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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013




Ph.D Student of Food Microbiology, Department of Food Science &
Technology, Ferdowsi University of Mashhad, Mashhad, Iran
Professor, Department of Food Science & Technology, Faculty of Agriculture,
Ferdowsi University of Mashhad, P. O. Box 91775-1163, Mashhad, Iran


The formation of secondary metabolites is regulated by nutrients, growth rate, feedback control, enzyme
inactivation, and enzyme induction. Regulation is influenced by unique low molecular mass compounds,
transfer RNA, sigma factors and gene products formed during post-exponential development. The synthases of
secondary metabolism are often coded by clustered genes on chromosomal DNA and infrequently on plasmid
DNA. Strategies for overproduction of microbial products can be based on microbial response (Elicitors,
quorum sensing), genetic engineering, metabolic engineering and ribosome engineering. Also molecular genetic
improvement methods include amplification of SM biosynthetic genes, inactivation of competing pathways,
disruption or amplification of regulatory genes, manipulation of secretory mechanisms, expression of a
convenient heterologous protein, combinatorial biosynthesis.

Key words: Secondary metabolites, Overproduction, Microbial, Strategies, Genetic.


Secondary metabolites are organic compounds that metabolites, strategies for overproduction of such
are not directly involved in the normal growth, metabolites with the aim of highlighting strategies
development or reproduction of an organism (Anon, based on genetically modificationmethods.
2008). Secondary metabolites often play an
important role in defense systems of different Regulation of secondary metabolites production
organisms (Stamp N, 2003). Humans use secondary The formation of secondary metabolites is regulated
metabolites as medicines, flavorings, and by nutrients, growth rate, feedback control,
recreational drugs. Microbial secondary metabolites enzymeinactivation, and enzyme induction.
include antibiotics, pigments, toxins, effectors of Regulation is influencedby unique low molecular
ecological competition and symbiosis, pheromones, mass compounds, transfer RNA, sigmafactors and
enzyme inhibitors, immune modulating agents, gene products formed during post-
receptor antagonists and agonists, pesticides, exponentialdevelopment.Thesynthesis of secondary
antitumor agentsand growth promoters of animals metabolites areoftencoded by clustered genes on
and plants. They have a majoreffect on the health, chromosomal DNA and infrequently on plasmid
nutrition and economics of our society. This paper DNA. Unlike primary metabolism, thepathways of
discusses in detail the regulation of secondary secondary metabolism are still not understood to a

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great degree and thus provide opportunities for 1. STRATEGIES FOR OVER-
basicinvestigations of enzymology, control and
differentiation. Secondary metabolism is brought on
by exhaustion of anutrient, biosynthesis or addition PRODUCTS
of an inducer, and/or by a growth rate decrease.
These events generate signals which affecta cascade 1.1. Microbial Response(Quorum Sensing,
of regulatory events resulting in chemical Elicitation)
differentiation (secondary metabolism) and 1.1.1. Elicitors
morphologicaldifferentiation (morphogenesis1). The Environmental abiotic and biotic stress factors have
signal is often a lowmolecular weight inducer which been proved to effect variety ofresponses in
acts by negative control, binding to and microbes. Elicitors, as stress factors, induce or
inactivating a regulatory protein enhance the biosynthesis ofsecondary metabolites
(repressorprotein/receptorprotein) which normally added to a biological system (Raina S et al, 2011).
prevents secondary metabolism and morphogenesis They are classified into variousgroups based on
during rapid growth andnutrient sufficiency. their nature and origin: physical or chemical, biotic
Nutrient/growth rate/inducer signalspresumably or abiotic.Initial studies on elicitation of secondary
activate a “master gene” which either acts at the metabolites werecarried out on plant cells and
level of translation by encoding a rare tRNA, or by extended, over the years, to bacteria, animal cell
encoding a positive transcription factor. Such cultures and filamentous fungi. Abiotic stress
master genes control bothsecondary metabolism and (abiotic elicitors) imposed bypH improves pigment
morphogenesis. At a second levelof regulatory production by Monascuspurpureus and antibiotic
hierarchy genes could exist which control production by Streptomyces spp.
onebranch of the cascade, i.e. either secondary Traditionallycarbohydrates have been used as
metabolism ormorphogenesis but not both. In the carbon sources in fermentation processes. They
secondary metabolismbranch, genes at a third level havealso been used widely in small amounts (mg L-
could control formation ofparticular groups of ) as elicitor molecules in bacterial andfungal
secondary metabolites. At a fourth levelthere may fermentations for overproduction of commercially
be genes which control smaller groups, and important secondarymetabolites.In one approach to
finally,fifth level genes could control individual improve production, the effect of carbohydratebiotic
biosynthetic pathways;these are usually positively elicitors(oligosaccharides, oligomannuronate,
acting but some act negatively.There are also oligoguluronate and mannan- oligosaccharides) on
several levels of hierarchy on the morphogenesis variety of fungal systems:
branch. The second level could include genes which Penicilliumspp.,Ganodermaspp., Corylopsisspp. and
controlaerial mycelium formation in filamentous bacterialcultures: Streptomyces spp., Bacillus spp.
organisms plus allthe sporulation genes lower in the for production of antibiotics, enzymes, pigments
cascade. Each third levellocus could control a and changes in morphology was investigated (Raina
particular stage of sporulation. Some ofthese loci S et al, 2011).
code for sigma factors. Feedback regulation also
1.1.2. Quorum Sensing
isinvolved in secondary metabolite control (Demain Quorum sensing is the communication between
AL, 1998). cells through the release of chemical signals when
celldensity reaches a threshold concentration
(critical mass. Under these conditions, they sense
the presence of other microbes. This process,
investigated for more than30 years, was first
Morphogenesis (from the Greek morphê shape and genesis creation, discovered in Gram-negative bacteria, and then in
literally, "beginning of the shape") is the biological process that causes
an organism to develop its shape. It is one of three fundamental
Gram-positivebacteria and dimorphic fungi (Raina
aspects of developmental biology along with the control of cell S et al, 2011). The quorum sensing signals differ in
growth and cellular differentiation(Anon, 2012).

