Genetic improvement of processes yielding microbial products
Jose L. Adrio1 & Arnold L. Demain2
1
Department of Biotechnology, Puleva Biotech, S.A., Granada, Spain; and 2Charles A. Dana Institute for Scientists Emeriti, Drew University, Madison, NJ, USA
Correspondence: Jose L. Adrio, Department
of Biotechnology, Puleva Biotech, S.A.,
Camino de Purchil, 66, 18004 Granada,
Spain. Tel.:134 958 24 02 27; fax:134 958
24 01 60; e-mail: jladrio@pulevabiotech.es
Received 3 March 2005; revised 18 August
2005; accepted 19 August 2005
First published online 17 October 2005.
Although microorganisms are extremely good in presenting us with an amazing
array of valuable products, they usually produce them only in amounts that they
need for their own benefit; thus, they tend not to overproduce their metabolites. In
strain improvement programs, a strain producing a high titer is usually the desired
goal. Genetics has had a long history of contributing to the production of
microbial products. The tremendous increases in fermentation productivity and
the resulting decreases in costs have come about mainly by mutagenesis and
screening/selection for higher producing microbial strains and the application of
recombinant DNA technology.
Editor: Alexander Boronin
Keywords
strain improvement; genetic recombination;
primary metabolites; secondary metabolites;
directed evolution; combinatorial biosynthesis.
Introduction
Microorganisms can generate new genetic characters (‘genotypes’) by two means: mutation and genetic recombination. In
mutation, a gene is modified either unintentially (‘spontaneous mutation’) or intentially (‘induced mutation’).
Although the change is usually detrimental and eliminated by
selection, some mutations are beneficial to the microorganism. Even if it is not beneficial to the organism, but beneficial
to humans, the mutation can be detected by screening and can
be preserved indefinitely. This is indeed what the fermentation
microbiologists did in the strain development programs that
led to the great expansion of the fermentation industry in the
second half of the twentieth century.
It was fortunate that the intensive development of microbial genetics began in the 1940s when the fermentative
production of penicillin became an international necessity.
The early studies in basic genetics concentrated on the
production of mutants and their properties. The ease with
which ‘permanent’ characteristics of microorganisms could be
changed by mutation and the simplicity of the mutation
techniques had tremendous appeal to microbiologists. Thus
began the cooperative ‘strain-selection’ program among workers at the U.S. Department of Agriculture Laboratories in
Peoria, the Carnegie Institution, Stanford University and the
University of Wisconsin, followed by the extensive individual
FEMS Microbiol Rev 30 (2006) 187–214
programs that still exist today in industrial laboratories
throughout the world. It is clear that mutation has been the
major factor involved in the hundred- to thousand-fold
increases obtained in production of microbial metabolites
and that the ability to modify genetically a microbial culture
to higher productivity has been the most important factor in
keeping the fermentation industry in its viable, healthy state.
Applications of mutation
Mutation has improved the productivity of industrial cultures (Vinci & Byng, 1999; Parekh et al., 2000). It has also
been used to shift the proportion of metabolites produced in
a fermentation broth to a more favorable distribution,
elucidate the pathways of secondary metabolism, yield new
compounds, and for other functions. The most common
method used to obtain high yielding mutants is to treat a
population with a mutagenic agent until a certain ‘desired’
kill is obtained, plate out the survivors and test each
resulting colony or a randomly selected group of colonies
for product formation in flasks. The most useful mutagens
include nitrosoguanidine (NTG), 4-nitroquinolone-1oxide, methylmethane sulfonate (MMS), ethylmethane sulfonate (EMS), hydroxylamine (HA) and ultraviolet light
(UV). The optimum level of kill for increased production of
antibiotics is thought to be in the range 70–95% (Simpson &
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doi:10.1111/j.1574-6976.2005.00009.x
Abstract
188
Mutants producing increased quantites of
metabolites
Genetics has had a long history of contributing to the production of microbial products. The tremendous increases in
fermentation productivity and the resulting decreases in costs
have come about mainly by mutagenesis and screening for
higher producing microbial strains. At least five different classes
of genes control metabolite production (Malik, 1979): (i)
structural genes coding for product synthases, (ii) regulatory
genes determining the onset and expression of structural genes,
(iii) resistance genes determining the resistance of the producer
to its own antibiotic, (iv) permeability genes regulating entry,
exclusion and excretion of the product, and (v) regulatory
genes controlling pathways providing precursors and cofactors.
Overproduction of microbial metabolites is effected by (i)
increasing precursor pools, (ii) adding, modifying or deleting
regulatory genes, (iii) altering promoter, terminator and/or
regulatory sequences, (iv) increasing copy number of genes
encoding enzymes catalyzing bottleneck reactions, and (v)
removing competing unnecessary pathways (Strohl, 2001).
It is now over 60 years since the first superior penicillinproducing mutant, Penicillium chrysogenum X-1612, was
isolated afer X-ray mutagenesis. This heralded the beginning
of a long and successful relationship between mutational
genetics and industrial microbiology (Hersbach et al., 1984).
The improvement of penicillin production by conventional
strain improvement resulted both from enhanced gene
expression and from gene amplification (Barredo et al.,
1989; Smith et al., 1989). Increased levels of mRNA corresponding to the three enzymes of penicillin G biosynthesis
were found in high-penicillin producing strains of P. chrysogenum as compared to wild-type strains (Smith et al.,
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1990). High-producing strains contained an amplified region; a 106-kb region amplified five to six times as tandem
repeats was detected in a high-producing strain, whereas
wild-type P. chrysogenum and Fleming’s original strain of
P. notatum contained only a single copy (Fierro et al., 1995).
Strain improvement has been the main factor involved in
the achievement of impressive titers of industrial metabolites.
The production titer of tetracycline as far back as 1979 was
reported to be over 20 g L 1 (Podojil et al., 1984), mainly due
to strain improvement. Later, titers of 30–35 g L1 were
reached for chlortetracycline and tetracycline. The production
titer of penicillin is 70 g L 1 and that of cephalosporin C over
30 g L 1 (Elander, 2003). The production titer of tylosin has
been reported to be over 15 g L1 (Chen et al., 2004) and that
of salinomycin is 60 g L 1 (Liu, 1982).
Morphological and pigment mutants
Although almost nothing is known about the mechanisms
causing higher production in superior random or morphological mutants, it is likely that many of these mutations involve
regulatory genes, especially as regulatory mutants obtained in
basic genetic studies are sometimes found to be altered in
colonial morphology. Thus, morphological mutants have been
very important in strain improvement. These include mutants
affected in mycelia formation, which produce colonies with a
modified appearance or a new color. Color changes have also
been important for pigment producers (Table 1).
Auxotrophic mutants
Very early in the development of the concepts of regulation,
geneticists realized that the end product of a biosynthetic
pathway to a primary metabolite excercises strict control
over the amount of an intermediate accumulated by an
auxotrophic mutant of that pathway. Only at a growthlimiting concentration of the end product would a large
accumulation of the substrate of the deficient enzyme occur.
This principle of decreasing the concentration of an inhibitory or repressive end product to bypass feedback inhibition
or repression was best accomplished by the use of auxotrophic mutants. Indeed, auxotrophic mutation has been a
major factor in the industrial production of primary products such as amino acids and nucleotides (Table 1). The
production of secondary products such as antibiotics is also
markedly affected by auxotrophic mutation, even when
auxotrophs are grown in nutritionally complete and even
complex media. Although the change in product formation
is usually in the negative direction, higher-producing auxotrophs are obtained from producers of antibiotics.
When several primary metabolites are produced by a
single branched pathway, mutation in one branch of the
pathway often leads to overproduction of the product of the
FEMS Microbiol Rev 30 (2006) 187–214
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Caten, 1979), although some industrial programs use much
higher levels, e.g. up to 99.99%. It is incorrect to condemn a
mutation and screening procedure because, on average, it
decreases production ability; indeed, this is the case for
successful mutagenesis. One should only be interested in the
frequency of improved mutants.
Although single cells or spores are preferred for mutagenesis, non-spore-forming filamentous organisms have been
mutated successfully by mutagenizing mycelia, preparing
protoplasts and regenerating on solid medium (Keller,
1983). Sonication is sometimes used to break up Streptomyces mycelia after mutagenesis and before screening for
improved mutants (Takebe et al., 1989). Poorly sporulating
filamentous organisms can be mutagenized after fragmentation or formation of protoplasts (Kim et al., 1983; Kurzatkowski et al., 1986).
More detailed information can be found in several authorative reviews on genetics and especially on mutation in actinomycetes (Baltz, 1986, 1995, 1998, 1999; Hopwood, 1999).
