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Metabolic Engineering 10 (2008) 281–292

Contents lists available at ScienceDirect

Metabolic Engineering

1096-71

doi:10.1

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journal homepage: www.elsevier.com/locate/ymben

Improving production of bioactive secondary metabolites in actinomycetesby metabolic engineering

Carlos Olano, Felipe Lombo, Carmen Mendez, Jose A. Salas �

Departamento de Biologıa Funcional e Instituto Universitario de Oncologıa del Principado de Asturias (IUOPA), Universidad de Oviedo, 33006 Oviedo, Spain

a r t i c l e i n f o

Article history:

Received 10 June 2008

Received in revised form

8 July 2008

Accepted 9 July 2008Available online 15 July 2008

Keywords:

Streptomyces

Metabolism

Antibiotic biosynthesis

Precursor engineering

Heterologous expression

Regulation

Ribosome engineering

Genome shuffling

76/$ - see front matter & 2008 Elsevier Inc. A

016/j.ymben.2008.07.001

esponding author. Fax: +34 985103652.

ail address: jasalas@uniovi.es (J.A. Salas).

a b s t r a c t

Production of secondary metabolites is a process influenced by several physico-chemical factors

including nutrient supply, oxygenation, temperature and pH. These factors have been traditionally

controlled and optimized in industrial fermentations in order to enhance metabolite production. In

addition, traditional mutagenesis programs have been used by the pharmaceutical industry for strain

and production yield improvement. In the last years, the development of recombinant DNA technology

has provided new tools for approaching yields improvement by means of genetic manipulation of

biosynthetic pathways. These efforts are usually focused in redirecting precursor metabolic fluxes,

deregulation of biosynthetic pathways and overexpression of specific enzymes involved in metabolic

bottlenecks. In addition, efforts have been made for the heterologous expression of biosynthetic gene

clusters in other organisms, looking not only for an increase of production levels but also to speed the

process by using rapidly growing and easy to manipulate organisms compared to the producing

organism. In this review, we will focus on these genetic approaches as applied to bioactive secondary

metabolites produced by actinomycetes.

& 2008 Elsevier Inc. All rights reserved.

1. Introduction

Microorganisms are the source of many drugs includingantibiotics, antitumor compounds, immunosuppressants, antivir-al, antiparasitic agents and enzyme-inactivating compounds.About 23,000 bioactive secondary metabolites produced bymicroorganisms have been reported, and only 150 of them arebeing used in pharmacology, agriculture or other fields. Over10,000 of these compounds are produced by actinomycetes,representing 45% of all bioactive microbial metabolites discov-ered, 80% if we only consider those compounds in practical use.Among actinomycetes, around 7600 compounds are produced byStreptomyces species (Berdy, 2005).

Drugs in commercial use are obtained at industrial scale eitherby fermentative production, chemical synthesis or semisyntheticprocesses. When fermentation is used, it requires microbialstrains producing high titers of compound. However, wild-typestrains isolated from nature usually produce only discreteamounts of a particular secondary metabolite, which in terms ofits isolation implies the need for production improvement to meetcommercial requirements. This improvement will, in addition,determine in most cases whether a new natural product goes tomarket or is abandoned, since it is clear that when production

ll rights reserved.

yields increase then costs are reduced. The large-scale productionof drugs from microbial fermentation has been the basis of theindustry since the development of penicillin in the 1940s. Thetiters of products made by industrial cultures nowadays are veryhigh after years of intense improvement programs using thetraditional ‘‘mutate-and-screen’’ method of strain improvementthat was early developed for the penicillin strain. Today, penicillinproducer Penicillium chrysogenum makes over 70 g/l of drugstarting from a strain capable of producing only 60 mg/l, whichrepresents a 1000-fold increase. Other impressive examples ofstrain improvement are the production of riboflavin by Ashbya

gossypii that has been improved 40,000 times and the productionof a 100,000-fold excess of vitamin B12 by Pseudomonas

denitrificans (Demain, 2006).Current methods to increase the productivity of industrial

microorganisms go from the classical random mutagenesisperformed in close association with optimization of large-scaleindustrial fermentations, to the use of more rational methods. Oneof these is metabolic engineering where, in order to maximizeproduct yields, primary metabolic fluxes are redirected bythe introduction of genetic modifications through recombinantDNA technology, in a manner that supports high secondarymetabolite productivities (Adrio and Demain, 2006; Nielsen,1998). In addition, the development of modern technologies suchas DNA sequencing, transcription profiling, genomics, proteomics,metabolomics, transcriptomics and metabolite profiling hascreated new opportunities to engineer microorganisms for the

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C. Olano et al. / Metabolic Engineering 10 (2008) 281–292282

production of natural products in high yields (Bro and Nielsen,2004).