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different microbialsystems; examples are acyl- receptor proteins which normally repress
homoserine lactones, modified or unmodified chemicaland morphological differentiation
peptides,complex γ -butyrolactone molecules and (secondary metabolism and differentiation intoaerial
their derivatives. mycelia and spores respectively) but, when reacted
A numberofphysiologicalactivities of microbes(e.g. with butanolide, can no longer function.
symbiosis, competence, conjugation, sporulation, Homoserine lactones of Gram-negative bacteria
biofilmformation, virulence, motility and the function athigh cell density and are structurally
production of various secondary metabolites) are related to the butanolides. They turn on plantand
regulated through the quorum-sensing.Thereis great animal virulence, light emission, plasmid transfer,
potential for the use of this communication process and production of pigments, cyanide and β-lactam
for industrial exploitation.Filamentous fungi are a antibiotics. They are made by enzymes homologous
main microbial source for production of to Lux1, excreted by the cell, enter other cells at
pharmaceutical andbiotechnological products. high density, bind to a Lux Rhomologue, the
However, until recently, very little was reported in complex then binding to DNA upstream of genes
theliterature regarding quorum sensing phenomena controlled by “quorum sensing”and turning on their
in these fungi. Scientistsexplored, for thefirst time, expression. Quorum sensing also operates in the
the possibility of overproduction of fungal case of thepeptide pheromones of the Gram-positive
metabolites inresponse to the supplementation of bacteria. Here, secretion is accomplishedby an ATP
liquid cultures by variety of quorum binding cassette (ABC transporter), the secreted
sensingmolecules.Bacillus licheniformisis widely pheromone beingrecognized by a sensor component
present in the environment. Its metabolic diversity of a two-component signal transduction system
hasresulted in its use for production of enzymes, (Demain AL, 1998).The pheromone often induces
antibiotics and fine chemicals. bacteriocin produced its own synthesis as well as those proteins
by B. licheniformisis a polypeptide antibiotic active involvedin protein/peptide antibiotic (including
against Gram positiveand some Gram-negative bacteriocins and lantibiotics) production,virulence
bacteria. bacteriocinis also used as animal feed and genetic competence. The B-factor of A.
additive (Raina S et al, 2011). Sclerotiorin Mediterraneiis an inducer ofansamycin (rifamycin)
synthesized by Penicilliumsclerotiorumis a formation (Demain AL, 1998).
phospholipase A2 inhibitorand has been classified
as an octaketide. Sclerotiorin has also been studied 2. GENETICENGINEERING
for itscholesterol ester transfer protein (CETP)
inhibitory activity and recently, the extracts from
Penicilliumsclerotiorum have been studied for their
Improvement of the productivity of commercially
activity against methicillin resistant Staphylococcus
viable microbial strains is an important field in
aureus (MRSA) (Raina S et al, 2011).
microbiology, especially since wildtype strains
Precursors often stimulate production of
isolated from nature usually produce only a low
secondary metabolites either byincreasing the
level (1–100 g/ml) of antibiotics. Therefore, a great
amount of a limiting precursor, by inducing a
deal of effort and resources have been committed to
biosynthetic enzyme (synthase) or both. These are
improving antibiotic-producing strains to meet
usually amino acids but other small molecules
commercial requirements.Although classical
alsofunction as inducers. The most well-known are
methods are still effective even without using
the auto-inducers which include butyrolactones
genomic information or genetic tools to obtain
(butanolides) of the actinomycetes, N-acyl-
highlyproductive strains, these methods are always
homoserine lactones of Gram-negativebacteria,
time and resource consuming. One of the current
oligopeptides of Gram-positive bacteria, and B-
topics is to use microorganisms for bioremediation.
factor (3’-[1-butylphosphoryl] adenosine) of
Environmental protection efforts have been focused
on the development of more effective processes for
Theactinomycetebutanolides exert their effects via