J. L. Adrio & A. L. Demain
189
Genetic improvement of processes yielding microbial products
Table 1. Mutations leading to increased product formation
Mutation type
Organism
5.7. Ashbya gossypii
5.8. Penicillium
chrysogenum
5.9. Amycolatopsis
mediterranei
6. Product resistance
6.1 Streptomyces
goldiniensis
6.2. Nocardia
uniformis
6.3. Streptomyces
kitasatoensis
6.4. Streptomyces
rimosus
7. Antibiotic resistance 7.1 Streptomyces
coelicolor and
Streptomyces lividans
FEMS Microbiol Rev 30 (2006) 187–214
Overproduced
compound
Reference
Wrinkled colonies
Histidine
Roth & Ames (1966)
‘Bald colonies’; then white colonies
Daunorobicin
Blumauerova et al. (1978)
Reddish-orange colonies with no
aerial mycelia
Pink colonies on agar containing
b-ionone or diphenylamine
Pink instead of brown mycelia
Beromycins
Blumaerova et al. (1980, 1980)
Astaxanthin
Lewis et al. (1990);
Chumpolkulwong et al. (1997)
Lee et al. (2003)
Guanine auxotrophy
Leucine auxotrophy
Leucine auxotrophy
Teichoplanins
5 0 -Inosinic acid (IMP)
and hypoxanthine
Bacitracin
Teshiba & Furuya (1983)
Haavik & Froyshov (1982)
Non-auxotrophic for aspartate
Cephamycin C and
penicillin N
Tylosin
Godfrey (1973)
Lee & Lee (1995)
Producing ability
Chlortetracycline
Dulaney & Dulaney (1967)
Producing ability
Aurodox
Unowsky & Hoppe (1978)
Resistance to ethionine
Methionine
Tani et al. (1988)
Resistance to thialysine
Cephamycins
Resistance to thialysine
Desferrioxamine
Mendelovitz
& Aharonowitz (1983)
Smith (1987)
Resistance to 2-ketobutyrate in
presence of valine or isoleucine
Resistance to valine hydroxamate
Monensins A & B
Pospisil et al. (1999)
Teichoplanins
Resistance to iron, to tubercidin,
to 2-DOG
Resistance to itaconic acid and
aminomethylphosphinic acid
Resistance to phenylacetic acid
(precursor)
Sequential resistance to tryptophan
(feedback inhibitor), phydroxybenzoate, and propionate
(precursor)
Resistance to aurodox
Riboflavin
Wang et al. (1996);
Jin et al. (2002a)
Stahmann et al. (2000)
Riboflavin
Stahmann et al. (2000)
Penicillin G
Barrios-Gonzalez et al. (1993)
Rifamycin B
Jin et al. (2002)
Aurodox
Unowsky & Hoppe (1978)
Resistance to nocardicin
Nocardicin
Elander & Aoki (1982)
Resistance to leucomycin
Leucomycin
Higashide (1984)
Resistance to oxytetracycline
Oxytetracycline
Gravius et al. (1994)
Resistance to streptomycin,
gentamicin, paromomycin,
rifamycin and combinations
Actinorhodin
Hosoya et al. (1998); Hesketh
& Ochi (1997); Okamoto et al.
(2003); Okamoto-Hosoya et al.
2000; Hu & Ochi (2001)
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1. Morphological and/ 1.1. Salmonella
or pigment change typhimurium
1.2. Streptomyces
coeruleorubidus
1.3. Streptomyces
glomeratus
1.4. Phaffia
rhodozyma
1.5. Actinoplanes
teichomyceticus
2. Auxotrophic
2.1. Brevibactetrium
ammoniagenes
2.2. Bacillus
licheniformis
2.3. Streptomyces
lipmanii
3. Reversion of
3.1. Streptomyces
auxotrophy
fradiae
4. Reversion of
4.1. Streptomyces
non-production
viridifaciens
4.2. Streptomyces
goldiniensis
5. Antimetabolite
5.1. Candida boidinii
resistance
5.2. Streptomyces
clavuligerus
5.3. Streptomyces
pilosus
5.4. Streptomyces
cinnamonensis
5.5. Actinoplanes
teichomyceticus
5.6. Candida flareri
Mutant characteristics
190
J. L. Adrio & A. L. Demain
Table 1. Continued.
Overproduced
compound
Organism
Mutant characteristics
8. Reversal of carbon
source repression
8.1. Saccharomyces
cerevisiae
8.2. Schwanniomyces
castelli
8.3. Pichia
polymorpha
8.4. Penicillium
chrysogenum
8.5 Aspergillus niger
8.6. Aspergillus niger
9.1. Streptomyces
aureofaciens
9.2. Streptomyces
griseus
10.1. Acremonium
chrysogenum
10.2. Streptomyces
viridifaciens
10.3. Bacillus subtilis
10.4. Aspergillus
nidulans
10.5. Aspergillus niger
10.6. Rhyzopus oryzae
10.7. Streptomyces
kasugaensis
10.8. Acremoniums
chrysogenum
10.9. Streotomyces
hygroscopicus
11.1. Brevibacterium
flavum
11.2. Brevibacterium
ammoniagenes
11.3. Escherichia coli
11.4. Corynebacterium
glutamicum
Resistance to 2-deoxyglucose
9. Reversal of
phosphate inhibition
10. Increased
production on agar
11. Change in
permeability
Resistance to 2-deoxyglucose
Cheese whey
hydrolysis
Isomaltase, amylase
Resistance to 2-deoxyglucose
Inulinase
McCann & Barnett (1984);
Sills et al. (1984)
Bajon et al. (1984)
Resistance to 2-deoxyglucose
Penicillin G
Chang et al. (1980)
Rapid growth on high sucrose
Resistance to 2-deoxyglucose
Small colonies on phosphate-limiting
agar
Production in excess-phosphate
medium
Increased clear zone around colony
Citric acid
Citric acid
Tetracycline
Schreferl-Kunar et al. (1989)
Kirimura et al. (1992)
Colombo et al. (1981)
Candicidin
Martin et al. (1979)
Cephalosporin C
Elander (1969)
Increased clear zone around colony
Chlortetracycline
Dulaney & Dulaney (1967)
Increased clear zone around colony
Increased clear zone around colony
Mycobacillin
Penicillin
Bannerjee & Bose (1964)
Ditchburn et al. (1974)
Increased clear zone around colony
Increased clear zone around colony
Increased clear zone around plugs
of agar
Increased clear zone around plugs
of agar
Increased clear zone around plugs
of agar
Inability to grow on glutamate
Citric acid
Lactate
Kasugamycin
Das & Roy (1981)
Longacre et al. (1997)
Ichikawa et al. (1971)
Cephalosporin C
Chang & Elander (1979)
Complex ‘165’
Gesheva (1994)
Glutamic acid
5 0 -Inosinic acid
Shiio et al. (1982); Mori
& Shiio (1983)
Teshiba & Furuya (1983)
Proline
Tryptophan
Rancount et al. (1984)
Ikeda & Katsumata (1995)
Increased sensitivity to deoxycholate
and lysozyme
Elimination of active praline uptake
Decrease in tryptophan uptake
other branch. Examples include the overproduction of
phenylalanine by tyrosine auxotrophs and vice versa, and
the overproduction of lysine by auxotrophs requiring threonine and methionine. In the case of branched pathways
leading to a primary metabolite and a secondary metabolite,
auxotrophic mutants requiring the primary metabolite
sometimes overproduce the secondary metabolite (Table 1).
Reversion of an auxotroph to prototrophy sometimes
leads to new prototrophs possessing higher enzyme activity
than present in the original ‘grandparent’ prototroph. Such
increased enzyme activity was probably the result of a
structural gene mutation producing a more active enzyme
or an enzyme less subject to feedback inhibition (Table 1).
Revertants of non-producing mutants
A high proportion of such mutants has been found to
produce increased amounts of antibiotics (Table 1).
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Reference
Bailey et al. (1982)
Antimetabolite-resistant mutants
Basic studies on regulation have shown that it is possible to
select regulatory mutants, which overproduce the end
products of primary pathways, using toxic metabolite analogues. Such antimetabolite-resistant mutants often possess
enzymes that are insensitive to feedback inhibition, or
enzyme-forming systems resistant to feedback repression.
The selection of mutants resistant to toxic analogues of
primary metabolites has been widely employed by industrial
microbiologists (Table 1).
A variation of the antimetabolite selection techniques is
possible when a precursor is toxic to the producing organism. The principle here is that the mutant most capable of
detoxifying the precursor by incorporating it into the
antibiotic will be the best grower in the presence of the
precursor (Table 1). When the secondary metabolite
FEMS Microbiol Rev 30 (2006) 187–214
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Mutation type
191
Genetic improvement of processes yielding microbial products
produced is itself a growth inhibitor of the producing
culture, as in the case of certain antibiotics, the metabolite
can sometimes be used to select resistant mutants that are
improved producers.
Certain streptomycin resistance mutations cause increased production of unrelated antibiotics. In addition to
improvement in secondary metabolite formation by mutation to streptomycin resistance, resistance to gentamicin or
to paromomycin is even more effective. Furthermore,
triple mutation to resistance to streptomycin, gentamicin
and rifampicin, each of which individually increased actinorhodin formation, was found to be the most effective
(Table 1).
Mutants resistant to nutritional repression
Improved production on agar
In many cases, fermentation performance on an agar plate is
related to production in submerged liquid culture, and the
method has application as a means of detecting superior
mutants. So-called ‘zone mutants’ have proven useful for
improving several different processes (Table 1).
A widely used modification involves the production of
antibiotics by colonies on separate plugs of agar followed by
placement of these plugs on a seeded assay plate and
measurement of the resultant clear zones. The use of this
‘agar piece method’ resulted in improvement of antibiotic
production (Table 1). Agar-piece screening of antibiotic
production in the presence of inhibitory levels of phosphate
(15 mM) led to the isolation of six markedly improved and
stable Streptomyces hygroscopicus strains producing the
macrolide antifungal complex ‘165’ (Gesheva, 1994).
Permeability mutants
Product excretion in overproducing strains often occurs
when uptake and/or catabolism is impaired. Thus, genetic
lesions eliminating active uptake can be used to specifically
enhance excretion of metabolites (Table 1). It is often of
benefit to isolate mutants unable to grow on the
FEMS Microbiol Rev 30 (2006) 187–214
Mutants showing qualitative changes in the mix
of fermentation products
As many organisms produce secondary metabolites as
mixtures of a chemical family or of several chemical families,
mutation has been used to eliminate undesirable products in
such fermentations. An example is that of Nakatsukasa and
Mabe (Nakatsukasa & Mabe, 1978), who found that streaking out a natural single colony isolate from Streptomyces
aureofaciens (producing the polyether narasin and the
broad-spectrum antibiotic enteromycin) on galactose led to
yellow and white sectoring. The effect was specific for
galactose. Of the four colony types obtained, one produced
only narasin and two produced only enteromycin.
