Production of microbial secondary metabolites ...

Critical Reviews in Microbiology, 2010; 36(2): 146–167

REVIEW ARTICLE

Production of microbial secondary metabolites: Regulation by the carbon source

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Beatriz Ruiz1, Adán Chávez1, Angela Forero1, Yolanda García-Huante1, Alba Romero1, Mauricio Sánchez1, Diana Rocha1, Brenda Sánchez1, Romina Rodríguez-Sanoja1, lizabeth Langley2 Sergio Sánchez1, and E Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510. México, and 2Instituto Nacional de Cancerología, México D.F. 14080. México.

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Abstract Microbial secondary metabolites are low molecular mass products, not essential for growth of the producing cultures, but very important for human health. They include antibiotics, antitumor agents, cholesterollowering drugs, and others. They have unusual structures and are usually formed during the late growth phase of the producing microorganisms. Its synthesis can be influenced greatly by manipulating the type and concentration of the nutrients formulating the culture media. Among these nutrients, the effect of the carbon sources has been the subject of continuous studies for both, industry and research groups. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. Their knowledge and manipulation have been useful either for setting fermentation conditions or for strain improvement. During the last years, important advances have been reported on these mechanisms at the biochemical and molecular levels. The aim of the present review is to describe these advances, giving special emphasis to those reported for the genus Streptomyces. Keywords: Carbon source regulation; secondary metabolites; strain improvement; Bld proteins; phosphoenolpyruvate: phosphotransferase system (PTS); catabolite control protein (CCpA); cyclic AMP (cAMP); carbon catabolite repressor (CreA)

Introduction Microbial secondary metabolites are low molecular mass products of secondary metabolism, usually produced during the late growth phase (idiophase) of a relatively small sort of microorganisms. Secondary metabolites are not essential for the growth of the producing cultures but serve diverse survival functions in nature (Demain and Fang, 2000). Although not essential for microbial growth, secondary metabolites are very important for the health, nutrition, and economics of our societies (Berdy, 2005). Probably the most important use of secondary metabolites has been as anti-infective drugs. In the year 2000, the anti-infective secondary metabolites marketed 55 billion dollars (Barber et al., 2004), but in the year 2007, the market for antibiotics was 66 billion dollars (Demain and Sanchez, 2009).

If modern medicine is to continue in its present form, novel families of antibiotics must enter the marketplace at regular intervals. Recently, new antibiotics have been introduced to the anti-infectious disease market, e.g., augmentin, ceftriaxone and clarithromycin. However, within the next 10 years, more aggressive screening programs for the selection of novel natural and chemical compounds are necessary to produce novel antibiotics against resistant bacteria. In addition to the search for new compounds with antibacterial activity, during the last decades the pharmaceutical industry has extended the screening programs to other disease areas (Cardenas et al., 1998; Kremer et al., 2000; Demain, 2002) such as cholesterol lowering drugs, e.g., statins (Nicholls et al., 2007), anticancer drugs e.g. bleomycin, dactinomycin, doxorubicin and staurosporin (Minotti et al., 2004), immunosuppressants to allow organ

Address for Correspondence: S. Sanchez, Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, México D.F. 04510. México. E-mail: [email protected] (Received 23 July 2009; revised 13 November 2009; accepted 16 November 2009) ISSN 1040-841X print/ISSN 1549-7828 online © 2010 Informa UK Ltd DOI: 10.3109/10408410903489576

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Carbon regulation of secondary metabolism 147 transplantation, e.g., cyclosporine, FK-506, rapamycin and ascomycin (Borel, 2002;), antimycotic agents, e.g., anidulafungin (Ikeda et al., 2007), antihelmintic compounds i.e. ivermectin, milbemycin and spinosyns (Kirst et al., 2002), and gastrointestinal motor stimulators, e.g., EM 574 and mitemcinal (McCallum and Cynshi, 2007). In addition, the National Cancer Institute (NCI) invested considerable effort in evaluating the inhibitory activity of HIV-1 virus by pepstatin A, a small pentapeptide produced by several Streptomyces species with a unique hydroxyamino acid (statine), that sterically blocks the active site of HIV-1 protease (Cragg and Newman, 2001; Yang et al., 2001). The outstanding role of microorganisms in the production of antibiotics and other drugs for treating certain serious diseases is notorious. Currently, with less than 1% of the microbial world having been explored, the advances in techniques for microbial cultivation and extraction of nucleic acids from soil and marine habitats are allowing access to a vast untapped reservoir of genetic and metabolic diversity (Sanchez and Olson, 2005). The main biosynthetic pathways involved in secondary metabolism are those forming aromatic compounds, isoprenes, oligosaccharides, peptides, polyketides, and ß-lactam rings. Knowledge of the pathways varies from cases in which the amino acid sequences of the enzymes and nucleotide sequences of the genes are known (e.g., for cephalosporins and penicillins), to those in which even the enzymatic steps are still unknown (Paradkar et al., 2003). Secondary metabolites are formed via enzymatic pathways that occur via individual proteins, free or complexed, or through parts of large multifunctional polypeptides carrying out a multitude of enzymatic steps, e.g., polyketide synthases and peptide synthetases (Demain, 1998). The genes encoding for enzymes of secondary metabolism are usually chromosomal. For example, the 30 biosynthetic str/sts and blu genes of streptomycin biosynthesis (and of related antibiotics such as 5’-hydroxystreptomycin and bluensomycin) that have been cloned from Streptomyces griseus and Streptomyces glaucescens were all found in a chromosomal region of 30–40 kb (Piepersberg and Distler, 1997). Only a few biosynthetic genes have been shown to be plasmidborne, e.g. those encoding synthesis of methylenomycin A of Streptomyces coelicolor and antibiotic AS-48 of Enterococcus faecalis S-48 (Martinez-Bueno et al., 1990). Whether chromosomal or plasmid-borne, the secondary metabolism genes are usually clustered, especially in prokaryotes, but not necessarily as single operons. Antibiotic clusters in actinomycetes have different sizes. For instance, sizes of, 20 kb or less have been reported for actinorhodin and other simple aromatic polyketides; 90 to 100 kb for rapamycin and rifamycin and other

complex polyketides. In the latter, 50–80 kb of DNA are needed for the modular PKSs themselves (Hopwood, 1999). Clusters of fungal biosynthetic genes have also been found for biosynthetic processes of sterigmatocystin by Aspergillus nidulans (Brown et al., 1996) and trichothecenes by Fusarium sporotrichioides (Hohn et al., 1995). Secondary metabolism usually occurs at the late growth phase of the producing microorganisms. The temporal nature of secondary metabolism is certainly genetic in nature but expression can be influenced greatly by environmental manipulations. Therefore, secondary metabolism is often brought on by exhaustion of a nutrient, or addition of an inducer and/or by a decrease in growth rate (Bibb, 2005). These events generate signals that cause a cascade of regulatory events resulting in chemical (secondary metabolism) and morphological differentiation (morphogenesis) of the microbial secondary metabolite producers. The signal is often a low molecular weight butyrolactone inducer that acts by binding to and inactivating a regulatory protein (repressor protein/ receptor protein) that normally prevents secondary metabolism and morphogenesis during rapid growth and nutrient sufficiency (Ohnishi et al., 2005). Formation of antibiotics is also regulated by nutrients (nitrogen, phosphorous and carbon source), metals, growth rate, feedback control and enzyme inactivation (Sanchez and Demain, 2002). Among the nutrients, the effect of carbon source on secondary metabolite production has been the subject of continuous study for both, industry and research groups, not only from the fermentation, but also from the biochemical and molecular biological stand point. In this work, we will review the regulation of the synthesis of microbial secondary metabolites by carbon source, as well as the mechanisms involved in it. Special emphasis will be given to secondary metabolites produced by the genus Streptomyces.

Regulation by the carbon source In microbial cells, glucose, usually an excellent carbon source for growth, interferes with the formation of many secondary metabolites “too much of a good thing can be bad” (Demain, 1989). In media containing a mixtures of a rapidly and slowly-used carbon sources, the former is used first to produce cells but little or no secondary metabolites are synthesized. After the rapidly-assimilated compound is depleted, the “second-best” carbon source is used for idiolite formation. Carbon regulation depends on the rapid utilization of the preferred carbon source. One distinctive characteristic of secondary metabolism is its association with low growth levels (Demain, 1989), therefore, low concentrations of the repressing carbon source in the


148 Ruiz et al. culture medium give low growth rates and eliminate the interference over antibiotic biosynthesis. Several mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. These mechanisms show important differences depending on the type of microorganism being considered. For a detailed review from the above mentioned mechanisms, refer to the works of Deustscher (2008) and Görke and Stülke (2008), respectively. Only a few characteristics will be mentioned in this review. However, in the present work major emphasis will be given to the advances in the mechanism of carbon catabolite repression (CCR) in the genus Streptomyces.

Carbon source regulation of secondary metabolite production in Gram-positive bacteria Gram-positive bacteria are characterized by having peptidoglycans as well as polysaccharides and/or teichoic acids as part of their cell wall structure. In addition, the guanine and cytosine (GC) content of the DNA significantly varies among these bacteria. Thus, the phylum Firmicutes corresponds to Gram-positive bacteria with low GC content and the phylum Actinobacteria contain high GC in its DNA structure. Firmicutes include the classes Bacilli, Chlostridia, and Mollicutes. Actinobacteria include the order Actinomycetales. Gram-positive bacteria have been reported as being able to produce secondary metabolites either through ribosomal or non-ribosomal mechanisms. In this regard the orders Actinomycetales, Bacillales, and Lactobacillales deserve special mention. Actinomycetales The Actinomycetales are filamentous, often branching and morphologically diverse bacteria (Madigan et al., 2003). They are mostly Gram-positive eubacteria, although some species with complex cell wall structures do not show clear Gram-staining. Most species are saprotrophic, but a few are pathogenic in plants or animals, including humans. In addition, they are primarily aerobic and chemo-organotrophic microorganisms with high GC-content. Actinomycetales include families like the corynebacteriaceae, pseudonocardiaceae, streptomycetaceae, nocardiaceae, etc. (Garrity et al., 2004) and have received special attention either because of their industrial applications or their capability for causing human diseases. Because of their importance as secondary metabolite producers, the streptomycetaceae, nocardiaceae, and corynebacteriae deserve special attention.