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the treatment of toxic wastes. Soil bacteria have a Relied on mutation, followed by random screening,
wide range of metabolic abilities that make them then careful fermentation tests are performed and
useful tools for mineralization of toxic compounds new improved mutants are selected. Physical
(Ochi K et al 2004). mutagens such as UV-light orchemicalmutagens
such as N-methyl-N’–nitro-N-nitrosoguanidineand
2.1. Overproduction of Primary Metabolites ethyl ethanesulphonate are used in these
Overproduction Of Primary Metabolitesbased on methods.Advantagesof Classical genetic methods
genetic engineering is regulated byfeed back are simplicity, no need to sophisticated equipment,
inhibition by the end product of a particular minimal specialized technical manipulation,
pathway is suppressed by generation of auxotrophs effectiveness (rapid titer increases) the only
(i.e. mutation to cause accumulation of metabolite drawback, is labour intensive.
of interest), mutants resistant to antimetabolites
through modification of enzyme structure at 2.3.2. MutationandRational Selection (Directed
allosteric site, modification of operator or regulator Selection Techniques)
gene to express the enzyme constitutively (Barrios Selection for a particular characteristic of the
Gonzalez J et al, 2003). desired genotype, different from the one of final
interest, but easier to detect. Eliminate all
2.2.Overproduction of Secondary Metabolites undesirable genotypes, allowing very high numbers
Overproduction Of Secondary Metabolites based on of isolates to be tested easily.Design of these
genetic engineering is regulated bythe structural methods requires: some basic understanding of the
genes (directly participating in their biosynthesis), product metabolism and pathway regulation.For
regulatory genes, antibiotic resistance gene example addition a toxic precursor of penicillin to
(immunizing responsible for their own metabolites) the agar medium of penicillin producing
and genes involved in primary metabolism microorganisms prevents the growth of sensitive
(affecting the biosynthesis of secondary strains and only resistant mutants with more
metabolites).Improvement strain advantages include penicillin production propagated. The other
increasing yields of the desired metabolite, removal example, addition of rose Bengal and thymol to the
of unwanted co-metabolites, improving utilization medium of carotenoid producing yeast exposed to
of inexpensive carbon and nitrogen sources, visible light has been used to select carotenoid over
alteration of cellular morphology to a form better producing strains(Barrios Gonzalez J et al, 2003).
suited for separation of the mycelium from the
product and/or for improved oxygen transfer in the 2.3.3. RecombinationMethods
fermenter(Barrios Gonzalez J et al, 2003).Genetic Recombination by protoplast fusion between related
engineering methods are divided into two species of fungi with genetic development (high
groupsnamely: Classical genetic methods and levels of a SM) and new isolate (low levels of a new
Molecular genetic improvement methods. SM) results in high productivity of the newly
identified SM from the two strains.
2.3.Classical Genetic Methods
I. mutation and random selection 2.4. Molecular Genetic ImprovementMethods
II. mutation and rational selection Requirementknowledge and tools to perform
III. Genetic recombination methods molecular genetic
Mutation: Mutant generation of the existing wild improvementinclude,identification of biosynthetic
strains is the most practiced strategy for enhancing pathway, adequate vectors and effective
the yield of primary and secondary transformation protocols.The main strategies being
metabolites.Mutant generation has improved the used in molecular genetic improvement of
yield of certain antibiotics by 15- 400 times in SMproducing strains are as follow:
comparison to wild strains.
2.3.1. Mutation and Random Selection

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2.4.1. Amplification ofSMBiosynthetic Genes Amplification of a regulatory gene (ccaR1) hasled to