Streptomyces griseus ssp. cryophilus makes four R3 sulfated
and four R3 unsulfated carbapenems. The sulfated forms are
less active than the unsulfated forms. To completely eliminate
the R3 sulfated forms, sulfate transport mutants were obtained. These were of two types: (i) auxotrophs for thiosulfate
or cysteine; and (ii) selenate-resistant mutants. Each type
produced completely unsulfated forms and titers were equivalent to the total titer of the parent (Kitano et al., 1985).
Eight avermectins are produced by Streptomyces avermitilis, of which only a small number are desirable. A nonmethylating mutant produced only four of the compounds
and a mutant that failed to make the 25-isopropyl substituent (from valine) produced a different mixture of components. By protoplast fusion, a hybrid strain was obtained
which made only two components, B2a and B1a (Omura
et al., 1991). Stutzman-Engwall and colleagues (StutzmanEngwall et al., 2003) introduced random mutations by PCR
into gene aveC and obtained a mutant that produced an
avermectin B1 : B2 ratio of 2.5, much improved over the 0.6
ratio of the parent S. avermitilis strain.
Mutation was used to eliminate the undesirable polyketides sulochrin and asterric acid from broths of the lovastatin producer, Aspergillus terreus (Vinci et al., 1991). Mutants
have also been employed to eliminate undesirable coproducts from the monensin fermentation (Pospisil et al.,
1984).
Mutants producing new antibiotics
Mutant methodology has been used to produce new molecules. The medically useful products demethyltetracycline
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Nutritional repression can also be decreased by mutation to
antimetabolite resistance. Examples of selection agents are
2-deoxyglucose (2-DOG) for enzymes and pathways controlled by carbon source regulation (Table 1), methylammonium for those regulated by nitrogen source repression, and
arsenate for phosphate regulation.
Mutants that use phosphate less efficiently for growth
sometimes show improved antibiotic production. Thus,
screening for small colonies on phosphate-limiting media
could be a useful strain improvement technique for phosphate-regulated products (Table 1).
desired product as sole carbon or energy source. Such
mutants are often impaired in their ability to takeup the
product and they contain lower intracellular levels of the
product, thus lessening feedback regulation. In certain
improved mutants, there is an increase in sensitivity
to deoxycholate and lysozyme, indicating a change in
permeability.
192
Use of mutants to elucidate biosynthetic
pathways
A further use of mutants has been the elucidation of
metabolic pathways. This has been exploited for the biosynthesis of tetracyclines (McCormick, 1965), novobiocin
(Kominek, 1972), erythromycin (Martin et al., 1966;
Martin & Rosenbrook, 1967), neomycin (Pearce et al.,
1978), tylosin (Baltz et al., 1983), other aminoglycosides
(Penzikova & Levitov M, 1970; Takeda et al., 1978; Fujiwara
et al., 1980; Kase et al., 1982; Hanssen & Kirby, 1983),
rosaramicin (Vaughn et al., 1982), daunorubicin (McGuire
et al., 1981), other anthracyclines (Motamedi et al., 1986;
Yue et al., 1986), actinomycin (Troost & Katz, 1979),
carbapenems (Nozaki et al., 1984; Kojima et al., 1988),
ansamycins (Kibby et al., 1980; Ghisalba et al., 1981),
patulin (Gaucher et al., 1981) and phenazines (Byng et al.,
1979).
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Genetic recombination
In contrast to the extensive use of mutation in industry, genetic
recombination was not much used at first, despite early claims
of success (Jarai, 1961; Mindlin, 1969), mainly due to the
absence or the extremely low frequency of genetic recombination in industrial microorganisms (in streptomycetes, it was
usually 106 or even less). Other problems were evident with
the b-lactam-producing fungi. Although Aspergillus exhibited
sexual and parasexual reproduction, the most commercially
interesting genera, Cephalosporium and Penicillium, were the
most difficult to work with as they only reproduced parasexually, which rarely resulted in recombination.
Recombination was erroneously looked upon as an alternative to mutation instead of a method that would complement mutagenesis programs. The most balanced and
efficient strain development strategy would not emphasize
one to the exclusion of the other; it would contain both
mutagenesis-screening and recombination-screening components. In such a program, strains at different stages of a
mutational line, or from lines developed from different
ancestors, would be recombined. Such strains would no
doubt differ in many genes and by crossing them, genotypes
could be generated which would never occur as strictly
mutational descendants of either parent. Recombination
was also of importance in the mapping of production genes.
Studies on the genetic maps of overproducing organisms
such as actinomycetes are relatively recent. The model for
such investigations was the genetic map of Streptomyces
coelicolor (Kieser et al., 1992), which was found to be very
similar to those of other Streptomyces species, such as
S. bikiniensis, S. olivaceous, S. glaucescens and S. rimosus.
Protoplast fusion
As mentioned above, genetic recombination was virtually
ignored in industry, mainly due to the low frequency of
recombination. However, use of protoplast fusion changed
the situation markedly. After 1980, there was a heightened
interest in the application of genetic recombination to the
production of important microbial products such as antibiotics. Today, frequencies of recombination have increased to
even greater than 101 in some cases (Ryu et al., 1983), and
strain improvement programs routinely include protoplast
fusion between different mutant lines. The power of recombination was demonstrated by Yoneda (Yoneda, 1980), who
recombined individual mutations, each of which increased aamylase production by two- to seven-fold in Bacillus subtilis.
A strain constructed by genetic transformation, which contained all five mutations, produced 250-fold more a-amylase.
Recombination is especially useful when combined with
conventional mutation programs to solve the problem of
‘sickly’ organisms produced as a result of accumulated
genetic damage over a series of mutagenized generations.
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and doxorubicin were discovered by simple mutation of
the cultures producing tetracycline and daunorubicin,
respectively. Later, the technique of ‘mutational biosynthesis’ (= mutasynthesis) was devised (Shier et al., 1969). In
this process, a mutant blocked in secondary metabolism is
fed analogs of the moiety whose biosynthesis is blocked. If
successful, the mutant (called an ‘idiotroph’) produces a
new antibiotic derivative (Nagaoka & Demain, 1975). The
hybramycins were the first compounds to be made this way
(Shier et al., 1969). Since then, mutational biosynthesis has
been used for the discovery of many new secondary metabolites (Lemke & Demain, 1976; Daum & Lemke, 1979;
Kitamura et al., 1982). The most well-known is the commercial antihelmintic agent doramectin, the production
of which employed a mutant of the avermectin producer
S. avermitilis (Cropp et al., 2000).
New anthracyclines and aglycones have been isolated
from blocked mutants of the daunorubicin and doxorubicin
producers (Cassinelli et al., 1980; McGuire et al., 1981). By
adding carminomycinone or 13-dihydrocarminomycinone
to an idiotroph of Streptomyces galilaeus (the producer of
aclacinomycin), the aglycones were glycosylated to form a
new trisaccharide anthracycline, trisarubicionol (Yoshimoto
et al., 1981).
New macrolide antibiotics have been produced from
blocked mutants of the tylosin-producer, Streptomyces fradiae (Kirst et al., 1983). Four new hybrid macrolide antibiotics were obtained by feeding erythronolide B to a
blocked mutant of the oleandomycin producer, Streptomyces
antibioticus (Spagnoli et al., 1983). A blocked-mutant of the
mycinamicin producer, Micromonospora polytrota, was fed
various rosaramicin precursors and converted them into
new rosaramicins (Lee et al., 1983).
J. L. Adrio & A. L. Demain
193
Genetic improvement of processes yielding microbial products
FEMS Microbiol Rev 30 (2006) 187–214
flocculation (Panchal et al., 1982), lactose utilization (Farahnak et al., 1986), the killer character (Bortol et al., 1986;
Farris et al., 1992), cellobiose fermentation (Pina et al.,
1986) and methionine overproduction (Brigidi et al., 1988).
Plasmids, transposons, cosmids and phage
Plasmid DNA has been detected in virtually all antibioticproducing species of Streptomyces. Some are sex plasmids
and constitute an essential part of the sexual recombination
process and others contain either structural genes or genes
somehow influencing the expression of the chromosomal
structural genes of antibiotic biosynthesis.
Most plasmids have no apparent effect on metabolite
production and only very few antibiotic biosynthesis processes are encoded by plasmid-borne genes. However, the
production of methylenomycin A is encoded by genes
present on plasmid SCP1 in Streptomyces coelicolor. When
the plasmid was transferred to other streptomycetes, the
recipients produced the antibiotic. For many years, plasmid
SCP1 was never observed or isolated as a circular DNA
molecule, because it was a giant linear plasmid. It was
initially difficult to separate such giant linear plasmids from
chromosomal DNA but this was later accomplished by
pulsed field gel electrophoresis or orthogonal field alteration
gel electrophoresis (OFAGE) (Kinashi & Shimaji, 1987).