Streptomyces The Streptomyces genus has been found in different environments, which frequently turn out to be complex and adverse. Therefore, in order to develop, they must compete with other microorganisms for the nutrients present in the environment. For this purpose, they have the ability to produce a wide range of secondary metabolites, such as antibacterial and antifungal substances (Xiong et al., 2004). In addition, nutrients in nature are usually present in a wide range of complexity so microorganisms must have the necessary tools to succeed in their utilization. For this purpose, 819 potentially secreted proteins have been predicted to operate in S. coelicolor. Among them, amylases, cellulases/endoglucanases, chitinases/chitosanases, proteases/peptidases, and pectate lyases are of special importance and many of these enzymes also have commercial interest (Bentley et al., 2002). Furthermore, due to their capability to degrade multiple natural polymers, the streptomycetes play an important role in soil ecology. Very often, the complex and changing environmental conditions can produce microbial stress. To face this, streptomycetes are armed with many proteins (614 for S. coelicolor), which help this genus survive adverse conditions. These protective proteins include transporters, drug efflux proteins and hydrolases (Bentley et al., 2002). The high physiological versatility of streptomycetes, their complex life cycle and ability to produce a great variety of secondary metabolites is reflected in their genome size. With sizes of 8.54, 8.7 and 9.03 Mb, the genomes of S. griseus, S. coelicolor and Streptomyces avermitilis, respectively, are among the largest genome sequences found in the microbial world (Ohnishi et al., 2008; Weber et al., 2003). They have a higher GC-content (more than 70%) than nearly all other organisms. Unlike most other eubacterial chromosomes, the chromosome of this genus is linear with terminal-inverted repeats and covalently bound terminal proteins at the 5’ end. Housekeeping genes are mainly found in the central section of the linear chromosome, whereas non-essential functions are often located near its ends (Hopwood, 1999). It is in this section where genes coding for secondary metabolites or transposons are located. Furthermore, other genes coding for proteins involved in biosynthesis of secondary metabolites are also present in large linear plasmids found in these bacteria (Mochizuki et al., 2003). The ability to produce a wide range of secondary metabolites including antibiotics and bioactive compounds is one of the most interesting properties of Streptomyces. This genus produces about 70% of the clinically useful antibiotics and anticancer agents (Demain, 1999). For secondary metabolite production, common intracellular intermediates like amino acids, sugars, fatty


Carbon regulation of secondary metabolism 149 acids, terpenes, etc. are condensed into more complex structures by defined biochemical pathways (Omura et al., 2001). Nearly 5% of the streptomycetes genome (between 23 and 30 gene clusters) is devoted to the synthesis of secondary metabolites (Ikeda et al., 2003). The highest percentage reported is 6.43% for the S. avermitilis genome, as this microorganism has the highest proportion of predicted secondary metabolite gene clusters of all bacterial genomes sequenced to date (Omura et al., 2001). In agreement with this information, S. avermitilis contains 25 clusters involving the biosynthesis of melanin, carotenoids, siderophores, polyketides, and peptide compounds (Ikeda et al., 2003). Streptomyces secondary metabolites are manufactured via microbial fermentation. The onset of biosynthesis is responsive to environmental cues including phosphate (Martín, 2004) and oxygen concentration, the nature and levels of the carbon and nitrogen source as well as typical variables like temperature, light, and pH. Several regulatory mechanisms are precisely involved for the onset, maintenance and conclusion of secondary metabolism (Sanchez and Demain, 2002). Among these mechanisms, carbon source regulation is one of the main factors required for controlling secondary metabolism. It is well known that several sugars are commonly used as carbon sources for growth and secondary metabolite production, but some of them are preferred by this genus. At the molecular level, the preferential use of one carbon source over the other and the synthesis of secondary metabolites responds either to prevention of transcriptional activation (Uguru et al., 2005) or to repression. Repression by carbon source is commonly known as CCR (Hodgson, 2000; Brückner and Titgemeyer, 2002; Titgemeyer and Hillen, 2002). This is a phenomenon usually caused by glucose, but in different organisms, other rapidly metabolized carbon sources can cause repression and, indeed, sometimes repress catabolism of glucose itself (Sanchez and Demain, 2002). More than 30 examples of secondary metabolites are reported to be suppressed by the presence of the carbon source. Glucose and other carbohydrates, such as glycerol, maltose, mannose, sucrose and xylose, have been reported to interfere with the synthesis of secondary metabolites. For instance, glucose depresses formation of aminoglycoside antibiotics (streptomycin, kanamycin, istamycin, neomycin, gentamicin), via repression of biosynthetic enzymes (Demain, 1989; Piepersberg and Distler, 1997). The sugar suppresses streptomycin and neomycin production by S. griseus and Streptomyces fradiae, respectively. The mechanism involves mannosidostreptomycinase and alkaline phosphatase repression for streptomycin and neomycin biosynthesis, respectively (Demain and Inamine, 1970; Bandyopadhyay

and Majumdar, 1974). For gentamicin production, the glucose effect seems to take place at a step beyond antibiotic intermediate 2-deoxystreptamine (Escalante et al., 1992). Additional examples of antibiotics whose production is regulated by carbon source include the β-lactam antibiotics and macrocyclic polyketides. Cephamycin C is a β-lactam antibiotic produced by Streptomyces clavuligerus whose synthesis is hindered by glycerol through the repression of the enzymes cephamycin C synthetase and expandase. Moreover, expandase activity is inhibited by phosphorylated intermediates of glycolysis like glucose 6-phosphate and fructose 1-6 bis-phosphate (Lebrihi et al., 1988). In addition to cephamycin C, S. clavuligerus produces clavulanic acid, a beta-lactamase inhibitor. Although structurally related, these two beta-lactams are derived from different biosynthetic precursors. Surprisingly, although glycerol abolishes cephamycin C production, it concomitantly increases clavulanic acid formation. Indeed, since this actinomycete cannot utilize glucose (Garcia-Dominguez et al., 1989), glycerol is a good carbon source for clavulanic acid fermentation (Saudagar and Singhal, 2007). Macrocyclic polyketides produced by type-I and II poliketyde synthetases (PKSs) are highly significant secondary metabolites because of their clinical applications. Examples produced by the type-I PKSs comprise rifamycin and erythromycin, useful against mycobacterial infections; FK506, rapamycin, monensin and avermectin are used as antitumor, immunosuppressant, and veterinary agents, respectively (Lal et al., 2000). Polyketides produced by type-II PKSs include actinorhodin, tetracenomycin, anthracyclines and tetracyclines (Lal et al., 2000). The production of polyketides is also suppressed by different carbon sources. For instance, glucose inhibits actinorhodin production in Streptomyces lividans by repressing the synthesis of afsR2 mRNA that encodes for a global regulatory protein involved in the stimulation of secondary metabolite biosynthesis. As expected, no repression is observed when glucose is substituted by glycerol in this microorganism (Kim et al., 2001). As in S. lividans, this gene is necessary in S. coelicolor for actinorhodin production. Using 2-D gel electrophoresis, it has recently been demonstrated that AfsR2 binds to SCO6569 suggesting that this protein is a AfsR2-dependent down regulator for actinorhodin biosynthesis in S. coelicolor (Im et al., 2009). The production of actinomycin by Streptomyces antibioticus is also subject to control by carbon source. In this microorganism, glucose transcriptionally represses hydroxykynureninase, an enzyme of the antibiotic pathway. In a control culture of S. antibioticus with galactose as the carbon source, the specific mRNA is low during the trophophase and high in idiophase. When this culture is compared to a glucose medium, the mRNA is much


150 Ruiz et al. lower and accordingly, the increase in enzyme activity seen in the control by 48 h is almost completely repressed by glucose or glycerol even after 96 h incubation (Brown et al., 1980). In addition to hydroxykynureninase, the phenoxazinone synthase is repressed by glucose. This 88,000 Mr enzyme encoded by a 2.3 kb gene, catalyzes the condensation of 4-methyl-3-hydroxyanthraniloyl pentapeptide to produce actinomycin. Compared to control cells grown in galactose, the specific activity of phenoxazinone synthase and its mRNA levels is decreased in a glucose medium (Jones, 1985). Table 1 shows some examples of secondary metabolites produced by actinomycetes repressed by different carbon sources as well as their target enzymes. There are more examples of secondary metabolites whose synthesis is impaired by carbon source, but with scarce information about the target enzymes. This is the case for the synthesis of retamycin, an anthracycline antitumoral complex produced by Streptomyces olindensis using a polyketide pathway. This compound can be produced in a chemostat using a phosphate limited defined medium. Anthracycline production from this microorganism can be repressed by 139 mM glucose. Under these conditions, an increase in the excretion of organic acids (pyruvate, citrate, succinate and lactate), with reduction in the biomass yield is observed, suggesting that flux through the glycolytic pathway plays an important role in the repression of the biosynthesis of this antitumoral agent (Inoue et al., 2007). Doxorubicin also belongs to the anthracycline family of antitumor compounds. Its synthesis can be hindered

by glucose and galactose in Streptomyces peucetius var. caesius, a strain derived by mutation from the daunorubicin producer Streptomyces peucetius. The glucose effect is observed when sugar is added at the beginning or after 24 h fermentation, but not when added during the stationary growth phase. Furthermore, in a resting cell system containing a protein synthesis inhibitor, anthracycline formation is not affected by high glucose concentrations, suggesting a repressive rather than an inhibitory effect as the possible regulatory mechanism (Escalante et al., 1999). Spiramycin is a macrolide antibiotic produced by Streptomyces ambofaciens, used to treat toxoplasmosis. Production of the 16-membered antibiotic is restrained by glucose and glycerol (Lounès et al., 1996a). Although glycerol increases growth rate and internal ATP, it depresses spiramycin production. Specific spiramycin production is increased 10-fold by fed-batch cultures with glycerol and ammonium feeding (Lounès et al., 1996b). In a batch culture under ammonium depletion, the excess glucose led to pyruvate and α-ketoglutarate accumulation and maintained antibiotic production phase on these acids after glucose exhaustion (Colombié et al., 2005). One of the conclusions from the above mentioned information is that not all secondary metabolite genes are equally sensitive to carbon source regulation control. For instance, actinorhodin production is sensitive to glucose concentrations in the range of 100 mM, however higher concentrations are necessary to repress doxorubicin production (Escalante et al., 1999).