(Targeted duplication or amplification of SM 3 fold overproduction of β-lactam compounds(in
production gene) streptomycesclavuligerus). Also disruption of
This strategy is divided into two approaches: negatively acting regulatory gene mmy of
• Targeted gene duplication (or amplification): methylenoomycin biosynthesis has led to 17 fold
Identify a neutral site in the chromosome where overproduction of actinorhodine. In another
genes can be inserted without altering the investigation, Introduction of a single copy of the
fermentation properties of the strain. Then the positively acting gene act II has led to 35 fold over
neutral site is cloned and incorporated into the production actinorhodine (instreptomycescoeli
vector with the antibiotic gene. After color) (Barrios Gonzalez J et al, 2003).
transformation, the gene is inserted into the
chromosomal neutral site by homologous 2.4.4. Manipulation of Secretory Mechanisms
recombination.Example for neutral site cloning: Several protein hyper producing yeast strains have
targeted duplication of the tylF gene that encodes been constructed by increasing specific genes of
the rate limiting O-methylation of macrocin in the secretion path (such askar2 and pdi1 genes) or by
tylosin biosynthesis in an industrial production disruption of genes like pmr1gene (Barrios
strain of streptomycesfradiae. Transformants that Gonzalez J et al, 2003).
contained two copies of the tylFgene produced 60%
more tylosin than the parental strain were developed 2.4.5. Expression of a Convenient Heterologous
(Barrios Gonzalez J et al, 2003). Protein2
Incorporation a new enzymae in the strain
• Whole pathway amplification (heterologous gene) that will lead to the formation
of a new related product of industrial interest
2.4.2. Inactivation ofCompeting Pathways (Barrios Gonzalez J et al, 2003).
Block a pathway that competes for a common
intermediate key precursors such as cofactors, 2.4.6. CombinatorialBiosynthesis
reducing power and energy supply are being used to Development of novel antibiotics, by using non-
perform this strategy. Improved strains by this conventional compounds as substrate for the
strategy could be able to channel the precursors to biosynthetic enzymes of the microorganism. These
the SM biosynthesis. Transposonmutagenesis in enzymes can be modified or mutated insuch a way
actinomycetes, gene disruption, or inserting an as to increase their affinity for those unnatural
antisense synthetic gene are used to perform substrates.Different activity modules of enzymes
suchstrategy.For example α-aminoadipic acidisa like polyketid synthases can be rearranged by
precursor of penicillin biosynthesis, also acts as genetic engineering to obtain a microbial strain that
branching point to lysine synthesis. Disruption of synthesizes an antibiotic with novel characteristics.
gene lys2 connects α-aminoadipic acid towards For Example expression of glycosyltransferase
lysine has generated auxotrophs of amino acid with genes from A.orientalis in Streptomyces
100% increase in penicillin yields (Barrios toyocaensis (producer of the non-glycosylated
Gonzalez J et al, 2003). hepta-peptid) generate novel monoglycosilated
derivative (Barrios Gonzalez J et al, 2003).
2.4.3. Disruption or Amplification
ofRegulatoryGenes 1
ccaRis A regulatory gene that located within the cephamycin gene
Regulation at a molecular level is more complicated cluster of Streptomyces clavuligerus, is linked to a gene (blp)
than identifying the biosynthetic pathway and encoding a protein similar to a b-lactamase-inhibitory protein.
cloning the corresponding genes.For example Expression of ccaRis required for cephamycin and clavulanic acid
biosynthesis in S. Clavuligerus( Pérez-Llarena F J et al 1997).
In cell biology and protein biochemistry, heterologous expression
means that a protein is experimentally put into a cell that does not
normally make (i.e., express) that protein(Anon, 2011).

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metabolic engineering can be performed to achieve

3. METABOLIC ENGINEERING desired goals(Yup Lee S et al, 2009).
Homofermentative lactic acid bacteria have a
relativelysimple metabolism completely focused on
Since the advent of recombinant DNA technology,
the rapid conversionof sugar to lactic acid. The
genetic engineering of cells, particularly
general habitat of lacticacid bacteria are nutritious,
microorganisms, has been successfully practiced for
high-sugar-containing environments.Under these
the development of strains capable of
conditions, the lactic acid bacteria havedeveloped a
overproducing recombinant proteins and small
typical metabolism that allowrapidsugar conversion
molecule chemicals. For the latter, strategies
and is devoid of most biosynthetic activities.Under
beyond simple genetic engineering are often
normal food fermentation conditions, the
required as they are synthesized through multiple
mainproduct of metabolism is lactic acid, but other
intracellular reactions, which are further
products areformed as by-products, such as acetic
complicated by various factors including cofactor
acid, acetaldehyde, ethanol, and diacetyl, all
balance and regulatory circuits. Metabolic
contributing to the specific flavor of fermented
engineering can be defined as purposeful
products. The main function of this
modification of cellular metabolism using
sugarmetabolism is to generate the energy necessary
recombinant DNA and other molecular biological
for rapidgrowth and for maintenance of intracellular
techniques. Metabolic engineering considers
pH during acidificationof theenvironment.The
metabolic and cellular system as an entirety and
biosynthetic capacity of these food microorganisms
accordingly allows manipulation of the system with
is very limited. The building blocks for growth
consideration of the efficiency of overall
generally originate from hydrolysis of food protein.
bioprocess, which distinguishes itself from simple
The bacteria possess an elaborate proteolytic system
genetic engineering. Furthermore, metabolic
centred on the complete breakdown of protein
engineering is advantageous in several aspects,
fragments into free aminoacids. The amino acids are
compared to simple genetic engineering or random
subsequently taken up andused for cell-protein
mutagenesis, since it allows defined engineering of
synthesis or for modification reactionsin the
the cell, thus avoiding unnecessary changes to the
biosynthesis of other nitrogen compounds, such
cell and allowing further engineering if
asvitamins and nucleotides.There is almost no
necessary.Many drugs and drug precursors found in
overlap between the energy (carbon)metabolism
natural organisms are rather difficult to synthesize
and the biosynthesis (nitrogen) metabolism inlactic
chemically and to extract in large amounts.
acid bacteria. This makes them ideal as targets
Metabolic engineering is playing an increasingly
formetabolic engineering. Either metabolism can be
important role in the production of these drugs and
changeddramatically without influencing the other
drug precursors. This is typically achieved by
as long as energygeneration or biosynthesis of cell
establishing new metabolic pathways leading to the
material is undisturbed.
product formation, and enforcing or removing the
existing metabolic pathways toward enhanced
3.1. Production of Exopolysaccharides
product formation. Recent advances in system
Although exopolysaccharide (EPS) production is a
biology and synthetic biology are allowing us to
result of abiosynthetic pathway, and not an energy
perform metabolic engineering at the whole cell
generating pathway,it is closely linked to the
level, thus enabling optimal design of a
general glycolysis reactions. The sugarmoieties in
microorganism for the efficient production of drugs
the polysaccharide repeating unit are supplied
and drug precursors. Among the many successful
bysequential addition reactions of ‘activated’ sugar–
examples of metabolic engineering, recent reports
nucleotidebuilding blocks by specific
on the efficient production of L-valine, L-threonine,
glycosyltranferases. Thesesugar–nucleotides, such
lycopene, antimalarial drug precursor, and
as UDP–glucose, UDP–galactose and TDP–
benzylisoquinoline alkaloids represent how
rhamnose, are all synthesized from glucose-1-