In this review, we focus on the different genetic approachesused for the production improvement of secondary metabolitesproduced by actinomycetes. In general, a particular metabolitecan be overproduced following different approaches: (i) alteringthe metabolic flux distribution of its different precursors,(ii) deregulating its specific biosynthetic pathway, (iii) increasingself-resistance or inducing resistance to several antibiotics,(iv) overexpressing structural genes coding for enzymes involvedin the biosynthesis of the metabolite, (v) using global geneticapproaches such as genome shuffling and (vi) by expressing thebiosynthetic gene cluster in a heterologous host or an industriallyoptimized strain. Examples of each of these approaches aredescribed below, summarized in Table 1 and schematized in Fig. 1.

2. Precursor engineering

The availability of biosynthetic precursors is a key factordetermining the productivity of secondary metabolites. Primarymetabolism is the supplier for those precursors that are generallyformed through the catabolism of various carbon substrates suchas fatty acids, monosaccharides or proteins. The identification andgenetic manipulation of key enzymes regulating carbon fluxthrough the metabolic network of the central carbon metabolismcan lead to an increase in the availability of a particular precursoras shown in Figs. 2 and 3.

2.1. Carbohydrate metabolism

In glucose catabolism the Embden–Meyerhof and pentosephosphate (PPP) pathways are interlinked to form the metabolicnetwork shown in Fig. 2. Key enzymes in the individual pathwaysregulate the carbon flux among them. The first intermediate inglucose catabolism, glucose-6-phosphate (G6P), is used as acommon substrate for phosphoglucose isomerase (Pgi), glucose-6-phosphate dehydrogenase (Zwf), and phosphoglucomutase(Pgm). Zwf channels glucose to the PPP. Pgm catalyzes thereversible interconversion of G6P and glucose-1-phospate (G1P),potentially leading to the deviation of carbon to the biosynthesisof glucose-based polymers such as glycogen (Ryu et al., 2006) orto the biosynthesis of 6-deoxyhexoses (6DOH) characteristic, butnot exclusive, of secondary metabolism and found in manybioactive secondary metabolites (Salas and Mendez, 2005). Pgileads carbohydrate catabolism through the Embden–Meyerhofpathway and subsequently to the tricarboxylic acid cycle, whereother precursors for secondary metabolite formation are provided.

Attempts of genetic manipulation of the initial steps in theEmbden–Meyerhof pathway and PPP have been reported for theenhanced production of clavulanic acid, actinorhodin and un-decylprodigiosin. The glycolytic pathway in Streptomyces clavuli-

gerus was genetically engineered by disruption of gap1 and gap2

genes coding for glyceraldehyde-3-phosphate dehydrogenasesinvolved in the conversion of D-glyceraldehyde-3-phosphate(G3P) into 1,3-diphosphoglicerate (1,3-BPG). Since G3P andL-arginine are precursors in the biosynthesis of clavulanic acid,the accumulation of G3P in the gap mutants correlated with asignificant increase in the production of the antibiotic, increasethat reached 3.1-fold when L-arginine was fed to the cultures(Li and Townsend, 2006). PPP was engineered by independentlyremoving two different sets of genes: zwf1 and zwf2 coding forisoenzymes of glucose-6-phosphate dehydrogenase, and devB

coding for a 6-phosphoglucolactonase in Streptomyces lividans.These deletions lead to channeling the precursors flux through theEmbden–Meyerhof instead of PPP, which in turn lead to the

increase of acetyl-CoA, precursor of undecylprodigiosin andactinorhodin, increasing production of these antibiotics (Fig. 2)(Butler et al., 2002).