Some products of unicellular bacteria are plasmid-encoded. These include aerobactin, a hydroxamate siderophore
and virulence factor produced by Escherichia coli (McDougall
& Neilands, 1984) and other Gram-negative bacteria (Enterobacter aerogenes, Enterobacter cloacae, Vibrio mimicus, and
species of Klebsiella, Salmonella and Shigella). Aerobactin is
synthesized by a plasmid-borne five-gene cluster, which is
negatively regulated by iron (Roberts et al., 1986); it can also
be produced via chromosomal genes (Moon et al., 2004). It
also appears that siderophore production by Arizona hinshawii is plasmid-encoded. A microcin, an antimetabolite of
methionine, which is produced by E. coli and acts as a
competitive inhibitor of homoserine-O-transuccinylase, is
encoded by a plasmid that occurs at 20 copies per genome
equivalent (Perez-Diaz & Clowes, 1980). The gene coding for
the parasporal crystal body (d-endotoxin) of Bacillus thuringiensis is plasmid-borne (Whiteley & Schnepf, 1986; De
Maagd et al., 2003) in most species but is on the chromosome in a few other species.
Instability in Streptomyces is brought about by environmentally stimulated macrolesions, e.g. deletions, transpositions, rearrangements and DNA amplification. They occur
spontaneously or are induced by environmental stresses
such as intercalating dyes, protoplast formation and regeneration, and interspecific protoplast fusion. Streptomycetes
are the only prokaryotes known to be subject to spontaneous
DNA amplification, sometimes amounting to several
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For example, a cross via protoplast fusion was carried out
with strains of Cephalosporium acremonium from a commercial strain improvement program. A low-titer, rapidly-growing, spore-forming strain which required methionine to
optimally produce cephalosporin C was crossed with a
high-titer, slow-growing, asporogenous strain which could
use the less expensive inorganic sulfate. The progeny included a recombinant which grew rapidly, sporulated, produced cephalosporin C from sulfate and made 40% more
antibiotic than the high-titer parent (Hamlyn & Ball, 1979).
Protoplast fusion was used to modify the characteristics
of an improved penicillin-producing strain of P. chrysogenum which showed poor sporulation and poor seed growth.
Backcrossing with a low-producing (12 g L1) strain yielded
a high-producing (18 g L 1) strain with better sporulation
and better growth in seed medium (Lein, 1986). Interspecific protoplast fusion between the osmotolerant Saccharomyces mellis and the highly fermentative S. cerevisiae yielded
stable hybrids fermenting high concentrations of glucose
(49% w w1) (Legmann & Margalith, 1983).
Another application of protoplast fusion is the recombination of improved producers from a single mutagenesis
treatment. By recombination, one could combine the yieldincrease mutations and obtain an even more superior
producer before carrying out further mutagenesis. Two
improved cephamycin-C producing strains from Nocardia
were fused and among the recombinants were two cultures
that produced 10–15% more antibiotic than the best parent
(Wesseling & Lago, 1981). Genetic recombination allows the
discovery of new antibiotics by fusing producers of different
or even the same antibiotics. A recombinant obtained from
two different rifamycin-producing strains of Nocardia mediterranei produced two new rifamycins (16,17-dihydrorifamycin S and 16,17-dihydro-17-hydroxy-rifamycin S) (Traxler et al., 1982). However, according to Hopwood
(Hopwood, 1983), these examples may reflect the different
expression of genes from parent A in the cytoplasm of parent
B, rather than the formation of hybrid antibiotics. Interspecific protoplast fusion between S. griseus and five other
species (Streptomyces cyaneus, Streptomyces exfoliatus, Streptomyces griseoruber, Streptomyces purpureus and Streptomyces
rochei) yielded recombinants of which 60% produced no
antibiotics and 24% produced antibiotics different from the
parent strains (Okanishi et al., 1996). New antibiotics can
also be created by changing the order of the genes of an
individual pathway in its native host (Hershberger, 1996).
A new antibiotic, indolizomycin, was produced by protoplast fusion between non-antibiotic producing mutants of
Streptomyces griseus and Streptomyces tenjimariensis (Gomi
et al., 1984). Osmotolerance of food yeasts such as
Saccharomyces cerevisiae and S. diastaticus was increased by
protoplast fusion with osmotolerant yeasts. Other traits
transferred between yeasts by protoplast fusion include
194
Improvement of microbial processes by
genetic engineering
Primary metabolites
New processes for the production of amino acids and
vitamins have been developed by recombinant DNA tech2005 Federation of European Microbiological Societies
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nology. Escherichia coli strains were constructed with plasmids bearing amino acid biosynthetic operons. Plasmid
transformation was accomplished in Corynebacterium, Brevibacterium and Serratia and, as a result, recombinant DNA
technology has been used routinely to improve such commercial amino acid-producing strains (Sahm et al., 2000).
A recombinant strain of E. coli (made by mutating to
isoleucine auxotrophy, cloning in extra copies of the thrABC
operon, inactivating the threonine-degrading gene tdh, and
mutating to resistance to high concentrations of L-threonine
and L-homoserine) produced 80 g L1 L-threonine in 1.5
days at a yield of 50% (Eggeling & Sahm, 1999). Cloning
extra copies of threonine export genes into E. coli led to
increased threonine production (Kruse et al., 2002).
The introduction of the proline 4-hydroxylase gene from
Dactylosporangium sp. into a recombinant strain of E. coli
producing L-proline at 1.2 g L1 lead to a new strain producing 25 g L 1 of hydroxyproline (trans-4-hydroxy-L-proline) (Shibasaki et al., 2000). When proline was added,
hydroxyproline reached 41 g L1, with a yield of 87% from
proline.
An engineered strain of Corynebacterium glutamicum
producing 50 g L1 of L-tryptophan was further modified
by cloning in additional copies of its own transketolase gene
to increase the level of the erythrose-4-phosphate precursor
of aromatic biosynthesis (Ikeda & Katsumata, 1999). A low
copy number plasmid increased production to 58 g L1,
whereas a high copy number plasmid decreased production.
L-Valine production by mutant strain VAL1 of C. glutamicum amounted to 105 g L1 (Radmacher et al., 2002;
Lange et al., 2003). The mutant was constructed by overexpressing biosynthetic enzymes via a plasmid, eliminating
ilvA encoding threonine dehydratase, and deleting two genes
encoding enzymes of pantothenate biosynthesis. The culture
was grown with limitation of isoleucine and pantothenate.
By introduction of feedback-resistant threonine dehydratases and additional copies of genes encoding branched
amino and biosynthetic enzymes, lysine- or threonineproducing strains were converted into L-isoleucine producers with titers up to 10 g L1 (Morbach et al., 1996;
Guillouet et al., 1999; Hashiguchi et al., 1999). Amplification of the wild-type threonine dehydratase gene ilvA in a
threonine-producing strain of Corynebacterium lactofermentum led to 15 g L1 of isoleucine overproduction (Colon
et al., 1995).
Biotin has been made traditionally by chemical synthesis
but recombinant microbes have approached a competitive
economic position. The cloning of a biotin operon
(bioABFCD) on a multicopy plasmid allowed E. coli to
produce 10 000 times more biotin than did the wild-type
strain (Levy-Schil et al., 1993). Sequential mutation of
Serratia marcescens to resistance to the biotin antimetabolite
acidomycin (= actithiazic acid) led to mutant strain SB412,
FEMS Microbiol Rev 30 (2006) 187–214
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hundred tandem copies, accounting for over 10% of total
DNA, in the absence of selection. Amplification seems to be
coupled to DNA deletion and may involve insertion sequence (IS)-like elements (Baltz, 1986). Ethidium bromide
cures plasmids in streptomycetes but also increases the
frequency of deletion mutations, especially in areas of the
chromosome that are already unstable (Crameri et al., 1986).
Transposable elements, DNA sequences encoding a transposase enzyme (Berg & Berg, 1983) that move from one
replicon to another without host recombination functions
or extensive homology with the site of integration, have
been extremely useful for the following reasons: (i) they
usually provide stable, nonreverting mutants; (ii) they can
be used to determine the order of genes in an operon; (iii) it
is easy to select for mutants because transposons contain
antibiotic- or mercury-resistance markers; (iv) they provide
portable regions of homology for chromosomal mobilization; (v) they provide markers for non-selectable genes and
allow the cloning of such genes which can then be used as
hybridization probes to fish out the wild-type gene from a
genomic library; and (vi) they often have unique restriction
sites, and thus are good markers for isolating defined
deletion derivatives or locating the precise position of a gene
by heteroduplex mapping.
In the daptomycin producer Streptomyces roseosporus,
some Tn 5099 transposition mutants produced 57–66%
more daptamycin than the parent, whereas others produced
less or the same (McHenney & Baltz, 1996; Baltz et al., 1997).
Transposition increased the rate-limiting step of tylosin
biosynthesis in Streptomyces fradiae, i.e. the conversion of
macrocin to tylosin. Transposing a second copy of tylF into a
neutral site on the S. fradiae chromosome increased its gene
product, macrocin O-methyltransferase, and tylosin production, while decreasing the concentration of the final intermediate (macrocin). Tylosin production was increased by up
to 60% and the total macrolide titer was unchanged (Solenberg et al., 1996). Transposon mutagenesis eliminated the
production of the toxic oligomycin by the avermectinproducing Streptomyces avermitilis (Ikeda et al., 1993).
Cloning a 34-kb fragment from Streptomyces rimosus via a
cosmid into Streptomyces lividans and Streptomyces albus
resulted in oxytetracycline production by the recipients (Binnie et al., 1989). Contrary to earlier reports, all the oxytetracycline genes were clustered together on the S. rimosus
chromosomal map (Butler et al., 1989).
J. L. Adrio & A. L. Demain
195
Genetic improvement of processes yielding microbial products
FEMS Microbiol Rev 30 (2006) 187–214
Cloning of aldehyde dehydrogenase of Acetobacter polyoxogenes on a plasmid vector into Acetobacter aceti ssp.
xylinum increased the rate of acetic acid production by over
100% (1.8 g L1 h 1 to 4 g L 1 h1) and titer by 40%
(68 g L1 to 97 g L1) (Fukaya et al., 1989).