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phosphate as a general precursor. The conversion of vitaminsbecause the growth ofthese bacteria is
theglycolytic intermediate glucose-6-phosphate to strictly dependent on the presence ofsmall amounts
glucose-1-phosphate, catalyzed by the enzyme of these compounds.Still, some lactic acid bacteria
phosphoglucomutase(PGM), and the synthesis of are able to produce vitaminsand it isinteresting to
UDP–glucose from glucose-1-phosphate, catalyzed speculate how production by these lacticacid
by GalU, could very well be controllingpoints in bacteria can be enhanced.
EPS production. Preliminary studies in
laboratoryhave already shown that overexpression 3.3. Proteolysis
of either thepgmor the galUgene results in increased Proteolysis is an essential process for growth of
accumulation of theUDP–glucose and UDP– lactic acidbacteria in milk. Already several years
galactose, respectively, in cells of L.lactis. ago, increased growthrate of L. lactisin milk was
Interestingly, EPS production by L. lactisis much observed upon overproduction ofthe cell-wall-
lower when growing on fructose than on glucose or associated proteinase PrtP. It was alsoreported that
lactose as the energy source. This could be a result in the whole process of protein breakdown
of the low activity of fructose biphosphatase, which topeptides and subsequently to free amino acids, the
is essential forgrowth (and EPS-production) on uptake oflarger peptides from the external medium
fructose but not on theother sugars. Overexpression to the inside of thecell, dictated by the oligopeptide
of the fbpgene in L. lactis, viathe NICE-system, led transporter (OPP), was acrucial step in growth ofL.
to increased intracellular levels ofnucleotide sugars, lactisin milk. This was evidentfrom the inability
accelerated growth and higher levels ofEPS during ofOPP-negative mutants to grow onculture medium
growth on fructose. with casein as the sole source of amino acids.The
complete breakdown of the oligopeptides to single
3.2. Nitrogen Metabolism amino acids, in lactic acid bacteria, is a result of the
Lactic acid bacteria that form the inherent flora simultaneousaction of a whole set of intracellular
infermented foods usually have an intricate peptidases.These peptidases have overlapping
machinery forbreakdown ofprotein. This has been substrate specificityand none of them are
most extensivelystudied in the dairy lactic acid individually essential for growth onthese peptides.
bacteria such as L. lactis and several Lactobacillus This was elegantly shown where the genes coding
spp. This proteolysis provides the lactic acid for the intracellularpeptidases were disrupted
bacteriawith the essential free amino acids for individually and incombinations of two, three, four
growth and, as a result, these bacteria have a very and five different peptidase-disruptions. Only when
limited capacity for thebiosynthesis of amino acids. three or more peptidaseswere disrupted
Some remnants of these reactions remain in specific simultaneously, the growth of L. lactisin milk was
strains, most evident as amino acidconverting clearly effected. The proteolysis and subsequent
reactions resulting in the generation of flavor amino acid conversion bylactic acid bacteria is an
components, for example, methanethiol as the essential process in flavor formation in cheese
product of methione metabolism. Some metabolic during the ripening process. Metabolicengineering
engineering on thelevel of increased proteolysis of lactic acid bacteria, on the level of proteolysis,
and/or flavor production hasbeen undertaken in has been attempted in numerous occasions to
lactic acid bacteria (Figure1).Becausemost of the improve flavor development in cheese. The most
fermentable substrates are rich invitamins, promisingresults, however, have not been gained by
nucleotides and minerals, the resident lacticacid increasedactivity of the enzymes involved, but by
bacteria generally have a limited biosynthetic increased releaseof some relevant enzymes into the
capacityfor these compounds. Lactobacillus is culture medium. By directly controlling lysis of the
especially knownfor its inability to synthesize lactic acid bacteria, resultingin release of
vitamins, such as folic acid, vitamin B12, intracellular peptidases and/or amino acidconverting
panthotenic acid, and so on. In fact, these enzymes, increased flavor formation has
bacteriaare used in the biological assays for these beenobserved. The most effective example of