2.2. Fatty acid precursors

Engineering the availability of coenzyme A (CoA) activatedfatty acid precursors has been reported for the enhancedproduction of several polyketides such as erythromycin, oligomy-cin, monensin B and actinorhodin in the producer organisms. Inthe case of erythromycin, two of its precursors are malonyl-CoAderived from the carboxylation of acetyl-CoA and methylmalonyl-CoA, which can be synthesized through different pathways suchas carboxylation of propionyl-CoA and rearrangement of succinyl-CoA (Fig. 3). Engineering the methylmalonyl-CoA metabolic nodein erythromycin producers Saccharopolyspora erythraea and Aero-

microbium erythreum leads to the enhanced production of theantibiotic depending on the fermentation medium used. Over-production of erythromycin was achieved by inactivating themethylmalonyl-CoA mutase (MCM) gene mutB and cultivation ofSac. erythraea or A. erythreum in a carbohydrate-based mediumwhile in an oil-based medium erythromycin production dimin-ished (Reeves et al., 2004, 2006). In a carbohydrate-basedmedium, the MCM reaction acts like a drain on the methylma-lonyl-CoA pool, but in an oil-based medium, the same reactionacts to fill the methylmalonyl-CoA pool (Reeves et al., 2006). Inaddition, overproduction of erythromycin was accomplished byduplication of the MCM operon (mutA, mutB, meaB and mutR) andcultivation of Sac. erythraea in an oil-based fermentation medium(Reeves et al., 2007). These experiments led to the conclusion thatthe carbon flow under oil-based growth conditions was fromsuccinyl-CoA to methylmalonyl-CoA (Reeves et al., 2007).

Antiparasitic avermectins and cell growth inhibitor oligomycinare macrocyclic lactones produced by Streptomyces avermitilis

(Omura et al., 2001). During the biosynthesis of the avermectins,two different starter units are used: isobutyryl-CoA and2-methylbutyryl-CoA produced by the degradation of branched-chain amino acids valine and isoleucine. Inactivation of bkdF

encoding a branched-chain a-keto acid dehydrogenase leads tothe abolition of avermectins production and in turn to theoverproduction of the macrolide oligomycin by enabling addi-tional extender units (malonyl-CoA, methylmalonyl-CoA andethylmalonyl-CoA) to enter the biosynthetic pathway of oligomy-cin (Cropp et al., 2001; Wei et al., 2006). In Streptomyces

cinnamonensis, inactivation of crotonyl-CoA reductase (CCR)involved in the reduction of crotonyl-CoA to butyryl-CoA leadsto the depletion of ethylmalonyl-CoA synthesized from butyryl-CoA and subsequently to the accumulation of monensin B insteadof monensin A. Monensin B requires only methylmalonyl-CoAwhile monensin A requires both methylmalonyl-CoA and ethyl-malonyl-CoA during its biosynthesis (Cropp et al., 2001). Actinor-hodin production by Streptomyces coelicolor is another example ofenhanced production of a polyketide by modification of itsprecursor supplies. In this case, the overexpression of the genesaccA2, accB and accE, coding for the different subunits of theenzyme acetyl-CoA carboxylase (ACC) in S. coelicolor, wassufficient to enhance carbon flux to malonyl-CoA, which is aprecursor of actinorhodin together with acetyl-CoA, leading to asix-fold increase in actinorhodin production (Ryu et al., 2006).

2.3. Cofactors

Additional elements of the central carbon metabolism takepart in the biosynthesis of secondary metabolites as precursorsand are also targets for metabolic engineering approaches. This is

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Table 1Increase in production of secondary metabolites produced by actinomycetes achieved by metabolic engineering

Compound Strain Engineering approach Increase (fold)

Actinomycin S. antibioticus Ribosome engineering 5.25

Actinorhodin S. coelicolor Fatty acid precursors 6

Up-regulation 2.6–40

Ribosome engineering 1.6–180

S. lividans Carbohydrate metabolism 4–5

Cofactors 4

Up-regulation 470

Down-regulation 3.5–5

C-1027 S. globisporus Biosynthetic structural genes 2–4

Cephamycin C S. clavuligerus Biosynthetic structural genes 2–4

Up-regulation 2–3

Chromomycin S. griseus Down-regulation 3

Clavulanic acid S. clavuligerus Carbohydrate metabolism 2.1–3.1

Biosynthetic structural genes 1.6–5

Up-regulation 1.5–23.8

Clorobiocin S. coelicolor Heterologous expression 1

Daunorubicin S. peucetius Biosynthetic structural genes 8–9

Up-regulation 2.4–10

6-dEB S. coelicolor Heterologous expression and fatty acid precursors 4

E. coli Heterologous expression and fatty acid precursors 1.8

Desosaminyl tylactone S. venezuelae Heterologous expression, PKS deletion and up-regulation 17.1