Genetic engineering of the inosine monophosphate
(IMP) dehydrogenase gene in a B. subtilis strain producing
7 g L1 of the desirable guanosine and 19 g L1 of the
undesirable inosine changed production to 20 g L 1 guanosine and 5 g L1 inosine (Miyagawa et al., 1986).
A recombinant E. coli strain was constructed that produced optically active pure D-lactic acid from glucose at
virtually the theoretical maximum yield, e.g. two molecules
from one molecule of glucose (Zhou et al., 2003). The
organism was engineered by eliminating genes of competing
pathways encoding fumarate reductase, alcohol/aldehyde
dehydrogenase and pyruvate formate lyase and by a mutation in the acetate kinase gene.
New technologies that have proven to be very useful for
increasing production of primary metabolites include genome-based strain reconstruction, metabolic engineering,
and whole genome shuffling (see section on Novel genetic
technologies).
Secondary metabolites
The application of recombinant DNA technology to the
production of secondary metabolites has been of great
interest (Baltz & Hosted, 1996; Diez et al., 1997). The tools
of the recombinant geneticist for increasing the titers of
secondary metabolites have included: (i) transposition mutagenesis, (ii) targeted deletions and duplications by genetic
engineering and (iii) genetic recombination by protoplast
fusion (Baltz, 2003). Recent additions to these techniques
include genomics, transcriptome analysis, proteomics, metabolic engineering, and whole genome shuffling (see section on Novel gene technologies).
One of the first indications that rDNA technology could
be applied to antibiotics and other secondary metabolites
was that it could be carried out in streptomycetes (Thompson et al., 1982). Plasmids were constructed from plasmid
SLP 1.2 of Streptomyces lividans and plasmid SCP2 from
Streptomyces coelicolor. In mating of plasmid-negative
S. lividans, ‘pocks’ (circular zones of sporulation inhibition
associated with plasmid transfer in the lawn of streptomycete growth arising from a regenerated protoplast population) were seen. This was due to looping out of a piece of S.
coelicolor DNA, which became a series of small S. lividans
plasmids (SLP 1.1 to 1.6) that were good cloning vehicles.
The genetic engineering of actinomycetes was limited for
a number of years by restriction barriers hindering DNA
introduction and by the inhibition of secondary metabolism
by self-replicating plasmid-cloning vectors (Baltz & Hosted,
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which produced 20 mg L1 biotin (Sakurai et al., 1994).
Further improvements were made by mutating selected
strains to ethionine-resistance (strain ET2, 25 mg L1), then
mutating ET2 to S-2-aminoethylcysteine resistance (strain
ETA23, 33 mg L1) and finally cloning in the resistant bio
operon (Sakurai et al., 1994) yielding a strain able to
produce 500 mg L1 in fed-batch fermentor culture along
with 600 mg L1 of biotin vitamers. Later advances led to
production by recombinant S. marcescens of 600 mg L1 of
biotin (Masuda et al., 1995).
A process for riboflavin production in Corynebacterium
ammoniagenes (previously Brevibacterium ammoniagenes)
was developed by cloning and overexpressing the organism’s
own riboflavin biosynthesis genes (Koizumi et al., 2000) and
its own promoter sequences. The resulting culture produced
15.3 g L1 riboflavin in 3 days. Genetic engineering of a
Bacillus subtilis strain already containing purine analogresistance mutations led to the improved production of
riboflavin (Perkins & Pero, 1993). An industrial strain of B.
subtilis was produced by inserting multiple copies of the rib
operon at two different sites in the chromosome, making
purine analog-resistance mutations to increase guanosine
triphosphate (GTP; a precursor) production and a riboflavin analog (roseflavin)-resistance mutation in ribC that
deregulated the entire pathway (Perkins et al., 1999).
Vitamin C (ascorbic acid) has traditionally been made in
a five-step predominantlychemical process by first converting glucose to 2-keto-L-gulonic acid (2-KGA) with a yield of
50% and then converting the 2-KGA by acid or base to
ascorbic acid. A novel process for vitamin C synthesis
involved the use of a genetically engineered Erwinia herbicola strain containing a gene from Corynebacterium sp. The
engineered organism converted glucose into 1 g L1 of 2KGA (Anderson et al., 1985; Pramik, 1986). A better process
was devised independently, which converted 40 g L1 glucose into 20 g L1 2-KGA (Grindley et al., 1988). This
process involved cloning and expressing the gene encoding
2,5-diketo-D-gluconate reductase from Corynebacterium sp.
into Erwinia citreus. Another process uses a recombinant
strain of Gluconobacter oxydans containing genes encoding
L-sorbose dehydrogenase and L-sorbosone dehydrogenase
from G. oxydans T-100. The new strain was an improved
producer of 2-KGA (Saito et al., 1997). Further mutation to
suppress the L-idonate pathway and to improve the promoter led to the production of 130 g L1 of 2-KGA from
150 g L1 sorbitol.
Carotenoids were overproduced by introducing carotenoid gene clusters from Erwinia uredovora into E. coli and
overexpressing E. coli deoxyxylulose phosphate synthase, the
key enzyme of the non-mevalonate isoprenoid biosynthetic
pathway (Matthews & Wurtzel, 2000). Lycopene accumulated to 1.3 mg g1 dry cell weight and zeaxanthin to
0.6 mg g1.
196
b-Lactam antibiotics
Cloning has been very important in understanding the
biosynthesis of b-lactam antibiotics (Demain & Elander,
1999), its genetics and improving the production processes.
Early common pathway
All producers of penicillins and cephalosporins, including
cephamycins, use the same two enzymes to start the
biosynthetic process. The steps involve the condensation of
L-a-aminoadipic acid, L-cysteine and L-valine to form the
tripeptide, d-(a-aminoadipyl)-L-cysteinyl-D-valine (ACV)
by ACV synthetase (ACVS), encoded by gene pcbAB (also
known as acvA in A. nidulans). This is followed by cycliza2005 Federation of European Microbiological Societies
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tion of ACV into isopenicillin N (IPN) by IPN synthase
(cyclase; encoded by pcbB). The cloning of the gene encoding ACVS from P. chrysogenum (Diez et al., 1990), C.
acremonium (Gutierrez et al., 1991) and Nocardia lactamdurans (Castro et al., 1988) contributed greatly to the
elucidation of the biosynthetic pathway. Overexpression of
acvA in A. nidulans, by replacing the normal promoter with
the ethanol dehydrogenase promoter (Kennedy & Turner,
1996), increased penicillin production up to 30-fold. The
cyclase genes from different microorganisms were all cloned
(Aharonowitz et al., 1992; Martin et al., 1997) and provided
pure enzyme for structural studies. Cloning multiple copies
of cyclase into C. acremonium yielded an improved cephalosporin C-producing strain (Skatrud et al., 1987).
The hydrophobic branch
Producers of penicillin use a single step branch involving
penicillin acyltransferase acting on IPN. Its gene penDE (also
known as iat, aat and acyA in A. nidulans) was cloned from
P. chrysogenum into C. acremonium, which led to the
production of penicillin G (in the presence of exogenous
phenylacetic acid) along with cephalosporin C (Gutierrez
et al., 1991). Without cloning, C. acremonium cannot
produce penicillin G.
The hydrophilic branch
All producers of cephalosporins and cephamycins employ a
series of enzymes leading from IPN. First, IPN is epimerized
to penicillin N by IPN epimerase (encoded by cefD). The
next steps include ring expansion of penicillin N by deacetoxycephalosporin C (DAOC) synthase (expandase, encoded by cefE) and hydroxylation by DAOC 3 0 -hydroxylase
(encoded by cefF) to deacetylcephalosporin C (DAC).
Although expandase and hydroxylase are separate enzymes
encoded by separate genes in bacteria, these two activities
are found on the same protein in fungi, which is encoded by
one gene cefEF. At the DAC stage, the overall pathway again
splits into two branches. In C. acremonium, DAC is acetylated to cephalosporin C by DAC acetyltransferase encoded
by cefG. This step is the terminal reaction in cephalosporinproducing fungi. By contrast, actinomycetes carbamoylate
DAC using three enzymes, encoded by cmcH, cmcI and cmcJ
genes to yield cephamycin C (Brewer et al., 1980).
When an industrial production strain of C. acremonium
394-4 was transformed with a plasmid containing the pcbC
and the cefEF gene from an early strain of the C. acremonium
mutant line, a transformant producing 50% more cephalosporin C than the production strain, as well as less penicillin
N, was obtained. Production in pilot plant (150 L) fermentors was further improved by 15% (Skatrud et al., 1989).
One copy of the cefEF had been integrated into chromosome
III, whereas the native gene is on chromosome II.
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1996), but these problems were mainly overcome. Early
reviews on cloning and expressing antibiotic production
genes in Streptomyces were by Martin and Gil (Martin & Gil,
1984) and Liras (Liras, 1988).
An interesting possibility was the transfer of operons
from one streptomycete to another in the hope that the
structural genes might be better able to express themselves
in another species. Clustering facilitated the transfer of an
entire pathway in a single manipulation. Studies revealed
that many antibiotic biosynthesis genes were arranged in
clusters including undecylprodigiosin, actinorhodin, chloramphenicol, rifamycin, cephamycin, erythromycin, tetracyclines and tylosin among others. Thus, the entire
undecylprodigiosin pathway (‘red’ pathway) of S. coelicolor
was transferred on a 37-kb fragment into Streptomyces
parvulus and the antibiotic was produced (Coco et al.,
1991). Similarly, the entire cephamycin C pathway was
cloned and expressed from a cephamycin-producing strain
of Streptomyces cattleya. When the 29-kb DNA fragment was
cloned into the non-b-lactam producer, S. lividans, one
transformant (out of 30 000) made cephamycin (Chen
et al., 1988). When the fragment was introduced into
another cephamycin producer, Streptomyces lactamgens, a
two- to three-fold improvement was obtained.