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acceleratingthe cheese ripening process by complete release of peptidases and ofother enzymes
metabolic engineering, sofar, has been the nisin- and a sharp increase in production of freeamino
induced expression of bacteriophagelysin and holin acids and flavor compounds in cheese.
in L. lactis, resulting in complete lysis of the cells,

Figure 1
Overview of metabolic engineering on the level of proteolysis and biosynthesis of nitrogen compounds.
hol/lys, the holin and lysin from bacteriophage origin; OPP, oligopeptide transfer; PepX, different
peptidases; PrtP, cell- wall proteinase

3.4. Vitamin Production BacillusorPseudomonas) as they could also be

As mentioned before, lactic acid bacteria have a implemented for in situproduction processes, such
very limitedbiosynthetic capability for the as food fermentations (Hugenholtz J and
production of vitamins; however, there are certain Kleerebezem M, 1999).
exceptions. The yogurt bacterium Streptococcus
thermophilus has been observed to producefolic 4. RIBOSOME ENGINEERING
acid which, in fact, stimulates the growth of
theother yogurt bacterium, Lactobacillus
The discovery of microorganisms capable of
bulgaricus. L.lactis also produces substantial
tolerating, or growing on, high concentrations of
amounts of folic acid during fermentation. Many of
organic solvents provides a potentiallyinteresting
the genes coding for the pathway of folic
avenue for development of genetically
acidbiosynthesis have been identified in the
engineeredorganisms for treating hazardous
genome of this bacterium.Also, genes for
wastes. Thus, strain improvement iscrucially
riboflavin (vitamin B2) and biotin (vitamin B6)
important to fully exploit the cell’s
biosynthesis have been identified in L. lactis. This
ability.Sinceresearchers found a dramatic
would make it possible to engineer the
activation of antibiotic production by a certain
productionof these vitamins in these food-grade
ribosomal mutation (a mutation in rpsL gene
bacteria, just asrecently reported for B. subtilis.
encoding the ribosomal protein S12), they had an
Vitamin productionprocesses by lactic acid
idea that bacterialgene expression may be changed
bacteria would have huge advantagesover the
dramatically by modulating the ribosomal proteins
current used processes (by

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or rRNA, eventually leading to activation of secondarymetabolism in this genus have come

inactive(silent) genes.In bacteria, the ribosome from the studies of antibiotic production in
plays a special role for their own geneexpression Streptomyces coelicolor A3 and its close relative
by synthesis of a bacterial alarm one, ppGpp. One Streptomyces lividans.S. coelicolor produces at
of the most important adaptation systems for least four antibiotics, including the blue-pigmented
bacteria is the stringentresponse, which leads to the polyketide antibiotic actinorhodin (Act).S. lividans
repression of stable RNA synthesisin response to normally does not produce Act, although the strain
nutrient limitation. The stringentresponse depends has acomplete set of Act biosynthetic genes.
on the transient increase of hyperphosphorylated However, Act production inthis organism can be
guanosinenucleotideppGpp, which is synthesized activated by the introduction of certain
from GDP and ATPby the relA gene product regulatorygenes or by cultivation under specific
(ppGppsynthetase) in response to binding conditions. A strain of S. lividans, TK24 has been
ofunchargedtRNA to the ribosomal A site. Since found to produce a largeamount of Act under
bacterial secondarymetabolism is often triggered normal culture conditions (Figure2). Genetic
by ppGpp when cells enter into stationaryphase, it analyses revealed that a streptomycin-resistant
is important to take the stringent response into mutation,str-6, in TK24 is responsible for
considerationin activating or enhancing the activation of Act synthesis andthat str-6 is a point
bacterial secondary metabolism. mutation in the rpsL gene encoding ribosomal
protein S12, changing Lys-88 to Glu (K88E
4.1. Antibiotic Overproduction by rpsL mutation). It was alsoshown that introduction of
(RibosomalProtein S12) Mutations streptomycin-resistant mutationsimproves Act
4.1.1. Activation ofActinorhodin Production production in wild-type S. coelicolor (Figure2) and
Members of the genus Streptomyces produce a circumvents the detrimental effects on Act
wide variety of secondarymetabolites that include production in certain developmental mutants (relA,
about half of the known microbialantibiotics. relC, and brgA) of S. coeliolor.
Advances in understanding the regulation of

Figure 2.
Activation of antibiotic production by rpsL (encoding ribosomal protein S12) mutations in Streptomyces
lividans 66 and Streptomyces coelicolorA3(2). Blue color represents an antibiotic, actinorhodin. K88E means
a mutation at lysine-88 altering glutamate.