Table 1. Carbon sources interfering with secondary metabolism in actinomycetes. Interfering carbon Idiolite Microorganism source Target Actinomycin IV S. parvulus Glc, Hydroxykynureninase Gly Kynurenineformamidase II Tryptophanpyrrolase (R) Actinorhodin S. coelicolor Glc AfsR2, SCO6569 Cephamicin C Cloramphenicol Erythromycin

S. clavuligerus S. venezuelae Sac. erythraea

Kanamycin Neomycin

S. Kanamyceticus S. fradiae

Gly Glc Glc, Gly Glc Glc Glc Glc

Puromycin

S. alboniger

Glc

Cephamycin C synthetase and expandase Arylamine synthetase (R) S-AMEMT (I) Methylmalonyl-CoA-mutase Type I polyketide synthase N-AKAH (R) Alcaline phosphatase (R)

Reference Brown et al., 1983 Kim et al., 2001, Im et al., 2009 Lebrihi et al., 1988 Bhatnagar et al., 1988 Bermudes et al., 1998 Reeve & Baumberg, 1998 Satoh et al., 1976 Bandyopadhyay & Majumdar, 1974 Sankaran & Pogell, 1975

O-Demethylpuromycin O-methyltransferase (R) Streptomycin S. aureofaciens Glc, Man Mannosidostreptomycinase (R) Demain & Inamine, 1970 Tetracycline S. aureofaciens Glc ATC oxygenase (I) Erban et al., 1983 Tylosin S. fradiae Glc MMCAC and PCAC (R) Vu-Trong et al., 1980 Glc: glucose; Gly: glycerol; Man: mannose; R: repression; I: inhibition; S-AMEMT: S-adenosylmethionine erythromycin O-methyl transferase; ATC oxygenase: Anhydrotetracyclineoxygenase; MMCAC: Methylmalonyl-CoAcarboxyltransferase; PCAC: Propionyl-CoAcarboxylase; N-AKAH:NAcetylkanamycinamidohydrolase.

Carbon regulation of secondary metabolism 151


Mechanism of CCR in Streptomyces In regard to the mechanism for CCR in the genus Streptomyces, this is not clearly understood and there is probably more than one mechanism involved. The possible mechanisms have been studied primarily in S. coelicolor, by far one of the best well characterized biological models (Hopwood, 1999; Chater, 2006). One of these mechanisms involves the phosphoenolpyruvate: phosphotransferase system (PTS). The PTS utilizes a protein phosphoryl transfer chain to transport and phosphorylate its sugar substrates (Saier, 1998; Stülke and Hillen, 1999). In S. coelicolor, two PTS energy-coupling enzymes, enzyme I (EI) and HPr, have been identified (Titgemeyer et al., 1995; Butler et al., 1999; Parche et al., 1999). It has been shown that these proteins are involved in the transport of D-fructose and N-acetylglucosamine (Titgemeyer et al., 1995; Wang et al., 2002). After entering the cell, N-acetylglucosamine (GlcNAc) is phosphorylated to produce N-acetylglucosamine 6-phosphate (GlcNAc-6-P). A GntR-family transcription factor (DasR) seems to control the GlcNAc regulon, including the pts genes (ptsH, ptsI, and crr), needed for uptake of GlcNAc (Rigali et al., 2004, 20). The fructose specific permease (enzyme II) and the enzymes involved in the transport and phosphorylation of GlcNAc have been suggested to be involved in CCR (Nothaft et al., 2003; Wang et al., 2002). Rigali et al. (2006), have observed that adding GlcNAc to rich growth conditions prevents S. coelicolor progression beyond the vegetative state and abolishes secondary metabolite production. This effect is absent in a mutant defective in the transport of GlcNAc (Rigali et al., 2006). Binding of DasR to its target genes is abolished by GlcNAc-6-P, a central molecule in GlcNAc metabolism, which is able to suppress morphological differentiation. Deletion of dasR or the pts genes result in a bald phenotype. A complete GlcNAc-dependent signaling cascade, was proposed for Streptomyces by Rigali et al. (2008), from the perspective of the environmental nutritional status to the onset of antibiotic production through DasR. The evidence provided points out the function of PTS proteins as key elements in CCR exerted by fructose and GlcNAc in Streptomyces (Jahreis et al., 2008). However, deletion of the ptsH gene encoding HPr has no effect on glucose repression of agarase, galactokinase, and glycerol kinase, indicating that PTS responds only to the presence of fructose and GlcNAc (Nothaft et al., 2003; Wang et al., 2002). Therefore, at least one additional mechanism is necessary to explain how glucose and other carbon sources exert CCR. In addition to the PTS system, the analysis of Streptomyces mutants insensitive to CCR has suggested the involvement of the products of a number of genes such as bld and reg1 in the mechanism of CCR

(Champness, 1988; Nguyen, 1999). In S. coelicolor the synthesis of antibiotic and the formation of aerial hyphae initiate at approximately the same time during microbial development, and there is some evidence suggesting that both events are coordinated at the molecular level. In this regard, BldB has been characterized as a protein involved in aerial hyphae formation (Champness, 1988). Interestingly, it has been observed that after rapid growth has ceased, culture supernatants become protein rich, supporting the idea that protein secretion is also a stationary phase phenomenon (Kim et al., 2005). It is known that on different carbon sources, BldB null mutants have a bald phenotype, i.e. lacking an obvious aerial mycelium, pleiotropically blocked for antibiotic biosynthesis (Champness, 1988) and defective in CCR (Kelemen and Buttner, 1998; Pope et al., 1998; Eccleston et al., 2002). Thus, bldB, which encodes a small protein containing a putative DNA-binding helix-turn-helix motif, seems to link secondary metabolite production and development with carbon source-dependent gene regulation. In addition to the bldB mutants from S. coelicolor, other bld mutants from this microorganism that fail to develop normal aerial mycelia and to produce antibiotics have been isolated by UV irradiation. The mutants are grouped into four distinct phenotypic classes. In each class, the pleiotropic phenotype is due to a single mutation and separately involves the bldA, bldB, bldG, and bldH genes. Among them, bldA, bldG and bldH are repressed on media containing glucose or cellobiose. Repression is partially relieved using alternative carbon sources (arabinose, galactose, glycerol, mannitol, and maltose) for bldA and bldG mutants and totally relieved for bldH mutants (Champness, 1998). Therefore, all bld mutants isolated exhibit carbon-source dependent differentiation. The bldA gene encodes for a tRNA capable of efficient translation of the uncommon UUA (leucine) codon, which is rarely present in the Streptomyces genome as TTA. Interestingly, it has been shown that the gene for this protein proved to be dependent on the TTAcontaining pleiotropic regulatory gene adpA (Kim et al., 2005). adpA, also known as bldH, mediates the effects of bldA on morphological differentiation and secondary metabolism (Champness, 1998). In regard to bldG, it has been proposed that this gene encodes a putative anti-anti-sigma factor that might control transcription of both, aerial mycelia formation and antibiotic production (Bignell et al., 2000). An additional regulatory protein involved in CCR has been reported in S. lividans (Nguyen, 1997). This protein, encoded by the gene reg1, belongs to the LacI/GalR family of transcriptional regulators. When reg1 is disrupted, two effects are observed, a loss of glucose catabolite repression of α-amylase genes and inability of maltose to induce these genes (Nguyen et al., 1997). This dual role of Reg1


