant mutants
of Streptomyces cinnamonensis resulting in 2-ketoisovalerate
excretion. JAppl Microbiol, 85, 9-16.
Punt P J et al, 1998. Analysis of the role of the gene bipA, en-
coding the major endoplasmic reticulum chaperone protein in
the secretion of homologous and heterologous proteins in
black Aspergilli. Appl Microbiol Biotechnol, 50, 447-454.
Queener S W et
al,
1985. A system for genetic transformation of
Cephalosporium acremonium. in Microbiology, edited by L
Lieve. American Society for Microbiology, Washington DC.
Pp 468-472.
Ramana M M V et
al,
1999. Cyclosporin A production by Toly-
pocladium infiatum using solid state fermentation. Proc
Bio-
chern, 34, 269-280.
Robinson T et
al,
2001. Solid-state fermentation: a promising
microbial technology for secondary metabolite production.
Appl Microbiol Bitechnol, 55, 284-289.
Rosenblitt A et al, 2000. Solid substrate fermentation of Monas-
cus purpureus; Growth, carbon balance and consistency
analysis. Biotechnol Prog, 16, 152-162.
Rowlands R T, 1984. Industrial strain improvement: mutagenesis
and random screening procedures. Enzyme Microbiol Tech-
nol, 6, 3-10.
Sagt C M et al, 1998. Impaired secretion of a hydrophobic cu-
tinase by Saccharomyces cerevisiae correlates with an in-
creased association with immunoglobulin heavy-chain bind-
ing protein (BiP). Appl Environ
Microbiol,
64, 316-324.
Saloheimo M et
al,
1999. The protein disulphide isomerase gene
of the fungus Trichoderma reesei is induced by endoplasmic
reticulum stress and regulated by the carbon source. Mol Gen
Genet, 262, 35-45.
Sanchez F et al, 1987. Transformation in Penicillium chryso-
genum. Gene, 51,97-102
Schroeder W A
&
Johnson E A, 1995a. Singlet oxygen and per-
oxyl radicals regulate carotenoid biosynthesis in Phaffia rho-
dozyma. JBioi Chern, 270, 18374-18379.
Schroeder W A
&
Johnson EA, 1995b. Carotenoids protect
Phaf-
fia rhodozyma against singlet oxygen damage.
J
Ind Micro-
bioi, 14, 502-507.
Sekar C
&
Balaraman K, 1998. Optimization studies of the pro-
duction of cyclosporine A by solid state fermentation. Bioi
Eng, 18, 293-296.
Serrano-Carreon L et
al,
1992. A simple protocol for extraction
and detection of pyrazines produced by fungi in solid state
fermentation. Biotechnol Tech, 6,19-22.
Shankaranand V S et al, 1992. Idiosyncrasies of solid-state fer-
mentation system in the biosynthesis of metabolites by some
bacterial and fungal cultures. Proc Biochem, 27, 33-36.
Skatrud P L et
al,
1987. Efficient integrative transformation of C.
acremonium. Curr Genet, 12,337-348.
Skatrud P L et
al,
1989. Use of recombinant DNA to improve
production of cephalosporin C by Cephalosporium acremo-
nium. BiolTechnology, 7,477-485.
Solenberg P J
&
Baltz R H, 1994. Hyper-transposing derivatives
of the streptomycete insertion sequence IS493. Gene, 147,
47-54.
Solenberg P J et
al,
1996. Transposition mutagenesis in Strepto-
myces fradiae: Identification of a neutral site for the stable
insertion of DNA by transposon exchange. Gene, 168, 67-72.
Solenberg P J et al, 1997 Production of hybrid glycopeptide anti-
biotics in vitro and in Streptomyces toycaensis. Chern. Bioi,
4, 195-202.
Suryanarayan S, 2001. Solid State Fermentation. US Pat
6197573BI. 6 March 2001.
Suryanarayan S, 2002. Current industrial practice in solid matrix
fermentations for secondary metabolite production. The Bio-
con India Experience. Biochem Eng Jof Solid State Ferm. (in
press).
Theilgaard H et
al,
2001. Quantitative analysis of Penicillium
chrysogenum Wis54-1255 transformants overexpressing the
penicillin biosynthetic genes. Biotechnol Bioeng, 72, 379-
388.
Tomasini A et al, 1997. Gibberellic acid production using differ-
ent solid state fermentation systems. World JMicrobiol Bio-
technol, 13, 203-206.
Trejo H M R et al, 1993. Spectra of ergot alkaloids produced by
Claviceps purpurea 1029C in solid state fermentation sys-
tem: Influence of the composition of liquid medium used for
BARRIOS-GONZALEZ et al: MICROBIAL SECONDARY METABOLITES
impregnating sugar cane pith bagasse. Proc Biochem, 28, 23-
27.
Veenstra A E et al, 1991. Strain improvement of Penicillium
chrysogenum by recombinant DNA techniques. JBiotechnol,
17,81-90.
Velasco J et al, 2000. Environmentally safe production of 7-
aminodeacetoxycephalosporanic acid (7-ADCA) using re-
combinant strains of Acremonium chrysogenum. Nat Bio-
technol, 18, 857-861.
Vinci V
&
Byng G, 1999. Strain improvement by non-
recombinant methods. in Manual of Industrial Microbiology
and Biotechnology, edited by A L Demain
&
JE Davies.
American Society for Microbiology, Washington DC, Pp
103-113.
333
Wang H H et al, 1984. Cephalosporin production by SSF of rice
grains. JMicrobiollmmunol, 17, 55-69.
Yang S S
&
Ling MY, 1989. Tetracycline production with sweet
potato residue by solid state fermentation. Biotechnol Bio-
eng, 33, 1021-1028.
Yang S S
&
Wang J Y, 1996. Morphegenesis, ATP content and
oxytetracycline production by Streptomyces rimosus in solid
substrate cultivation.
J
Appl Bacteriol, 80, 545-550.
Yang S S
&
Yuan S S, 1990. Oxytetracycline production by
Streptomyces rimosus in solid state fermentation of sweet
potato residue. WJMicrobiol Biotechnol, 6, 236-244.
Yelton MM et al, 1984. Transformation of Aspergilllus nidulans
by using a trpC plasmid. Proc Natl Acad Sci USA, 81, 1470-
1474.