Doramectin S. avermitilis Biosynthetic structural genes 4–23

Doxorubicin S. peucetius Biosynthetic structural genes 3–74

Up-regulation 4

Erythromycin A. erythreum Fatty acid precursors 2–4

Sac. erythraea Fatty acid precursors 1.25–1.5

Expression of heterologous genes 2–2.5

Plasmid integration 2–5

Expression in industrial strains 50

Fredericamycin S. chattanoogensis Ribosome engineering 26

Formycin S. lavendulae Ribosome engineering 5.2

GE2270 P. rosea Ribosome engineering 1.8

Hydroxycitric acid Streptomyces U121 Genome shuffling 5

Kanamycin S. kanamyceticus Self-resistance 3.5

Megalomycin Sac. erythraea Heterologous expression and 6DOH metabolism 3.4

15-Methyl-6-dEB S. coelicolor Heterologous expression and plasmid co-integration 4–25

Mithramycin S. argillaceus Up-regulation 2–16

Monensin B S. cinnamonensis Fatty acid precursors 1.76

Nanchangmycin S. nanchangensis Biosynthetic structural genes 3

Neomycin S. fradiae Self-resistance 6

Nikkomycin X S. ansochromogenes Biosynthetic structural genes 1.8–2

Up-regulation 2

Novclobiocin 122 S. coelicolor Heterologous expression and 6DOH metabolism 8–26

Novobiocin S. coelicolor Heterologous expression 1

S. coelicolor Heterologous expression and up-regulation 3

Nystatin S. noursei Up-regulation 3.25

Biosynthetic structural genes 1.6

Oligomycin A S. avermitilis Fatty acid precursors 23

Pikromycin S. venezuelae Up-regulation 1.6–2.6

Pimaricin S. nataliensis Up-regulation 2.4

Down-regulation 1.8

Pristinamycin IIA S. pristinaespiralis Biosynthetic structural genes 1.25

Rapamycin S. hygroscopicus Up-regulation 1.2–1.4

e-Rhodomycinone S. peucetius Up-regulation 7–100

Salinomycin S. albus Ribosome engineering 1.5–2.3

Shengjimycin S. spiramyceticus Biosynthetic structural genes 2

Spinosyn Sac. spinosa Carbohydrate metabolism 3

Tetracenomycin D3 S. glauscescens Biosynthetic structural genes 20–30

Tylactone S. venezuelae Heterologous expression, PKS deletion and up-regulation 2.7

Tylosin S. fradiae Up-regulation 1.2–4.9

Down-regulation 1.5

Genome shuffling 6–8

Undecylprodigiosin S. coelicolor Up-regulation 31

S. lividans Carbohydrate metabolism 4

Down-regulation 11–12

Ribosome engineering 1.9–2.9

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Fig. 1. Scheme representing the different approaches used for improvement of

secondary metabolite production.

Fig. 2. Glucose catabolism and engineered steps involved in the biosynthesis

diphosphoglycerate; Cas2, clavaminate synthase; Cvm1, enzyme involved in the biosyn

pathways; F6P, fructose-6-phosphate; Gap1 and Gap2, glyceraldehyde-3-phosphate d

glucose-6-phosphate; G3P, glyceraldehyde-3-phosphate; Gtt, NDP-glucose synthase;

phosphoglucose isomerase; 6PG, 6-phosphogluconate; 6PGL, 6-phosphoglucolactone;

phosphate pathway; SAM, S-adenosyl-L-methionine; SanO, non-ribosomal peptide syn

dehydrogenases.

C. Olano et al. / Metabolic Engineering 10 (2008) 281–292284

the case of the cofactor S-adenosyl-methionine (SAM). Actinorho-din production was reported to be enhanced both in S. lividans andin S. coelicolor by overexpression of Streptomyces spectabilis metK

gene coding for S-adenosyl-L-methionine synthetase that cata-lyzes the biosynthesis of SAM from ATP and L-methionine. Thesame effect was obtained by addition of SAM to the culturemedium. The increase in actinorhodin production is the conse-quence of inducing the expression of pathway-specific transcrip-tional activator actII-orf4 (Kim et al., 2003; Okamoto et al., 2003).A similar effect on the production of pikromycin in Streptomyces

venezuelae was reported by expression of metK1-sp gene fromStreptomyces peucetius. In this case an increase in transcripts of thepathway-specific transcriptional activator pikD and a keto-synthase gene were observed (Maharjan et al., 2008).