152 Ruiz et al. resembles that of GylR, the repressor of the gyl operon in S. coelicolor, which is also involved in the CCR of this operon (Hindle et al., 1994). In addition, to the α-amylase genes, in reg1 mutants the CCR of chitinase genes is also abolished (Nguyen et al., 1997). Moreover, Reg1 binds to the promoter region of chitinase, xylanase and cellulase (Nguyen,, 1999). Reg1 has a 95% identity with MalR, a repressor of maltose utilization and 31% with CcpA, the carbon catabolite protein of Bacilli (Nguyen et al., 1997). Van Wezel et al. (1997a) observed that transcription of the malE gene (involved in maltose utilization and part of the malEFG operon), is repressed by MalR. malR mutants result in glucose-insensitive expression of malE, indicating that this repressor plays a role in glucose repression as well as maltose induction. However, the sensitivity of agarase to CCR is retained in malR mutants (Van Wezel et al., 1997a) and therefore MalR cannot be considered a functional homologue to CcpA. Surprisingly, MalR crossreacts with CcpA antibodies (Van Wezel et al., 1997b). D-glucose is one of the easiest utilizable carbon sources for growth in the genus Streptomyces. High concentrations of the sugar elicit CCR over the use of alternative carbon sources and the synthesis of several secondary metabolites. Glucose repression in S. coelicolor operates at the transcriptional level to repress enzymes involved in the utilization of glycerol, arabinose, fructose and galactose (Hodgson, 1982) and this effect seems to be due to either intermediates of carbohydrate catabolism, e.g., fructose 1,6-diphosphate and glucose 6-phosphate (Ramos et al., 2004; Borodina et al., 2008) or to enzymes of the glucose catabolic pathway such as glucose kinase (Ikeda et al., 1984; Angell et al., 1992; Kwakman and Postma, 1994; Saito et al., 1998). In this regard, it has been reported that mutants of S. coelicolor resistant to the non-utilizable glucose analogue, 2-deoxyglucose (DOG), appear to be generally deficient in glucose repression (Hodgson, 1982). DogR mutants can utilize glycerol, arabinose, fructose and galactose in the presence of glucose. Of these mutants, 85% could not grow on glucose. The loss of glucose repression in such mutants (DogR) has been correlated with the absence of an ATP-dependent glucose kinase (Glk) activity (Seno and Chater, 1983). Complementation of these mutants with the Glk gene (glkA) restores not only GlkA activity, but also DOG sensitivity and partially restores glucose repression (Ikeda et al., 1984; Angell et al., 1992). Similarly, when the S. coelicolor glkA gene, is introduced into a S. lividans DogR mutant (unable to utilize glucose and whose chitinase production is resistant to glucose repression), sensitivity to DOG and the ability to utilize glucose are restored, but glucose repression of chitinase production is only partially recovered (Saito et al., 1998). Interestingly, when S. coelicolor DogR mutants were complemented with the glk gene of the Gram-negative Zymomonas mobilis, Glk activity and glucose utilization

are restored, but not glucose repression (Angell et al., 1994). These results suggested a regulatory role, different from Glk catalytic activity, necessary for CCR in this microorganism. The regulatory role for Glk in glucose repression of S. coelicolor was further supported by the experiments of Kwakman and Postma (1994). These authors demonstrated that CCR of glycerol kinase and agarase is relieved in cultures of DogR mutants, grown in a series of carbon sources, such as galactose and glycerol, which are not metabolized via Glk. These results strongly suggested that catabolite repression is not regulated by the flux through Glk and that the protein itself has a regulatory role in CCR (Kwakman and Postma, 1994). In regard to the possible mechanism by which Glk exerts a regulatory effect on CCR, it has been observed that this enzyme does not contain obvious DNA binding sites and thus, it is unfeasible that it could directly regulate transcription. Therefore, it has been proposed that GlkA could transmit its CCR-signal via interaction with transcription factors (Angell et al., 1992). These may include pathway-specific regulators like MalR and GylR, repressors of the maltose and glycerol operons, respectively, or pleiotropic regulators like BldB (Pope et al., 1996; van Wezel et al., 1997b). To verify such relationship, MalR, GylR, and BldB proteins were heterologously overproduced, purified, and assayed for interaction with GlkA. This was approached using several means including surface plasmon resonance, pull-down assays, a peptide library server, immunoprecipitation and in vivo with two-hybrid technology (Mahr et al., 2000). However, these attempts did not allow identification of a GlkA interacting protein,making interaction between Glk and these transcriptional regulators unlikely. On the other hand, apparent binding between Glk and the glucose transporter GlcP has been reported (van Wezel et al., 2007), suggesting that a Glk-GlcP complex is required for efficient glucose metabolism. Thus, there is mounting evidence that Glk itself is not sufficient to elicit CCR in the genus Streptomyces. For instance, it has been previously shown in S. coelicolor that resistance to DOG, yields DogR mutants with decreased glucose kinase activity and reversal of glucose repression (Hodgson, 1982). A derivative of the temperate phage φC31 containing glkA alone is not enough to completely restore the wild type phenotype when used to lysogenize a S. coelicolor glk null mutant with the DogR phenotype. Complete restoration to the wild type phenotype is observed with the temperate phage φC31 KC896 containing both the glkA and sco2127 genes (Angell et al., 1992). Additional evidence has been supplied by Flores et al. (1993), showing no differences in the Glk levels between the parental strain of Streptomyces kanamyceticus with a mutant derived from this microorganism, insensitive to CCR for α-amylase and kanamycin formation. Therefore, these results suggest the necessity

motifs. Dot blot analysis of the S. peucetius var. caesius DogR mutant suggested that sco2127 encodes for a protein which stimulates transcription of glk and probably that of the glucose permease gene as well (Guzman et al., 2005a). In conclusion, these data suggest participation of an integral regulatory system that is initiated by an increase in glucose incorporation and its metabolism, resulting in increased synthesis of catabolites, which may be involved in eliciting CCR in this microorganism. In agreement with this possibility, among several products of glucose metabolism, fructose 1,6 bis-phosphate and phosphoenolpyruvate exert CCR on anthracycline formation in S. peucetius var. caesius (Ramos et al., 2004) with fructose 1,6 bis-phosphate being the most effective (Figure 1). Therefore, it is feasible that phosphorylated sugars can mediate CCR. In this regard, it is known that 2-deoxyglucose but not 3-O-methylglucose interferes with tylosin production by S. fradiae in a process sensitive to glucose and phosphate. It has been reported that 2-deoxyglucose is translocated and phosphorylated by most bacteria, while 3-O-methylglucose is imported but rarely phosphorylated, suggesting glucose phosphorylation as a necessary step for CCR (Demain, 1989).

of additional mechanisms in addition to Glk to explain the CCR mechanism exerted by glucose. In addition to DOG resistant mutants from S. coelicolor (Hodgson, 1982) and S. lividans (Saito et al., 1998), DogR mutants have also been isolated from S. peucetius var. caesius. This microorganism, with an 8.16-Mb genome size, is a doxorubicin producer (Segura et al., 1996). In addition to presenting CCR insensitivity and low Glk activity, the DogR mutants also show difficulties in transporting glucose (Escalante et al., 1999). As previously reported for a DogR mutant from S. coelicolor (Ikeda et al., 1984), a partial reversion of the DogR phenotype is observed when transforming the mutant with glkA. However, a complete reversion of this phenotype is obtained when the mutant is transformed with the sco2127 region alone (Guzman et al., 2005a; Guzman et al., 2005b). Thus, in the recombinant strain, glucose uptake and Glk activity values are reverted back to the level of the original strain or even higher, and the recombinant regains sensitivity to CCR. These results were quite unforeseen considering that the sco2127 region does not seem to encode for either a glucose permease (van Wezel et al., 2005) or a Glk (Angell et al., 1992), and also lacks DNA binding

SCO2127

glkA

Glucose (out)

glcP1

Permease

malR

gylR

bldB

Glucose (in) Transcriptional activator SCO2127

ATP

Glk Glucolysis


Carbon regulation of secondary metabolism 153

Aporepressorproteins

ADP Glucose 6-P Fructose 1,6 bis-P PEP Pyruvate

Active repressor Corepressors

Acetyl-CoA No transcription

Krebs cycle

Gene A

Gene B

Gene C

α-ketoglutarate Genes sensitive to carbon catabolite repression

Figure 1. Hypothetical model explaining the stimulatory effect of SCO2127. GlcP1: Glucose permease, Glk: glucose kinase, PEP: phosphoenolpyruvate, malR: gene encoding for maltose aporepressor, gylR: gene encoding for glycerol aporepressor, bldB: gene encoding for aerial hyphae formation aporepressor.


154 Ruiz et al. The sco2127 region (576 pb) is located upstream of the glkA gene and no evident function has been conferred to its possible expression product. Since sco2127 lacks DNA binding motifs a direct regulation of the glk promoter cannot be expected. Recently, the protein was overexpressed, purified and polyclonal anti-SCO2127 antibodies were produced. The antibodies were able to detect SCO2127 from crude extracts of both, S. coelicolor and S. peucetius var. caesius, giving a single protein band of 34 kDa in western blot analyses (Chávez et al., 2009). SCO2127 and its corresponding ortholog from S. peucetius var. caesius show a 61% identity in amino acid composition (Sohng, J.K. personal communication). When the protein was monitored in S. coelicolor cultures grown in the presence of 50 mM glucose, SCO2127 was detected at the beginning of fermentation and during the logarithmic growth phase, but decreased at later stages of microbial growth. Its production during logarithmic growth agrees with its predicted involvement in CCR, as SCO2127 expression is closely linked to glucose concentration present in the culture medium (Chávez et al., 2009). Detection of SCO2127 at the beginning of the fermentation may be related to its possible role in carbohydrate transport (Guzman et al., 2005b) since the seed cultures were grown in the presence of 50 mM mannitol. On the other hand, cultures grown in 100 mM glucose showed SCO2127 even at later stages of microbial growth coinciding with the presence of 40% of the initial available glucose. It seems reasonable to think that stimulation of both activities allow efficient glucose consumption with the concomitant production of glucose catabolites, compounds presumably involved in eliciting CCR (Ramos et al., 2004). However, a direct effect of SCO2127 on the activation of Glk or GlcP cannot be discarded. In this regard, the apparent binding of Glk to the major glucose transport system of S. coelicolor (GlcP) reported by van Wezel et al. (2007) might be mediated by SCO2127, increasing the efficiency of glucose metabolism. In regard to the possible role of cAMP in the CCR mechanism of Streptomyces For a long time, the existence of cAMP in Streptomyces was in doubt. However, cAMP and its binding protein are present in this genus. In addition, cAMP, 3,5-cyclic guanosine monophosphate (cGMP) is also present in streptomycetes (Gersch et al., 1978). There are several examples suggesting the participation of cAMP in the CCR mechanism. In S. kanamyceticus, cAMP relieves glucose repression of N-acetylkanamycin amidohydrolase (Satoh et al., 1976). In addition, dibutiryl cAMP supplementation to cultures of S. fradiae, under conditions of growth limitation by ferric ammonium citrate, stimulates tylosin production by 25–30% (Tata and Menawat, 1994). In the same line, variants of this microorganism with high