In fungi making penicillin G, the three structural genes
(ACVS, cyclase and penicillin acyltransferase) are clustered
on a single chromosome of Penicillium chrysogenum (Smith
et al., 1990) and of Aspergillus nidulans (MacCabe et al.,
1990). In these fungi, the genes of the cluster are separately
transcribed. By contrast, fungal genes coding for cephalosporin biosynthesis are distributed among different chromosomes. The deacetylcephalosporin C acetyltransferase gene
from Cephalosporium acremonium (cefG) is closely linked to
the expandase (cefEF) gene (Gutierrez et al., 1992; Matsuda
et al., 1992) and both are on chromosome II, whereas the
early genes of the pathway (pcbAB, pcbC) are located on
chromosome VI.
J. L. Adrio & A. L. Demain
197
Genetic improvement of processes yielding microbial products
Microbial enzymes
Genes encoding many microbial enzymes have been cloned
and the enzymes expressed at levels hundreds of times
FEMS Microbiol Rev 30 (2006) 187–214
higher than those naturally produced. Recombinant DNA
technology has been used (Falch, 1991): (i) to produce in
industrial organisms enzymes obtained from microbes that
are difficult to grow or handle genetically; (ii) to increase
enzyme productivity by use of multiple gene copies, strong
promoters, and efficient signal sequences; (iii) to produce in
a safe host useful enzymes obtained from a pathogenic or
toxin-producing microorganism; and (iv) to improve the
stability, activity or specificity of an enzyme by protein
engineering. The industrial enzyme business adopted rDNA
methods to increase production levels and to produce
enzymes from industrially-unknown microorganisms in
industrial organisms such as species of Aspergillus and
Trichoderma, as well as Kluyveromyces lactis, S. cerevisiae,
Yarrowia lipolytica and Bacillus licheniformis. Virtually all
laundry detergents contain genetically-engineered enzymes
and much cheese is made with genetically-engineered enzymes. Indeed, over 60% of the enzymes used in the
detergent, food and starch processing industries are recombinant products (Cowan, 1996).
Scientists at Novo Nordisk isolated a very desirable lipase
for use in detergents from a species of Humicola. For production purposes, the gene was cloned into Aspergillus oryzae,
where it produced 1000-fold more enzyme (Carlsen, 1990)
and is now a commercial product. Such lipases are used for
laundry cleaning, interesterification of lipids, and esterification
of glucosides producing glycolipids which have applications as
biodegradable non-ionic surfactants for detergents, skin care
products, contact lens cleaners and as food emulsifiers.
The a-amylase gene from Bacillus amyloliquefaciens was
cloned using multicopy plasmid pUB110 in B. subtilis
(Palva, 1982). Production was 2500-fold that in wild-type
B. subtilis and five-fold that of the B. amyloliquefaciens
donor. An exoglucanase from the cellulolytic Cellulomonas
fimi was overproduced after cloning in E. coli to a level of
over 20% of cell protein (O’Neill et al., 1986). The endo-bglucanase components of the cellulase complexes from
Thermomonospora and Clostridium thermocellum were
cloned in E. coli as was the cellobiohydrolase I gene of
Trichoderma reesei (Shoemaker et al., 1983; Teeri et al.,
1983). Pichia pastoris, a methanol-utilizing yeast, was engineered to produce S. cerevisiae invertase and to excrete it
into the medium at 100 mg L1 (Van Brunt, 1986). Interestingly, in S. cerevisiae, the invertase is periplasmic. Selfcloning of the xylanase gene in S. lividans resulted in sixfold overproduction of the enzyme (Mondou et al., 1986).
Many enzymes are made by filamentous organisms,
which are slow-growing and difficult to handle in fermentors. The transfer of these genes to rapidly-growing unicellular bacteria means that rapid growth and more
reproducible production can be achieved. Other advantages
are more rapid nutrient uptake due to a greater surface/
volume ratio, better oxygen transfer, better mixing and thus
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Transformation of P. chrysogenum with the Streptomyces
lipmanii cefD and Streptomyces clavuligerus cefE genes allowed the production of the intermediate DAOC (Cantwell
et al., 1992) at titers of 2.5 g L1. DAOC is a valuable
intermediate in the commercial production of semi-synthetic cephalosporins. Also, cloning of cefE from S. clavuligerus or cefEF and cefG (see next paragraph) from C.
acremonium into P. chrysogenum grown with adipic acid as
side-chain precursor (Crawford et al., 1995) resulted in
formation of adipyl-6-aminopenicillanic acid (adipyl-6APA) and adipyl-7-aminodeoxycephalosporanic acid (adipyl-7-ADCA) in the case of cefE and adipyl-6APA, adipyl7ADCA, adipyl-7-DAC and adipyl-7-aminocephalosporanic
acid (7-ACA) in the case of cefEF and cefG.
Disruption and one-step replacement of the cefEF gene
of an industrial cephalosporin C production strain of A.
chrysogenum yielded strains accumulating up to 20 g L 1 of
penicillin N. Cloning and expression of the cefE gene from S.
clavuligerus into those high-producing strains yielded recombinant strains producing high titers of DAOC (Velasco
et al., 2000). Production levels were nearly equivalent (80%)
to the total b-lactams biosynthesized by the parental strain.
Weak acetyltransferase promoter activity appears to be
the cause of DAC accumulation in broths of C. acremonium.
Cloning of cefG increased its copy number and cefG mRNA,
tripled acetyltransferase activity, and increased cephalosporin C titers in a dose-dependent manner (Matsuda et al.,
1992; Mathison et al., 1993). Cloning of the gene with its
own promoter had no effect on the low level of DAC
acetyltransferase normally observed in C. acremonium (Gutierrez et al., 1997). However, the use of foreign promoters
(the gpd promoter from A. nidulans, the bla promoter from
A. niger or the pbcC promoter from P. chrysogenum) had a
major effect on the level of cefG transcripts, DAC acetyltransferase protein level and activity, and antibiotic production; cephalosporin C production rose by two- to three-fold.
Of the cephalosporins produced, the undesirable DAC
decreased from 80% of the total to 30–39%, whereas
cephalosporin C increased by a similar amount.
Transformation of early strain P. chrysogenum Wis541255 with individual genes, pairs of genes, and all three
genes of the penicillin pathway showed that the major
increases occurred when all three genes were overexpressed
(Theilgaard et al., 2001). The best transformant contained
three extra copies of pcbAB, one extra copy of pcbC and two
extra copies of penDE and produced 299% of control shake
flask production and 276% of control productivity in
continuous culture.
198
Polymers, fuels, foods and beverages
Microbially-produced xanthan gum is not only an acceptable food-thickener but is one of the most promising agents
for enhanced oil recovery in the petroleum industry. Recombinant DNA manipulation of Xanthomonas campestris
increased titers of xanthan by two-fold and increased
pyruvate content by over 45% (Bigelas, 1989; Tseng et al.,
1992). The yield was 0.6 g g1 of sucrose utilized (Letisse
et al., 2001). Ten to twenty thousand tons of xanthan are
produced annually for use in the oil, pharmaceutical,
cosmetic, paper, paint and textile industries (Becker et al.,
1998).
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Escherichia coli was converted into a good ethanol producer (4.3%, v v 1) using recombinant DNA technology
(Ingram et al., 1987). Alcohol dehydrogenase II and pyruvate decarboxylase genes from Zymomonas mobilis were
inserted in E. coli and became the dominant system for NAD
regeneration. Ethanol represented over 95% of the fermentation products in the genetically-engineered strain. By
cloning and expressing the same two genes into Klebsiella
oxytoca, the recombinant was able to convert crystalline
cellulose to ethanol in high yield when fungal cellulase was
added (Doran & Ingram, 1993). The maximum theoretical
yield was 81–86% and titers as high as 47 g L 1 of ethanol
were produced from 100 g L 1 of cellulose.
Cloning of its ace (acetone) operon gene adc (encoding
acetoacetate decarboxylase), ctfA and ctfB (two genes encoding coenzyme A transferase) on a plasmid containing the adc
promoter into Clostridium acetobutylicum resulted in a 95%
increase in production of acetone, a 37% increase in butanol,
a 90% increase in ethanol, a 50% increase in solvent yield
from glucose and a 22-fold lower production of undesirable
acids (Mermelstein et al., 1993). The introduction of the
acetone operon from C. acetobutylicum into E. coli led to
high acetone production by the latter (Bermejo et al., 1998).
Beer wort contains barley b-glucans which reduce the
filtrability of beer and lead to precipitates and haze in the
final product. The gene coding for endoglucanase was
transferred from T. reesei to brewer’s yeast and the engineered yeast strain efficiently hydrolyzed the b-glucans
(Penttilä et al., 1987). Similiar technology created starchutilizing S. cerevisiae strains and wine yeast strains producing lower acidity and enhanced flavor. Brewing yeasts were
modified using recombinant DNA technology so that they
could produce A. niger amyloglucosidase and break down
unfermentable dextrins for light beer production (Van
Brunt, 1986; Hammond, 1988). The glucoamylase gene
from Aspergillus awamori was cloned and expressed stably
in polyploid distiller’s yeast. A high level of glucoamylase
was secreted. Almost all (95%) of the carbohydrates in the
25% starch substrate were utilized and high levels of ethanol
were produced. The engineered strain outperformed S.
diastaticus (Cole et al., 1988).
