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

OVERPRODUCTION STRATEGIES FOR MICROBIAL SECONDARY METABOLITES: A REVIEW

NAFISEH DAVATIA AND MOHAMMAD. B HABIBI NAJAFIB*

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

ABSTRACT

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.

INTRODUCTION

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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 great degree and thus provide opportunities for 1. STRATEGIES FOR OVER- basicinvestigations of enzymology, control and PRODUCTION OF MICROBIAL 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, i.e.by 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 havealso been used widely in small amounts (mg L- secondary metabolismbranch, genes at a third level 1 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 1Morphogenesis (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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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) (STRAINIMPROVMENT) 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 Amycolatopsismediterranei. on the development of more effective processes for Theactinomycetebutanolides exert their effects via

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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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). 2 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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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- TO INCREASE SECONDARY 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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Review Article ISSN 2250-0480 Vol 3/Issue 1/Jan-Mar 2013 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

Table1 Genetic techniques used to increase secondary metabolite production

CONCLUSIONS

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

REFERENCES

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4. Anon. Morphogenesis. (cited 2012 September Production. Advances in Applied 12). Available fromhttp://en.wikipedia.org/ Microbiology. 2004;56:155-184. wiki/Morphogenesis. 10. Pérez-Llarena F J, Liras P, Rodríguez-García 5. Barrios Gonzalez J, Fernandez FJ and Tomasini A and Martín J F. A Regulatory Gene (ccaR) A. Microbial secondary metabolites production Required for Cephamycin and Clavulanic Acid and strain improvement. Indian Journal of Production in Streptomyces clavuligerus: Biotechnology. 2003; 2:322-333. Amplification Results in Overproduction of 6. Demain AL. Induction of microbial secondary Both b-Lactam Compounds. Journal of metabolism. International Microbiol. Bacteriology. 1997;179(6):2053–2059. 1998;1:259-264. 11. Raina S, Murphy T, De Vizio D, Reffatti P, 7. Fraenkel Gottfried S. The raison d'Etre of Keshavarz T. Novel Strategies for secondary plant substances. Science. 1959; 129 Overproduction of Microbial Products. (3361):1466–1470. Chemical Engineering Transactions.2011; 24: 8. Hugenholtz J and Kleerebezem M. Metabolic 847-852. engineering of lactic acid bacteria: overview of 12. Stamp N. Out of the quagmire of plant defense the approaches and results of pathway rerouting hypotheses.The Quarterly ReviewofBiology. involved in food fermentations. Current 2003; 78 (1): 23–55. Opinion in Biotechnology. 1999;10:492–497. 13. Yup Lee S, Uk Kim H, Hwan Park J, Myung 9. Ochi K, Okamoto S, Tozawa Y , Inaoka T, Park J and Yong Kim T. Metabolic engineering Hosaka T, Xu J and Kurosawa K. Ribosome of microorganisms: general strategies and drug Engineering and Secondary Metabolite production. Reviews Drug Discovery Today. 2009;14: Numbers 1/2.

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