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These streptomycin-resistant mutations result in the Antibiotic biosynthesis pathways and their genetic
alterationof the Lys-88 to Glu (K88E) or regulatory cascadescomprise one of the most
Arg(K88R) and Arg-86 to His (R86H) intherpsL attractive fields in Streptomyces geneticsand are
gene. In addition to these streptomycin-resistant important in considering strain improvement. Onset
rpsLmutations, aparomomycin-resistant rpsL of themorphological differentiation and the
mutation (P91S) also can activateAct production in secondary metabolism, including antibiotic
S. coelicolor.Thesefindings indicate that the production, are thought to be coupled and
antibiotic production (secondary metabolism) in influenced bya variety of physiological and
streptomycetes is significantly controlled by the environmental factors. Antibiotic production in
translationalmachinery, that is, the Streptomycetes is generally growth phase
‘‘ribosome.’’Much progress has been made in dependent. Thus, the signal molecule for growth
elucidating the organization ofantibiotic rate control, ppGpp, is suggested to play a central
biosynthesis gene clusters in several Streptomyces role in triggering the onset ofantibiotic production
species, and a number of pathway-specific in Streptomyces. Namely, the ribosomes play
regulatory genes have been identified, which are anessential role in adjusting gene expression levels
required for the activation of their cognate by synthesizing ppGpp in response to nutrient
biosyntheticgenes. In the Act biosynthetic gene limitation. There is a positive correlation between
cluster, actII-ORF4 plays such a pathway-specific ppGpp and antibiotic biosynthesis: disruption of the
regulatory role, and the expressionlevel of this gene ppGpp synthetase gene, relA, or a deletion mutation
directly determines the productivity ofAct. Western (designated as relC) in the ribosomal L11 protein
blot analysisusing anti-ActII-ORF4 antibody gene has been shown to lead to a deficiency in
showed that the expression of ActIIORF4protein ppGpp accumulation after amino acid depletion
was strongly enhanced in the Act-high-producing (socalled‘‘relaxed’’ phenotype) accompanied by
rpsL mutant strains. Furthermore, RT-PCR impairment in antibioticproduction. The expression
experiments revealed that the increase ofthis level ofmany genes is regulated by ppGpp, either
regulatory protein can be attributed to the enhanced positively or negatively.Many genetic studies in E.
expressionof actII-ORF4 mRNA. Thus, certainrpsL coli suggested that RNA polymerase (RNAP) is the
mutations enhance expression of the actII-ORF4 target for ppGpp regulation. Genetic analysis
gene, leading tomassive production of Act. reveals that fourmajor functional domains exist in
the RNAP β-subunit (Figure 3). TheppGpp-
sensitivity domain is close to another important
domain of theRNAP β -subunit, the rifampicin
(Rif)-binding domain.
4.2. Antibiotic Overproduction by rpoB (RNA
Polymerase) Mutations

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Figure 3.
Functional map of E.coli RNA polymerase β-subunit and location of rif cluster I within RNA polymerase β-
subunit in relation to the previously suggested ppGpp-binding site in E.coli. Positions of the rif mutations
found in the present study are designated by arrows. Numbering begins at the start codon of the open
reading frame. ML, Mycobacterium leprae; SC, Streptomyces coelicolorA3(2); BS, Bacillus subtilis; EC,
Escherichia coli.

The crystal structure clearly revealed that Rif- (Figure3). More impressively, gene expression
cluster I isinvolved in the E. coli RNAP active analysis revealedthat the restoration of
center. Therefore it is reasonableto consider that actinorhodin production in the relrif double mutant
certain mutations in the Rif-binding domain strains is accompanied by increased expression
couldaffect the activity of RNAP and then may ofthe pathway-specific regulatory gene actII-
affect the function of the adjacent ppGpp-binding ORF4, which normally decreased in the rel
domain. Researcher spostulated that the impaired mutants. Accompanying the restoration of
ability to produce antibiotic due to the relA or relC antibiotic production, the relrif mutants also
mutation may be circumvented by introducing exhibited a lower rate ofRNA synthesis compared
certainRif-resistant (rif) mutations into the RNAP- to the parental strain when grown in anutritionally
subunit. This hypothesisis based on a notion that rich medium. Since the dependence of S.
the mutated RNAPs may behave like coelicolorA3 on ppGpp to initiate antibiotic
‘‘stringent’’RNAP without ppGpp binding. The production can apparently bebypassed by certain
results from rel mutants of S. coelicolor A3 and S. mutations in the RNAP, the mutant RNAP
lividans strongly supported this hypothesis. The mayfunction by mimicking the ppGpp-bound form
Rif-resistant isolates from the rel mutants regained (Figure4). This proposalcan be supported by the
the ability to produce the colored antibiotic fact that the mutant RNAP behaved like
actinorhodin, and various types of point mutation ‘‘stringent’’ RNAP with respect to RNA synthesis,
were mapped inthe so-called Rif-cluster I in the as demonstratedusing cells growing in a
rpoB gene that encodes the RNAP β-subunit nutritionally rich medium (Ochi K et al 2004).