Brewing yeasts were engineered to produce acetolactate
decarboxylase from Enterobacter aerogenes or A. aceti. This
enzyme eliminated diacetyl and the requirement for the
three- to five-week flavor maturation period which normally
follows a one-week fermentation stage (Sone et al., 1988).
The resulting beer suffered no loss of quality or flavor
(Holzman, 1994).
Bioconversions
Recombinant DNA techniques have been useful in developing new bioconversions and improving old ones. Using a
FEMS Microbiol Rev 30 (2006) 187–214
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more reliable control of pO2, pCO2 and pH, and a better
organism for mutagenesis.
Aspartase production was increased by 30-fold by cloning
in E. coli (Komatsubara et al., 1986). Captopril esterase of
Pseudomonas putida, used in preparing the chiral captopril
sidechain, was cloned in E. coli with a 38-fold increase in
activity (Elander, 1995). A 1000-fold increase in phytase
production was achieved in A. niger using recombinant
technology (Van Hartinsveldt et al., 1993). Cloning of the
benzylpenicillin acylase gene of E. coli on multicopy (50)
plasmids resulted in a 45-fold increase as compared to
uninduced wild-type production. Interestingly, the cloned
enzyme is constitutive (Mayer et al., 1980). Cloning additional penicillin V amidase genes into wild-type Fusarium
oxysporium increased enzyme titer by 130-fold (Komatsubara et al., 1986).
The properties of many enzymes have been altered by
genetic means. ‘Brute force’ mutagenesis and random
screening of microorganisms over the years led to changes
in pH optimum, thermostability, feedback inhibition, carbon source inhibition, substrate specificity, Vmax, Km and Ki.
This information was later exploited by the more rational
techniques of protein engineering. Single changes in amino
acid sequences have yielded similar types of changes in a
large variety of enzymes. Today, it is no longer necessary to
settle for the natural properties of an enzyme; these can be
altered to suit the needs of the investigator or the process.
For example, a protease from Bacillus stearothermophilus
was increased in heat tolerance from 86 1C to 100 1C, being
made resistant to boiling. The enzyme was developed by
site-directed mutagenesis (Van den Burg et al., 1998). Only
eight amino acids had to be modified. Temperature stability
at 100 1C was increased 340-fold and activity at lower
temperature was not decreased. All eight mutations were
far from the enzyme’s active site. Washing powders have
been improved in activity and low temperature operation by
the application of recombinant DNA technology and sitedirected mutagenesis to proteases and lipases (Falch, 1991;
Wackett, 1997).
J. L. Adrio & A. L. Demain
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Genetic improvement of processes yielding microbial products
Novel genetic technologies
A new genomic technique called ‘genome-based strain
reconstruction’ allows one to construct a strain superior to
the production strain because it only contains mutations
crucial to hyperproduction, but not other unknown mutations which accumulate by brute-force mutagenesis and
screening (Ohnishi et al., 2002). This approach was used to
improve the lysine production rate of Corynebacterium
glutamicum by comparing high producing strain B-6 developed by Hirao and coworkers (Hirao et al., 1989) (production rate slightly less than 2 g L 1 h 1) and a wild-type
strain. Comparison of 16 genes from strain B-6, encoding
enzymes of the pathway from glucose to lysine, revealed
mutations in five of the genes. Introduction of three of these
mutations into the wild-type created a new strain which
FEMS Microbiol Rev 30 (2006) 187–214
produced 80 g L 1 in 27 h, at a rate of 3 g L 1 h 1, the
highest rate ever reported for a lysine fermentation.
‘Metabolic engineering’ is the directed improvement of
product formation or cellular properties through the modification of specific biochemical reactions or introduction of
new ones using recombinant DNA technology (Stephanopoulos, 1999; Nielsen, 2001). Its essence is the combination
of analytical methods to quantify fluxes and the control of
fluxes with molecular biological techniques to implement
suggested genetic modifications. Flux is the focal point of
metabolic engineering. Different means of analyzing flux
are: (i) kinetic based models; (ii) control theories; (iii) tracer
experiments; (iv) magnetization transfer; (v) metabolite
balancing; (vi) enzyme analysis and (vii) genetic analysis
(Eggeling et al., 1996). Metabolic control analysis revealed
that the overall flux through a metabolic pathway depends
on several steps, not just a single rate-limiting reaction
(Kacser & Acerenza, 1993).
Metabolic engineering has been applied to antibiotic
production (Khetan & Hu, 1999, 1999; Thykaer & Nielsen,
2003). The increases in metabolic flux were carried out by
enhancing enzymatic activity, manipulating regulatory
genes, enhancing antibiotic resistance and heterologous
expression of novel genes. Table 2 summarizes several
examples of progress on the production of those secondary
metabolites.
The production of amino acids shows many examples of
this approach. A useful review of metabolic engineering in
C. glutamicum, especially in relation to L-lysine production,
was published by Sahm and colleagues (Sahm et al., 2000).
Metabolic flux studies of wild-type C. glutamicum and four
improved lysine-producing mutants available from the
ATCC showed that yield increased from 1.2% to 24.9%
relative to the glucose flux. Other recent examples are on
overproduction of aromatic amino acids and derivatives
(Bongaerts et al., 2001), L-lysine (Wittmann & Heinzle,
2002) and glutamate (Kimura, 2003).
There are many other successful applications of metabolic
engineering for products such as 1,3-propanediol (Nakamura & Whited, 2003), carotenoids (Rohlin et al., 2001;
Visser et al., 2003; Wang & Keasling, 2003), organic acids
(Kramer et al., 2003), ethanol (Nissen et al., 2000), vitamins
(Zamboni et al., 2003; Sybesma et al., 2004) and complex
polyketides in bacteria (Pfeifer et al., 2001; Khosla &
Keasling, 2003).
During the last few years, an expanded view of the cell has
been possible due to impressive advances in all the ‘omics’
techniques (genomics, proteomics, metabolomics, etc.) and
high-throughput technologies for measuring different
classes of key intracellular molecules. ‘Systems biology’ has
recently emerged as a term to describe an approach that
considers genome-scale and cell-wide measurements in
elucidating processes and mechanisms (Stephanopoulos
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plasmid containing tryptophan synthase plus induction
with 3-indole acrylate, recombinant E. coli was able to
produce 180 g L 1 of L-tryptophan from indole plus
L-serine in 8 h (Yukawa et al., 1988). Whereas S. cerevisiae
normally produces 2 g L 1 of malic acid from fumaric acid,
a recombinant strain containing a cloned fumarase gene was
able to produce 125 g L 1 with a yield of almost 90%
(Neufeld et al., 1991).
An oxidative bioconversion of saturated and unsaturated
linear aliphatic 12–22 carbon substrates to their terminal
dicarboxylic acids was developed by gene disruption and
gene amplification (Picataggio et al., 1992). Product concentrations reached 200 g L 1 and problematic side-reactions such as unsaturation, hydroxylation and chainshortening did not occur.
3-0-Acetyl-4 0 0 -0-isovaleryltylosin (AIV) is useful in veterinary medicine against tylosin-resistant Staphylococcus aureus. It is made by first producing tylosin with Streptomyces
fradiae and then using Streptomyces thermotolerans (producer of carbomycin) to bioconvert tylosin into AIV. A new
direct fermentation organism was constructed by transforming S. fradiae with S. thermotolerans plasmids containing acyl transferase genes (Arisawa et al., 1996).
Recombinant Candida pasteurianum can carry out the
conversion of glycerol to 1,3-propanediol (Luers et al.,
1997). A more economical process involving conversion of
the less expensive glucose to 1,3-propanediol has been
achieved with a recombinant E. coli strain (Nakamura &
Whited, 2003). The project is a collaborative effort by
Genencor International and DuPont (Potera, 1997). The
recombinant strain contains two metabolic pathways, one
for conversion of glucose to glycerol and the other for
conversion of glycerol to 1,3-propanediol (Tong et al.,
1991; Laffend et al., 1996). The 1,3-propanediol (also known
as trimethylene glycol or 3G) is used as the building block to
produce a new biodegradable polyester (3G1).
200
J. L. Adrio & A. L. Demain
et al., 2004). Progress in strain development will depend, not
only on all the technologies mentioned above, but also on
the development of mathematical methods that facilitate the
elucidation of mechanisms and identification of genetic
targets for modification.
A genome-wide transcript expression analysis called
‘massive parallel signature sequencing’ (Brenner et al.,
2000) was used successfully to discover new targets for
further improvement of riboflavin production by the fungus
A. gossypii (Karos et al., 2004). The authors identified 53
Table 2. Metabolic engineering of antibiotics
Target
1.Manipulation of
structural genes
2. Manipulation of
regulatory genes
Result
Reference
1.1. Amplifying an entire pathway
2.3-fold increase in cephamycin C
1.2. Amplifying a segment of a pathway 7-fold increase in daunorubicin
30% increase in tetracenomycin C
3- to 4-fold increase in spinosyn
1.3. Enhancing resistance
7- fold increase in neomycin
2.1. Amplifying positive regulatory genes
Pathway specific regulators
3.2. Increasing expression of rate-limiting 30-fold increase in penicillin increase in
enzymes
tylosin
3.3. Eliminating accumulation and
excretion of intermediate
3.4. Deleting gene leading to a side
product
3.5. Biosynthesizing compounds
previously made semisynthetically
3.6. Biosynthesizing new compounds
3.7. Increasing oxygen availability
3.8. Enhancing precursor uptake
Elimination of excretion of penicillin N;
15% increase in cephalosporin C
Elimination of oligomycin production
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Hwang et al. (2003); Lee et al. (2000)
Chater & Bruton (1985); Mao et al.