tylosin production contain 20–50% more cAMP than the parental strain (Demain, 1989). Spore germination, aerial mycelium formation and actinorhodin production are also dependent on cAMP in S. coelicolor (Süsstrunk, 1998). Disruption of cya, the gene encoding adenylate cyclase in S. coelicolor generated a mutant (BZ1) unable to produce cAMP and defective in aerial mycelium formation (Süsstrunk et al., 1998). Both, the wild type and mutant cultures showed acidification of the medium from 7 to 4.5 but only the parent could partially neutralize the medium during formation of aerial mycelia. Addition of exogenous cAMP or the use of a pH 7 buffer allows the mutant to make aerial mycelia and increases actinorhodin production. On the contrary, synthesis of undecylprodigiosin did not respond to cAMP in the presence or absence of buffer. This effect fits well with the selective repression of actinorhodin production in cultures grown in high glucose (Kang et al., 1998). In addition, the existence of CRP in S. coelicolor has been demonstrated. Thus, SCO3571 (crp) encodes for a protein of 224 amino acids that clusters to the CRP subfamily (Derouaux et al., 2004). As reported for cya mutants from S. coelicolor (Süsstrunk et al., 1998), strains defective in crp also revealed a lack of germination and actinorhodin production. However, in contrast to cya mutants, addition of exogenous cAMP did not restore the crp mutant phenotype, indicating that this protein is necessary in the CCR mechanism (Derouaux et al., 2004). Another nucleotide that has been involved in regulation of secondary metabolite biosynthesis is the ATP. Li et al. (2008) reported the effect of extracellular ATP (exATP) on antibiotic formation by S. coelicolor. When this microorganism is grown in the presence of 10 mM exATP, actinorhodin concentration increases 90% compared to a culture grown in the absence of nucleotide. Conversely, only a 23% increase in undecylprodigiosin is observed. These authors also claim that stimulation by exATP is extended to antibiotics produced by other streptomycetes, including S. lividans, S. griseus, S. violaceoruber, and S. avermitilis (Li et al., 2008). The authors suggest that exATP acts as a signaling molecule at the bacterial surface leading to intracellular signaling events and induction of transcriptional activators of antibiotic production. However, in higher exATP, cultures exhibit high intracellular ATP levels and a decreased production in both, actinorhodin and undecylprodigiosin. The nucleoside adenosine also seems to have a role on the production of actinorhodin. Adenosine kinase (ADK) phosphorylates adenosine to produce adenosine monophosphate (AMP) using adenosine triphosphate (ATP) as a main phosphoryl donor. In S. lividans mutants lacking ADK, no AMP is produced and adenosine is accumulated (Rajkarnikar et al., 2007). Under these conditions, production of actinorhodin is suppressed in the mutant and a loss of sporulation is observed. On the contrary,


Carbon regulation of secondary metabolism 155 production of undecylprodigiosin (the red antibiotic) is enhanced. A promoter-probe assay verified the repressive effect of adenosine on the transcription of the pathwayspecific activator, actII-ORF4 of actinorhodin production and induction of the undecylprodigiosin promoter (Rajkarnikar et al., 2007). No connection between the ATP and adenosine effects on antibiotic production are visualized since S. coelicolor cya mutants, unable to produce cAMP, are expected to accumulate intracellular ATP which might phosphorilate adenosine avoiding repression of actinorhodin production. Conversely, accumulation of intracellular ATP could be expected to occur in a S. lividans adk mutant, favoring cAMP and actinorhodin formation, which is not the case. Saccharopolyspora Saccharopolyspora erythraea is used for the industrialscale production of the antibiotic erythromycin A. Derivatives of this antibiotic play a crucial role in medicine for the treatment of infectious diseases and as a gastrointestinal motor stimulator (Demain and Sánchez, 2009). Its chromosome comprises 8.2 Mb pairs, predicted to encode 7,264 genes (Oliynyk et al., 2007). As reported for other actinomycetes, Sac. erythraea has a circular chromosome. Its genome contains at least 25 gene clusters for production of known or predicted secondary metabolites, and at least 72 genes have been predicted to confer resistance to a range of common antibiotic classes (Oliynyk et al., 2007). Erythromycin is produced from propionyl-CoA or succinyl-CoA, through a notable process of assembly that involves at least 28 active sites arranged along three large proteins. This is followed by hydroxylation and glycosilation processes involving 18 additional proteins. Finally, the microbial ribosomes are protected from the high antibiotic specific toxicity by specific methylation of the rRNA (Challis and Hopwood, 2003). Glucose transiently represses antibiotic formation (Escalante et al., 1982) probably by repressing methylmalonyl-CoA-mutase (Bermudes et al., 1998). While this enzyme is repressed by glucose, methylmalonylCoA decarboxylase production is not affected by carbon source, suggesting that the negative effect of glucose on erythromycin production could be due, in part, to lower pools of succinyl-CoA and methylmalonyl-CoA (Bermudez et al., 1998). Actually, engineering Sac. erythraea through duplication of the methylmalonyl-CoA mutase led to a 50% increase in erythromycin production (Reeves et al., 2007). Moreover, glucose or glycerol inhibits S-adenosylmethionine erythromycin O-methyl transferase activity. Reeve and Baumberg (1998) measured the effect of glucose on the transcription of the eryAI gene, encoding the type I polyketide synthase. With an increase in glucose concentration they found a decrease in the lag before the onset of erythromycin production,

but a drop in the final level of the eryAI expression.The substitution of glucose by cane molasses accompanied with a reduction of ammonium sulphate concentration and supplementation of the medium with n-propanol has been utilized for increasing erythromycin production and reduction in cost of antibiotic production (El-Enshasy et al., 2008). In Sac. erythraea, the erythromycin biosynthetic cluster lacks a regulatory gene (Chng et al., 2008). However, a 17.7 kDa bldD ortholog has been recently described, which positively regulates all the promoters in the erythromycin production cluster, suggesting the existence of a transcriptional activator of the ery gene cluster (Chng et al., 2008). In S. coelicolor, BldD negatively regulates expression of key developmental genes (Elliot et al., 2001). bldD mutants pleiotropically affect both, formation of aerial hyphae and antibiotic production in this microorganism (Elliot et al., 2003). Similarly Sac. erythraea bldD mutants produce a bald phenotype and 7-fold less erythromycin than the Sac. erythraea wild type strain (NRRL2338) (Chng et al., 2008). Other secondary metabolites produced by Saccharopolyspora spinosa are the macrocyclic lactones, spinosyns. This new class of compounds shows insecticidal activity with a high level of selectivity against crop pests such as tobacco budworm (Heliothis virescens) and southern armyworm (Spodoptera eridania). These compounds contain a tetracyclic core formed by a 12-membered macrocyclic lactone fused to a 5,6,5-cisanti-trans tricyclic ring system. Attached to the tetracyclic core are two sugars, an amino sugar (forosamine) and a neutral sugar (2,3,4-tri-O-methylated rhamnose). So far, more than 25 spinosyns have been isolated and identified from Sac. spinosa that vary in methyl substitution patterns on the forosamine nitrogen, the 2′-, 3′-, 4′-methyl positions of the rhamnose, and at the C6, C16, and C21 positions of the tetracycle (Crouse et al., 2001). The most abundant spinosyns isolated from the fermentation broth of Sac. spinosa are spinosyn A and spinosyn D. Spinosyns are assembled from acetate and propionate via a polyketide pathway that ultimately leads to the introduction of three intramolecular C-C bonds to form spinosyn tetracycle. The neutral sugar (rhamnose) and the amino sugar (forosamine) are coupled to the tetralyde at C9 and C17, respectively. The sugars are methylated by O-methyltransferases from S-adenosyl-methionine. Recently, the entire spinosyn biosynthetic gene cluster was determined in Sac. spinosa through gene sequencing and functional analysis of the gene products (Waldron et al., 2001). Spinosyns are produced by submerged fermentation of Sac. spinosa. Glucose is critical for growth and insecticidal production. A high glucose concentration (>79.6 g/L) inhibits mycelial growth and

156 Ruiz et al. spinosyn production (Jin et al., 2006a). Therefore, the c oncentration of this nutrient should either be limited during the initial growth phase of the culture or continuously fed in non-suppressive concentrations.


Nocardia This genus is able to produce a wide variety of secondary metabolites. Nocardicin A and B (Nocardia sp.), ryfamicin (Nocardia mediterranea), ansamitocin (Nocardia brasiliensis), 3′-O-demethyl mutactimycin (Nocardia transvalensis), neo-nocardin (Nocardia kuroishi), and cephamycin C (Nocardia lactamdurans) are some examples of antibiotics produced by this genus. Although reported as a good antibiotic producer, studies on the effect of the carbon source on idiolite formation are limited in this genus. For instance, it has been reported that various sugars and their metabolites inhibit cephamycin C production by resting cells of N. lactamdurans (Cortes et al., 1984). At the biochemical level, glucose-6-phosphate and fructose-1,6-diphosphate inhibit deacetoxycephalosporin C synthase, one of the pathway enzymes dealing with antibiotic formation. In regard to the cAMP levels, a similar behavior to that reported for several streptomycetes is observed, i.e., high during growth and low during antibiotic production, suggesting this nucleotide is not involved in derepressing secondary metabolism. Corynebacterium Corynebacterium kutscheri and Corynebacterium xerosis produce antimicrobials effective against bacteria and fungi. It has been established that antibiotic production in these species is greatly influenced by variation in the carbon sources. Among them, ribose and lactose suppress the antimicrobial activity of C. kutscheri and C. xerosis, respectively (El-Banna, 2006). Another Gram-positive microorganism of clinical importance is the enterotoxin producer Corynebacterium diphtheriae, which is the responsible agent of the infectious disease, diphtheria (Barksdale, 1970). This microorganism can utilize numerous carbon sources including maltose, glucose and fructose. Under submerged fermentation, toxin production is negatively affected by high glucose concentrations (Singer et al., 1967). The bacteria have genes for the PTS regulatory mechanism of CCR. Two energycoupling proteins, E1 and Hpr and permeases for glucose and fructose are present. Moreover, it contains two distinct genes encoding a IIABPtx-like and a novel HPr-type protein of unknown function. In addition, a possible PTS gene target was elucidated as a possible regulatory gene encoding an antiterminator protein (Parche et al., 2001).