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Figure 4
Hypothesis for ppGpp-independent antibiotic (actinohordin) production. ActII-ORF4 is the gene encodding
a pathway specific regulatory protein Act-ORF4. βrif represents mutated β-subunit. relA and relC are
mutations that block the synthesis of ppGpp.

4.3. Effect of str and rpoB Mutations in 4.3.2. Enzyme Overproduction

Various Bacteria Introduction of drug-resistant mutations has also
4.3.1. Antibiotic Overproduction been verified to beeffective in improving enzyme
Members of the genera Streptomyces, Bacillus, productivity. Several str mutants ofB. subtilis were
and Pseudomonasare soil bacteria that produce a shown to produce an increased amount (20–30%)
high number of agriculturally andmedically of α-amylase and protease. It is shown that rpoB
important antibiotics. The development of rational mutations are alsoeffective for overproduction
approachesto improve the production of antibiotics (1.5-fold to 2-fold) of extracellular enzymessuch as
from these organismsis therefore of considerable amylase and protease. Thus these methods may
industrial and economic beapplicable for overproduction of other enzymes
importance.Theimpairment in antibiotic production produced by variousmicroorganisms, especially at
resulting from a relA or relC mutation(that causes late growth phase(Ochi K et al 2004).
a failure to synthesize ppGpp) could be
completelyrestored by introducing mutations 4.4. Future ProspectsforRibosomeEngineering
conferring resistance to streptomycin(str). It is Researchersdemonstrated that a cell’s function can
apparent that acquisition of certain strmutations be altereddramatically by modulating the ribosome
allows antibiotic production to be initiated without using a drug-resistance mutationtechnique. Their
therequirement for ppGpp. This offers a possible approach is characterized by focusing on
strategy for improvingthe antibiotic ribosomalfunction at late growth phase (i.e.,
productivity.Indeed, in addition to stationary phase). In summary, their novel
actinorhodinproductionby S. coelicolor and S. breeding approach is based on twodifferent
lividans, introduction of a strmutationwas effective aspects, modulation of the translational apparatus
in enhancing antibiotic production by various by inductionof str and gen mutations, and
bacteria(Ochi K et al 2004). modulation of the transcriptionalapparatus by
induction of a rif mutation (Figure 5). Modulation

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ofthese two mechanisms may function elongation factor G and ribosomal protein L11,
cooperatively to increase antibioticproductivity. respectively.However, no mutations were found
Introduction of mutations conferring resistance to within the genes encoding elongationfactor G or
fusidic acid (fus) or thiostrept on (tsp), also causes ribosomal protein L11. It is therefore highly likely
activation of antibiotic production as well as str that these fus and tsp mutations are located on the
mutation. Moreover, these fus and tsp mutations genes encoding rRNAs. This is important because
were found to give rise to anaberrant protein it implies the existence of a new wayto modulate
synthesis activity, as did the str mutant ribosome. ribosomal function, in addition to ribosomal
Resistance to fusidic acid and thiostrept on is protein mutations (Ochi K et al 2004).
known to come frequently from a mutationin

Figure 5.
Scheme of ‘‘ribosome engineering’’ to activate cell’s ability

5. GENETICTECHNIQUES USED nutrition and economics of our society. The best-

known are the antibiotics. These remarkable
groups of compounds form a heterogeneous
METABOLITE PRODUCTION assemblage of biologically active molecules with
different structures and modes of action. They
Genetic techniques used to increase secondary attack virtually every type of microbial activity
metabolite productionare shown in table 1.These such as DNA, RNA, and protein synthesis,
compounds have a major effect on the health, membrane function, electron transport, sporulation,

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germination and many others. Other secondary agonists, pesticides, antitumor agents, immune
metabolites are pesticides, pigments, toxins, suppressives, cholesterol-lowering agents, plant
effectors of ecological competition and symbiosis, protectants and growth promotants of animals and
pheromones, enzyme inhibitors, immune plants (Adrio JL and Demain AL, 2010).
modulating agents, receptor antagonists and

Genetic techniques used to increase secondary metabolite production


The combination of complementary technologies metabolic engineering, ribosome engineering,

such as mutation and genetic recombination has combinatorial biosynthesis and molecular breeding
led to remarkable improvements in the productivity techniques.Functional genomics, proteomics and
of many primary and secondary metabolites as metabolomics are now being exploited for the
well as protein biopharmaceuticals and enzymes. discovery of novel valuable small molecules for
New genetic approaches for the development of medicine and enzymes for catalysis (Adrio JL and
overproducing strains are continuously emerging. Demain AL, 2010).
Among those that have proven to be successful are


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