(1999); Kennedy et al. (1999)
Brian et al. (1996)
Malmberg et al. (1995)
Kennedy & Turner (1996)
Cox et al. (1987); Fishman et al.
(1987)
Skatrud (1992)
Ikeda et al. (1993)
Production of 7Velasco et al. (2000)
aminodeacetoxycephalosporanic acid
in Acremonium chrysogenum
Production of adipyl-7-ADCA, adipyl-7- Crawford et al. (1995)
ACA in P. chrysogenum
60% increase in erythromycin production Brunker et al. (1998); Minas et al.
(1998)
4-fold increase in deoxyerythronolide B Lombo et al. (2001)
and 8, 8a-deoxyoleoandolide
Integrating transcriptional and metabolite profiles from
21 strains of A. terreus producing different levels of lovastatin and another 19 strains with altered (1)-geodin levels led
to an improvement in lovastatin production of over 50%
(Askenazi et al., 2003). The approach, named ‘association
analysis’, was used to reduce the complexity of profiling data
sets to identify those genes in which expression was most
tightly linked to metabolite production. Such an application
is suitable to all industrially useful organisms for which
genome data are limited.
c
Geistlich et al. (1992)
Lombo et al. (1999)
Voegtli et al. (1994)
genes of known function, some of which could clearly be
related to riboflavin production. This approach also allowed
the finding of sites within the genome with high transcriptional activity during riboflavin biosynthesis that are suitable integration loci for the target genes found.
Gene expression analysis of wild-type and improved
production strains of Saccharopolyspora erythraea and S.
fradiae using microarrays of S. coelicolor revealed that
regulation of antibiotic biosynthetic enzymes as well as
enzymes involved in precursor metabolism were altered in
FEMS Microbiol Rev 30 (2006) 187–214
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3. Engineering of
well-known pathways
5-fold increase in spiramycin
1.6-fold increase in mithramycin
Global regulators
Increase in actinorhodin and
undecylprodigiosin
2.2. Disrupting negative regulatory genes 1.5-3.5 fold increase in avermectin
Pathway specific regulators
increase in methylenomycin; in mitomycin C; 7- to 10-fold in lovastatin
Global regulators
increase in actinorhodin and
undecylprodigiosin
3.1. Kinetic analysis
2- to 5-fold increase in cephamycin C
Chen et al. (1988)
Otten et al. (1990)
Decker et al. (1994)
Madduri et al. (2001)
Crameri & Davies (1986)
201
Genetic improvement of processes yielding microbial products
FEMS Microbiol Rev 30 (2006) 187–214
mutations at a very low controlled rate (Stemmer, 1994;
Zhao & Arnold, 1997). Unlike site-directed mutagenesis,
this method of pooling and recombining parts of similar
genes from different species or strains has yielded remarkable improvements in enzymes in a very short amount of
time (Patten et al., 1997). A step forward in this technique
was breeding a population with high genetic variability as a
starting point to generate diversity (DNA Family Shuffling).
This approach led to a 240- to 540-fold improvement in
cephalosporinase activity when four cephalosporinase genes
were shuffled as a starting point (Crameri et al., 1998).
When each of these genes was shuffled independently, only
eight-fold improvements were obtained. Innovations that
expand the formats for generating diversity by recombination include formats similar to DNA shuffling and others
with few or no requirements for parental gene homology
(Kurtzman et al., 2001; Lutz et al., 2001).
Random redesign techniques are currently being used to
generate enzymes with improved properties such as: activity
and stability at different pH values and temperatures (Ness
et al., 1999), increased or modified enantioselectivity (Jaeger
& Reetz, 2000), altered substrate specificity (Suenaga et al.,
2001), stability in organic solvents (Song & Rhee, 2001),
novel substrate specificity and activity (Raillard et al., 2001),
increased biological activity of protein pharmaceuticals and
biological molecules (Patten et al., 1997; Kurtzman et al.,
2001) as well as novel vaccines (Marshall, 2002; Locher et al.,
2004). Proteins from directed evolution work were already
on the market by 2000 (Tobin et al., 2000). These were green
fluorescent protein of Clontech (Crameri et al., 1996) and
Novo Nordisk’s LipoPrimes lipase.
‘Whole genome shuffling (WGS)’ is a novel technique for
strain improvement combining the advantage of multiparental crossing allowed by DNA shuffling with the recombination of entire genomes. This method was applied
successfully to improved tylosin production in S. fradiae
(Zhang et al., 2002). Historically, 20 cycles of classical strain
improvement at Eli Lilly and Co. carried out over 20 years
employing about one million assays improved production
six-fold. In contrast, two rounds of WGS with seven early
strains each were sufficient to achieve similar results in one
year and involved only 24 000 assays. Such recursive
genomic recombination has also been used to improve the
acid-tolerance of a commercial lactic acid-producing Lactobacillus sp. (Patnaik et al., 2002).
‘Combinatorial biosynthesis’ is being used for the discovery of new and modified drugs (Hutchinson, 1998;
Reeves, 2003). In this technique, recombinant DNA techniques are utilized to introduce genes coding for antibiotic
synthases into producers of other antibiotics or into nonproducing strains to obtain modified or hybrid antibiotics.
The first demonstration of this technology involved gene
transfer from a streptomycete strain producing the
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those mutated strains (Lum et al., 2004). The S. erythraea
overproducer expressed the entire erythromycin gene cluster
for several more days than the wild-type. It seemed that the
eryA gene and protein expression differences observed for
the overproducer could account for over 50% of the total
erythromycin titer increase. The S. fradiae mutant expressed
the tylosin biosynthetic genes in a similar way to the wildtype strain; however, two genes, aco (encoding acyl-CoA
dehydrogenase) and icmA (encoding isobutyryl-CoA mutase), were expressed more highly than in the wild-type
strain. The induction of these two genes could increase the
flux of metabolites from fatty acids to tylosin precursors in
the overproducer.
These recent technologies and mathematical approaches
will all contribute to the generation and characterization of
microorganisms able to synthesize large quantities of commercially important metabolites. The ongoing sequencing
projects involving hundreds of genomes, the availability of
sequences corresponding to model organisms, new DNA
microarray and proteomics tools, as well as the new techniques for mutagenesis and recombination described above
will accelerate strain improvement programs. The development and combined application of these technologies will
help to develop what was already succinctly described
several years ago as ‘Inverse netabolic engineering’ (Bailey
et al., 1996), which means to identify, construct or calculate
a desired phenotype, identify the molecular basis of that
desirable property, and incorporate that phenotype into
another strain or other species by genetic and environmental
manipulations.
‘Directed evolution’ (= applied molecular evolution = directed molecular evolution) is a rapid and inexpensive way of finding variants of existing enzymes that work
better than naturally occurring enzymes under specific
conditions (Kuchner & Arnold, 1997; Skandalis et al., 1997;
Arnold, 1998). The process involves evolutionary design
methods using random mutagenesis, gene recombination
and high-throughput screening (Arnold, 2001). Diversity is
initially created by in vitro mutagenesis of the parent gene
using repeated cycles of mutagenic polymerase chain reaction (error-prone PCR) (Leung et al., 1989), repeated
oligonucleotide-directed mutagenesis (Reidhaar-Olson
et al., 1991), mutator strains (Bornscheuer et al., 1998) or
chemical agents (Taguchi et al., 1998). A key limitation of
these strategies is that they introduce random ‘noise’ mutations into the gene at every cycle and hence improvements
are limited to small steps. This strategy has been used
successfully in different applications (Zhao et al., 2002).
‘Molecular breeding techniques’ (DNA shuffling, Molecular BreedingTM) come closer to mimicking natural
recombination by allowing in vitro homologous
recombination (Ness et al., 2000). These techniques not
only recombine DNA fragments but also introduce point
202
Concluding remarks
Microorganisms produce many compounds of industrial
interest. These may be very large materials such as proteins,
nucleic acids, carbohydrate polymers, or even cells, or they
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c
can be smaller molecules that can be essential for vegetative
growth or inessential, i.e. primary and secondary metabolites, respectively. The power of the microbial culture in the
competitive world of commercial synthesis can be appreciated by the fact that even simple molecules are made by
fermentation rather than by chemical synthesis. Most natural products are so complex that they probably will never
be made commercially by chemical synthesis. Strains isolated from nature produce only tiny amounts of product.
This is because they need these secondary metabolites for
their own competitive benefit, and they do not overproduce
these metabolites. Regulatory mechanisms have evolved in
microorganisms which enable a strain to avoid excessive
production of its metabolites, thus, strain improvement
programs are required for commercial application. The goal
is to isolate cultures exhibiting desired phenotypes. Most
commonly, the ability of a strain to improve titer is what is
desired, although the other traits may also be improved on.
The tremendous increases in fermentation productivity and
the resulting decreases in costs have come about mainly by
using mutagenesis. In recent years, recombinant DNA
technology has also been applied. The promise of the future
is via extensive use of new genetic techniques such as: (i)
metabolic engineering accomplishing quantification and
control of metabolic fluxes and including inverse metabolic
engineering and transcript expression analyses such as
association analysis and massive parallel signature sequencing; (ii) directed evolution; (iii) molecular breeding including DNA shuffling and whole genome shuffling; and
(iv) combinatorial biosynthesis. These efforts will facilitate
not only the isolation of improved strains but also the
elucidation and identification of new genetic targets to be
used in strain improvement programs.
Acknowledgements
The authors thank the following colleagues for supplying
information: Richard H. Baltz, Graham S. Byng, Richard P.
Elander, David A. Hopwood, Daslav Hranueli, Krishna
Madduri, Jaraslav Spizek, William R. Strohl and J. Mark
Weber.
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