Bacillales The order Bacillales belongs to the class Bacilli and produces a full range of secondary metabolites with antimicrobial and toxin activity. These compounds are often, but not always, polypeptides. Known antibiotic producers from the Bacillaceae family whose synthesis is subject to CCR include Bacillus cereus that synthesizes cerexin and zwittermicin, Bacillus circulans and Brevibacillus laterosporus produce circulin, Bacillus licheniformis bacitracin, Bacillus pumilus pumulin and Bacillus subtilis makes polymyxin, difficidin, subtilin, and mycobacillin. Paenibacillus polymyxa produces both, polymyxin and colistin, Brevibacillus brevis makes gramicidin and tyrothricin, B. laterosporus produces laterosporin. Toxins whose synthesis is regulated by CCR include both, the hemolytic HBL and the nonhemolytic Nhe, enterotoxins produced by B. cereus (Ouhib et al., 2006). The mechanism of CCR in B. subtilis and other Firmicutes like Staphylococcus, Streptococcus, Enterococcus and Lactobacillus, differs from that of enterobacterias. These bacteria possess essentially the same protein constituents of the PTS as are found in E. coli. Therefore, the PTS components form a protein phosphorylation cascade, which uses PEP as phosphoryl donor. In addition, these Gram-positive bacteria possess a bifunctional enzyme, the HPr kinase/phosphorylase (HprK/P), which can be activated by several metabolites (fructose 1,6-diphosphate, gluconate-6-P and 2-phosphoglycerate) when growing in the presence of glucose. Activated HprK/P catalyses phosphorylation of HPr at Ser-46 and dephosphorylation of P-Ser-HPr. Phosphorylated HPr binds to a pleiotropic regulator, catabolite control protein A (CcpA), to allosterically promote catabolite repression and to prevent inducer accumulation by uncoupling sugar transport from H+ symport (Deutscher, 2008). CcpA functions as a pleiotropic regulator by binding to the so-called catabolite-responsive elements (cre), which are located either upstream in the promoter regions, or in open-reading frames (Lulko et al., 2007). It is interesting to note that polyclonal antibodies against CcpA from Bacillus megaterium share antigenic determinants with CcpA in many other Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes (Küster et al., 2006).

Lactobacillales In the lactobacillales order (class Bacilli), the production of bacteriocin (a bioactive peptide with antimicrobial activity towards gram positive bacteria) by Lactococcus lactis subsp. lactis is sensitive to carbon source regulation by fructose and glucose (De Vuyst and Vandamme, 1992;


Carbon regulation of secondary metabolism 157 Cheigh et al., 2002). In addition, batch culture production of pediocin (another peptide antibiotic) by Pediococcus acidilactici NRRL B-5627 is inhibited by increasing glucose concentrations in the growth medium (Guerra et al., 2007). In lactic acid bacteria, the transport and phosphorylation of glucose is undertaken by the mannose PTS (HPr, EI, and an EIIMan complex) (Chaillou et al., 2001). Mutations in EIIMan complex inactivate it and elicit loss of the preferential consumption of glucose over other carbon sources like lactose in Lactobacillus casei or xylose in Tetragenococcus halophila. The use of mutants affecting expression of mannose PTS in Streptococcus salivarius has a pleiotropic effect over various metabolic enzymes as well as on urease activity and on an inducible fructose PTS activity (Chaillou et al., 2001). From this function in lactic acid bacteria, it may be assumed that the activity of this PTS affects CCR. In addition, a role for CcpA in the transcriptional regulation of the xyl regulon in Lactobacillus pentosus has been demonstrated. However, no information on the relationship between the EIIMan complex activity and CcpA-dependent CCR mediated by glucose is available for S. salivarius. Therefore, the mechanisms by which the EIIMan complex are involved in regulatory functions are not well understood.

Carbon source regulation of secondary metabolite production in Gram-negative bacteria Production of secondary metabolites by Gram-negative bacteria is subject to different types of carbon source regulation. In some cases, production is sensitive to carbon source suppression. One example is carbapenem biosynthesis in Erwinia carotovora (Coulthurst et al., 2005). In this microorganism, glycerol suppresses carbapenem biosynthesis by repressing transcription of the carI gene responsible for the synthesis of N-3-(oxohexanoyl)-Lhomoserine lactone (OHHL), a quorum sensing molecule that induces antibiotic formation. Limitation of OHHL impacts expression of the carbapenem biosynthetic genes (carA-H) by preventing formation of the complex between CarR (DNA-binding transcriptional activator of carA-H) and OHHL. The formation of this complex (CarR-OHHL) is a necessary step for transcriptional activation of the carbapenem biosynthetic genes in E. carotovora (McGowan et al., 1995; Veselova et al., 2003). On the other hand, glycerol does not affect transcription of either carR or hor (transcriptional activator of antibiotic and pigment production) genes (Coulthurst et al., 2005). Carbapenem-like antibiotics, are also produced by other Gram-negative bacteria like Erwinia herbicola, Serratia sp. strain ATCC 39006 and Photorhabdus luminescens strain TT01 (Derzelle et al., 2002). Interestingly,

in P. luminescens the carR gene is not present, suggesting a different mechanism for regulation of antibiotic formation. Additionally, in contrast to E. carotovora, P. luminescens carbapenem-like synthesis is not induced by quorum sensing molecule N-acyl-homoserine lactone (AHL) (Derzelle et al., 2002). One additional example of carbon regulation has been reported in the ethanol-utilizing strain S389 of Serratia marcesens. Cang et al. (2000) observed in this microorganism, that addition of a low glucose concentration (27.7 mM) to the growth medium, almost completely repressed formation of the red tripirrole antibiotic, prodigiosin. Other carbon sources like galactose, fructose and sucrose also depressed antibiotic production. As mentioned above, Serratia also produces the antibiotic carbapenem. It is known that biosynthesis of both compounds (carbapenem and prodigiosin) are under quorum sensing control (Thomson et al., 2000; Williamson et al., 2006). Therefore, these secondary metabolites are controlled by the smaR quorum sensing locus, which represses both compounds when levels of AHL are low. Quorum sensing controls the production of prodigiosin, at least in part, by modulating transcription of three genes encoding regulatory proteins. Surprisingly, one of these proteins is a novel putative adenylate cyclase (Fineran et al., 2005), which likely renders a decrease in cellular cAMP (3,5-cyclic adenosine monophosphate) concentration affecting pigment production. Another example of carbon regulation in Gramnegatives is the production of coronatine by Pseudomonas syringae. Coronatine is a polyketide phytotoxin whose production is decreased by glycerol, inositol, fructose and xylose (Palmer et al., 1993). Conversely, phytotoxin production is favored by glucose. Pseudomonas aeruginosa produces rhamnolipids that are considered secondary metabolites with surface active properties. It is known that these biosurfactants solubilize hydrophobic substrates like hexadecane by reducing water surface tension. The strain produces surfactants in alkanes containing 12 carbons. However no production is observed in alkanes containing 10, 13, 14, and 16 carbons. Other carbon sources like glycerol, mannitol and glucose favor rhamnolipid production. However fructose does not (Robert et al., 1989). Among the various mechanisms of CCR in Gramnegative bacteria, the PTS can be mentioned as one of the most important. In Escherichia coli, PTS consists of four high-energy phosphoprotein intermediates and five protein domains. One of these proteins, EIIAglc is phosphorylated by a phosphoprotein (HPr) on His90. Later, EIIA glc∼P transfers its phosphate from His75 to a high affinity protein EIIB/C that occurs in the cell membrane as a homodimer. The amino acid chain of domain IIC crosses the membrane eight times harboring the sugar binding site. The hydrophilic domain IIB transfers the


158 Ruiz et al. phosphate group from EIIAglc∼P to glucose, rendering glucose 6-phosphate (Deutscher, 2008). In addition to transferring the phosphate group to EIIB/C, EIIAglc∼P activates adenylate cyclase. The activated form of adenylate cyclase synthesizes cAMP, necessary for synthesis of inducible enzymes and its intracellular levels mediate CCR. To activate transcription, cAMP binds to the DNA promoter region via cAMP receptor protein (CRP). In the presence of glucose, the sugar is transported into the cell and then phosphorylated. This event causes dephosphorylation of EIIAglc∼P, mediates inducer exclusion and deactivates adenylate cyclase. Inactivation of adenylate cyclase causes the cytoplasmic cAMP concentration to diminish and promotes dissociation of the cAMP-CRP complex from the DNA and deactivation of transcriptional initiation. The gene for EIIAglc is called crr, because mutants of E. coli lacking this gene are resistant to CCR (Deutscher, 2008). By examining the role of the N-acyl amino acid synthase (NASP)-associated cAMP binding domain in the regulation of the antibiotic N-acylphenylalanine from an uncultured β-Proteobacterium in E. coli, Clardy and Brady (2007) discovered that antibiotic biosynthesis is

cAMP dependent. In addition, using affinity chromatography, these authors confirmed a direct interaction between NASP and cAMP.

Carbon source regulation of secondary metabolite production in fungi As observed for actinomycetes, fungi also synthesize secondary metabolites in response to physiological stresses such as nutrient limitation (carbon, nitrogen or phosphate sources). Table 2 shows some examples of secondary metabolites produced by fungi that are repressed by different carbon sources, as well as their target enzymes. In Penicillium chrysogenum, glucose represses the transcription of penicillin biosynthetic genes pcbAB, pcbC and penDE (Gutiérrez et al., 1999) which encode for δ–(Lα-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, isopenicillin N (IPN) synthase and IPN acyltransferase, respectively (Figure 2). While glucose negatively affects the pcbAB, pcbC, and penDE gene promoters, alkaline pH exerts a small positive effect on these promoters (Gutiérrez et al., 1999).

Table 2. Carbon sources interfering with secondary metabolism in fungi. Interfering Idiolite Microorganism carbon source Cephalosporin C A. chrysogenum Glc Gly Mal Ergot alkaloids C. purpurea Glc Lovastatin M. pilosus cAMP A. terreus Glc Penicillin P. chrysogenum Glc,

Target Reference Deacetoxycephalosporin C synthetase (expandase) (R) Jekosch & Küch, 2000a Zhang & Demain, 1992 δ-ACVS (I), cyclase (R) DATS (I) Kruprinski et al., 1976 Protein kinase A (R) Miyake et al., 2006b DKS Hajjaj, et al., 2001 ACVS (R), IPN cyclase (R) Theilgaard et al., 1997 IPN acyl-transferase (R) Gutiérrez et al.,1999 A. nidulans Glc ACVS (R), IPN synthase (R) Brakhage et al., 2004. IPN acyl transferase (R) Litzka et al., 1995. Glc: glucose; Gly: glycerol; Mal: maltose; R: repression; I: inhibition; ACVS: δ-(L-α-aminoadipyl)-L-cysteinyl-L-valine synthethase; DATS: Dimethylallyltryptophan synthase; DKS: diketide synthetase.

Glucose

pcbAB

Glucose

pcbC

penDE

Alkaline pH Figure 2. Penicillin cluster in Penicillium chrysogenum and the effect of glucose and alkaline pH on the transcription of pcbAB, pcbC, and penDE genes. Positive effects are shown with black arrows and negative effects with dotted lines. Direction of transcription is shown by white arrows. Intergenic sequences are shown in dotted boxes. While alkaline pH exerts a small positive effect on the pcbAB, pcbC, and penDE gene promoters, glucose negatively affects both promoters.


Carbon regulation of secondary metabolism 159 In the same manner, the genes aatA and ipnA but not acvA (homologues to penDE, pcbC and pcbAB) are repressed by glucose in A. nidulans (Brakhage et al., 2004; Litzka et al., 1995). The consensus sequence 5’-SYGGRG-3’ is located in the upstream region of the ipnA gene and the binding of CreA (carbon catabolite repressor) to this sequence has been confirmed in vitro (Espeso et al., 1994). However, it has been elucidated that A. nidulans creA mutants cultured in glucose, still presented repression of ipnA transcription (Espeso et al., 1992). Mutations in creB and creC have shown little effect on carbon regulation of penicillin biosynthesis. All these results point to a second mechanism of carbon repression, which is creA independent (Espeso et al., 1995). In Acremonium chrysogenum, glucose represses cephalosporin C formation (Jekosch and Kück, 2000a). Since this organism contains cre1 as well as cre1-binding sites upstream of the isopenicillin N synthase (pcbC) and deacetoxycephalosporin C/deacetylcephalosporin C synthase (cefEF) genes, it is considered that cre1 might be involved in repression of antibiotic formation. Indeed, in the wild-type strain, glucose increased the level of cre1 transcripts six-fold. As expected, in a commercial highproducing A. chrysogenum strain, glucose did not affect the cre1 transcript levels. Therefore, it is feasible that cre1 might be involved in glucose repression of cephalosporin C production and that during the program of strain improvement this control mechanism could be deregulated (Jekosch and Kück, 2000b; Janus et al., 2008). Lovastatin is an inhibitor of the enzyme hydroximethylglutaryl coenzyme A reductase that catalyzes the reduction of hydroximethylglutaryl coenzyme A to mevalonate in cholesterol biosynthesis, reducing the blood cholesterol levels (Miyake et al., 2006a). Lovastatin can be produced by Penicillum and Monascus species and by Aspergillus terreus. Several authors have reported the negative effect of glucose on lovastatin production in Monascus and A. terreus, where lactose is the preferred carbon source (Casa-López et al., 2003; Hajjaj et al., 2001; Lai et al., 2007). The lovastatin biosynthetic gene cluster in A. terreus consists of 18 putative ORFs, which were assigned as lov genes by alignment with other related genes. Among those, lovE and ORF13 are similar to regulatory proteins (Kennedy et al., 1999). Analysis of the gene cluster showed the presence of two SYGGRG consensus sequences for CreA binding in the 5’ proximal region of ORF13 and in the putative promoter of ORF8 and lovE. The presence of CreA binding sites in two putative regulatory genes suggest that glucose repression of lovastatin biosynthesis could be mediated by CreA (Hajjaj et al., 2001). It is likely that CreA interacts with LovE and ORF13 promoters preventing biosynthetic enzymes to be synthesized (Barrios-González et al., 2008). Numerous species of Monascus produce several secondary metabolites such as γ-aminobutiric acid

(anti-hypertension agent), red pigments (antibacterial activity), citrinin (toxin), and lovastatin (Miyake et al., 2006b, Cheng et al., 2008). Feeding cAMP to Monascus pilosus and Monascus purpureus, showed a significantly reduction in secondary metabolite production. These results suggest that the negative effect of carbon sources such as glucose, on the production of red pigments and lovastatin could be due, at least in part, to the cAMP signaling pathway (Miyake et al., 2006b). In the same line, it has been observed that exogenous cAMP represses the production of lovastatin, red pigments, and citrinin in Monascus. Interestingly, in a MK-1 mutant with increased lovastatin and red pigment production (Miyake et al., 2006a) cAMP showed no influence on the production of these compounds, indicating that this mutant possibly lacks the cAMP signaling. Gibberellins are diterpene plant hormones, which control several aspects of plant growth and development. There are few species that produce gibberellins, such as Neurospora crassa, five species of Sphaceloma and Gibberella fujikuroi (MacMillan, 2002). In G. fujikuroi, putative CreA binding sites have been detected in some of the gibberellin biosynthetic genes. However, the transcription of CreA protein is not affected by glucose concentration as occurs in A. nidulans and Trichoderma reesei, which suggests the participation of additional regulatory factors in hormone production (Tudzynski et al., 2000). Direct inhibition of the action of secondary metabolic enzymes may also be involved in carbon source control. Thus, glucose and a number of its phosphorylated metabolites inhibit δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS), the first enzyme of cephalosporin synthesis, in crude extracts of A. chrysogenum and S. clavuligerus (Zhang and Demain, 1992). Because no inhibition is observed with the purified enzyme, it appears that this phenomenon might be caused by competition for ATP between primary (Embden-Meyerhof pathway) and secondary metabolism. Accordingly, it has been found that inhibition of ACVS by sugars could be prevented by addition of more ATP. Conversely, both the crude and the purified forms of ACVS are inhibited by glyceraldehyde3-phosphate (G-3-P). This effect seems to be due to the ability of G-3-P to chemically complex and remove cysteine, a substrate of ACVS. The in vivo significance of such inhibitory phenomena remains to be determined. In regard to the mechanism of CCR, in A. nidulans carbon catabolism is a tightly regulated process, whereby the most energetically favorable carbon sources are used preferentially over less readily metabolized carbon sources. It has been demonstrated that a Glk (GlkA4) and a hexokinase (Hxk) participate equally in sugar signaling. Single glkA4 and hxkA mutants were able to substitute for each other in their function in catalysis and in CCR. However, double mutants in glkA4 and hxkA genes


160 Ruiz et al. showed derepression in genes such as facA for acetate catabolism, xlnA for xylan degradation and alcA and alcR for ethanol utilization (Flipphi et al., 2003). These observations suggest that glucose phosphorylation could be the relevant signal for glucose repression in A. nidulans (Flipphi et al., 2003). In addition, the creA, creB, creC, and cre1 genes (Arst et al., 1990) were identified as coding for regulatory proteins involved in CCR in A. nidulans and Trichoderma reesei. CreA is a regulatory protein required for the repression of a large range of genes by binding to the consensus DNA sequence 5’-SYGGRG-3’ (where S could be C or G, Y could be C or T and R could be A or G) in promoters of glucose-repressible genes, turning off their expression. CreA and Cre1 contain two Cys2His2 DNA-binding zinc fingers similar to the S. cerevisiae glucose-repressor protein Mig1 (Cubero and Scazzochio, 1994; Cubero et al., 2000; Roy et al., 2008). creB encodes a deubiquitinating enzyme and creC encodes a WD40 protein that contains five WD40 repeats and a prolinerich region. CreC forms a complex with CreB (Todd et al., 2000; Lockington and Kelly, 2001). The proposed model for CCR via CreA modification or stability involves the CreB deubiquitination enzyme complexing with the CreC protein to modify or stabilize CreA under carbonrepressing conditions (Lockington and Kelly, 2002). An additional gene implicated in the mechanism of A. nidulans CCR is creD. Mutations in this gene suppress the creB15 and creC27 mutant phenotypes, showing derepression of facA and alcA genes. The creD gene encodes a protein that contains arresting domains and PY motifs and interacts with the HECT ubiquitine ligase (Boase and Kelly, 2004). Even though the elements of ubiquitination and deubiquitination networks have been disclosed, there still remains to determine their direct targets (Roy et al., 2008). Recently, it was found that the mechanism of action of CreA in CCR does imply neither its degradation nor its cellular localization, but seems to require some modification or interaction with another protein in order to act as a repressor (Roy et al., 2008). In a study elucidating the kinetics of glucose uptake in A. nidulans, two energy-requiring glucose transport systems were identified: a high affinity glucose-repressible system and one of low affinity inducible by glucose (MstE). Besides glucose, mstE expression is induced in the presence of other repressing carbon sources, and is dependent on the function of the transcriptional repressor CreA (Forment et al., 2006). Glucose repression has been widely studied in the yeast Saccharomyces cerevisiae. Addition of glucose to S. cerevisiae cells growing in non-fermentable carbon sources leads to an early repression, which requires participation of several hexose kinases and a late response achieved only when one hexokinase is present. Galactose, maltose, glycerol, and acetate do not cause the early effect (Sanz et al., 1996). Cyclic AMP (cAMP)

changes seem to play no role in the early repression response, since the same phenomenon is also observed in a cAMP-deficient mutant. In S. cerevisiae a glucose transporter family composed by six genes (HXT1-HXT4, HXT6, HXT7) encode the major proteins responsible for its transport on a broad range of glucose concentrations. These transporters exhibit different affinities for glucose and the genes are induced and/or repressed by different levels of glucose. Glucose must be taken up by cells to cause repression, and none of the major transporters serves a sensing/signaling function for glucose repression (Reifenberger et al., 1997). Glucose repression is mediated, at least in part, by the glucosedependent repressors Mig1 and Mig2, which are zincfinger proteins that bind to several promoters. Genetic analysis suggests that Mig2 plays a minor role relative to Mig1 (Carlson, 1999). When glucose is low, Mig1 is deactivated by phosphorylation by Snf1 (Cat1) protein kinase. Snf1 is required primarily for the adaptation of yeast cells to glucose limitation and for growth on carbon sources that are less preferred than glucose, but is also involved in responses to other environmental stresses (Schüller, 2003). Snf1 complexes with its regulatory partner (Snf4) and several beta-subunits for derepression of the structural genes repressed in the presence of high glucose concentrations (Hedbacker and Carlson, 2008). Recently, NRG (Negative regulator of glucose-repressed genes) has also been shown to be required for glucose repression. Interactions between NRG and Mig1 have been reported (Zhou and Winston, 2001).

Strain improvement for secondary metabolites production by avoiding or removing CCR Although it has been more than 60 years since the first penicillin overproducing mutant was obtained by X-ray treatment of P. chrysogenum X-1612 (Adrio and Demain, 2006), the conventional breeding (random mutagenesis and massive tracing of strains) remains the preferred method when the molecular genetic tools have not been developed for the producer microorganism (Khetan and Hu, 1999). Nutritional repression can be decreased by mutation to antimetabolite resistance. An example of a selective agent is DOG, utilized for enzymes and secondary metabolites whose synthesis is controlled by carbon source regulation. This can be illustrated with the generation of spontaneous mutants from S. peucetius var. caesius resistant to DOG (Segura et al., 1996). When one of these mutants is recombined with a mutant isolated as resistant to growth inhibition by high doxorubicin concentrations, the recombinant offspring show resistance to the mentioned compounds,


Carbon regulation of secondary metabolism 161 insensitivity to CCR and improvement in antibiotic formation (Segura et al., 1997). Jin et al. (2006b) selected mutants resistant to the growth inhibition by DOG and rhamnose. Following UV mutagenesis, they isolated a mutant with a 121% improvement in the spinosyn A and D yield. After optimization of the fermentation process, the yield reached 458 mg/L, which corresponded to a 71% increase compared to that observed in flask fermentations. In the case of M. pilosus the regulatory mutant MK-1 previously described, retains its ability to produce high lovastatin titers and red pigments in a medium supplemented with glucose (Miyake et al., 2006b). In addition, the production of lovastatin is not affected by the cAMP levels (Miyake et al., 2006b). It has been observed that deletion of the glucose 6-phosphate dehydrogenase and phosphoglucomutase genes in S. coelicolor and overexpression of the acetyl coenzyme A carboxylase genes, results in rapid glucose utilization and increased actinorhodin production in this microorganism (Ryu et al., 2006). In a similar work, it was shown that deletion of the pfkA2 gene, corresponding to one of the three reported homologue phosphofructokinases in S. coelicolor, increased actinorhodin and undecilprodigiosin production approximately four times as compared to the wild type strain (Borodina et al., 2008). Previously some key enzymes for secondary metabolism that are regulated or repressed by the carbon source have been mentioned (see Table 1). Some of these enzymes have been used as targets for strain improvement. For instance, in the case of cephalosporin production, a lead enzyme, deacetoxycephalosporin C synthase (DAOCS or expandase), which is affected by CCR, has been chosen as a target to increase production of these antibiotics. In A. chrysogenum, an initial approach was performed by Basch and Chiang (1998) by increasing the copy number of the expandase gene (cefEF) encoding for deacetoxycephalosporin C synthetase. In the resulting transformants, it is observed a 40–70% reduction in DAOC levels and an increase in the cephalosporin C titer. In S. clavuligerus, expandase was modified by site-directed mutagenesis to promote the conversion of penicillin G to 7-phenylacetylamino-deacetoxycephalosporonic acid, precursor of 7-aminodeacetoxycephalosporonic acid (7-ADCA). The resulting mutants showed increased kcat/Km values and 13-fold increase in relative activity toward penicillin G (Wei et al., 2003). Addition of different side chains to 7-ADCA converts this compound into the oral cephalosporins: cephradine, cephalexin, and cephadroxil. S-Adenosyl-L-methionine (SAM) is most commonly used in living systems as a methyl donor involved in methylation reactions. The synthesis of SAM, is catalyzed by an enzyme called SAM synthetase (MetK,

ATP: L-methionine S-adenosyltransferase, EC 2.5.1.6). Overexpression of SAM especially enhances production of secondary metabolites including actinorhodin and calcium-dependent antibiotics in S. coelicolor, pristinamycin in Streptomyces pristinaespiralis, granaticin in Streptomyces violaceoruber, oleandomycin in S. antibioticus and avermectin in S. avermitilis. (Kim et al., 2003; Okamoto et al., 2003; Huh et al., 2004). Although the overall mechanism underlying this activity is not completely understood, the obvious methylation of intermediates leading to the biosynthesis of antibiotics involving SAM provides a strong explanation for the high yield of these compounds (Kim et al., 2000). There have been notorious advances about the regulatory mechanisms of secondary metabolism at the biochemical and molecular levels. Thorough investigation of these mechanisms could allow us to increase the efficiency of metabolic pathways involved in secondary metabolite production, making the genetic improvement of industrial strains a more efficient and oriented task, compared with the classical strategies (Demain and Adrio, 2008).

Concluding remarks The outstanding role of microorganisms in the production of antibiotics and other drugs for treating certain serious diseases is notorious. By far, most of the industrially important secondary metabolites are produced by the genus Streptomyces. The complex life cycle, high physiological versatility and ability to produce a great variety of secondary metabolites by this genus, is reflected in its large genome size. Secondary metabolites are usually formed during the late growth phase via enzymatic pathways carrying out various enzymatic steps. They are often brought on by exhaustion of nutrients, a situation that generates signals that cause a cascade of regulatory events resulting in chemical and morphological differentiation. Among the nutrients, the effect of the carbon source on secondary metabolite production has been the subject of this review. Regulation by the carbon source depends on the rapid utilization of the preferred carbon source. Different mechanisms have been described in bacteria and fungi to explain the negative carbon catabolite effects on secondary metabolite production. These mechanisms show important differences depending on the microbe being considered. With the available information, some proteins and protein complex systems have been found to be crucial for the carbon source effect on secondary metabolite formation. These proteins and systems can be grouped as follows: (a) The PTS, one of the most important mechanisms utilized for Gram-negative and Gram-positive bacteria with low


162 Ruiz et al. GC-content, (b) The CarI (for OHHL synthesis), a key factor used for Erwinia, (c) The BlD and Reg1, transcriptional regulators in Streptomyces, (d) Other Bld proteins (BldA, BlDG, and BlDH) for Streptomyces, (e) The Glk enzyme for the genus Streptomyces, (f) The SCO2127 gene, that propels production of metabolic signals responsible for CCR in Streptomyces, (g) cAMP and its binding protein, very important for Gram-negative bacteria and also for the synthesis of some antibiotics in Streptomyces, (h) The CreA zinc-finger repressor in fungi, (i) The CcpA binding to cre elements in Bacillus. In addition, direct inhibition by carbon sources of key enzymes involved in secondary metabolite pathways has been also documented. In order for a desired secondary metabolite to become a commercial reality, the genetic improvement of its production is a necessary step before getting the required titers for industrial production. For every new strain developed genetically, there are further opportunities to raise its titers. The relevance in the knowledge of CCR mechanisms will provide not only scientific profit, but also rational strategies for selection of secondary metabolite overproducing strains. Both, strain improvement and nutritional modification will preclude the scaling up into the pilot scale by biochemical engineers and later, into factory size bioreactors.

Acknowledgements This work was written to honor Professor Arnold L. Demain, on the 60th anniversary of his very productive scientific and personal life. During this time, he has been strongly devoted to the study of secondary metabolism and has guided more than one hundred students throughout the industrial microbiology and biotechnology world. In 1995, most of these students gathered to celebrate the First Arny’s Army and Friends Symposium at MIT in the USA, an academic event that has been repeated every two years for the last 14 years. The authors would like to thank the assistance of Marco A. Ortíz and Laura Escalante during the elaboration of this work.

Declaration of interest This work was supported in part by the grant P46469Z from Consejo Nacional de Ciencia y Tecnología, Mexico.

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