3. Engineering regulatory networks

Genes for the biosynthesis of secondary metabolism pathwaysare commonly grouped together in clusters on the chromosomeincluding their pathway-specific regulatory genes. Pathway-speci-fic regulators can have either positive (activators) or negative(repressors) effects on the expression of gene cluster elements.There are clusters containing different number of pathway-specific

of several secondary metabolites produced by actinomycetes. 1,3-BPG, 1,3-

thesis of antipodal clavams; DevB, phosphoglucolactonase; 6DOH, 6-deoxyhexose

ehydrogenases; Gdh, NDP-glucose dehydratase; G1P, glucose-1-phosphate; G6P,

LAT, lysine e-aminotransferase; MetK, S-adenosyl-L-methionine synthetase; Pgi,

Pgm, phosphoglucomutase; Pah, proclavaminate amidino hydrolase; PPP, pentose

thetase; SanU and SanV, glutamate mutase; Zwf1 and Zwf2, glucose-6-phosphate

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Fig. 3. Fatty acid precursors and engineered steps involved in the biosynthesis of several secondary metabolites produced by actinomycetes. ACC, acetyl-CoA carboxylase;

BkdF, branched-chain a-keto acid dehydrogenase; ICM, isobutyryl-CoA mutase; IST, 400-O-acyltransferase; MMT, methylmalonyl-CoA transcarboxylase; MutB,

methylmalonyl-CoA mutase; PPC, propionyl-CoA carboxylase.

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positive regulatory genes ranging from one in actinorhodin path-way (Fernandez-Moreno et al., 1991) to three in daunorubicinpathway (Stutzman-Engwall et al., 1992; Otten et al., 1995, 2000).In addition, some clusters contain both activators and repressorssuch as in the tylosin pathway that contains two activator and tworepressor-coding genes (Stratigopoulos et al., 2004). However, insome pathways no regulatory genes have been identified, as is thecase of the erythromycin pathway (Rawlings, 2001). Moreover,other regulatory genes generally located outside the biosyntheticgene cluster may play a regulatory role in the cluster, in many casesshowing pleiotropic effects on the production of multiple second-ary metabolites. The best-known example is S. coelicolor thatproduces several antibiotics (actinorhodin, calcium-dependentantibiotic, undecylprodigiosin and methylenomycin) and wherethe onset of their biosynthesis is controlled by specific regulators(actII-orf4, cdaR, redD and redZ), while there are several pleiotropicgenes (i.e. afs, abs and bld) affecting antibiotic production and, inaddition, the morphological development of the bacteria (Huang etal., 2005). Taking into account all previous observations it seemsobvious that deregulation of the expression of secondary metabo-lite pathways, by overexpression of pathway-specific positiveregulators or by inactivation of pathway repressors, is the mostintuitive approach for the improvement of their production (Fig. 1).

3.1. Up-regulation

The vast majority of the pathway-specific activators inactinomycetes belong to the Streptomyces Antibiotic Regulatory

Protein family (SARP) characterized by the presence of a wingedhelix-turn-helix (HTH) motif towards the N-termini (Wietzorrekand Bibb, 1997). Overexpression of SARP positive regulators hasbeen reported to increase the production of different secondarymetabolites such as actinorhodin and undecylprodigiosin in S.

coelicolor by actII-orf4 and redD (Narva and Feitelson, 1990;Fernandez-Moreno et al., 1991), undecylprodigiosin in S. lividans

and S. parvulus by redD (Malpartida et al., 1990), nikkomycin in S.

ansochromogenes by sanG (Liu et al., 2005) and clavulanic acid in S.

clavuligerus by ccaR (Perez-Llarena et al., 1997; Hung et al., 2007).The production of other secondary metabolites such as tylosin,daunorubicin and mithramycin was reported to be up-regulatedeither using SARP activator coding genes or, in addition, otherpathway-specific positive regulators. The production of tylosin byStreptomyces fradiae was reported to be increased by overexpres-sion of tylS or tylR encoding a SARP regulator and a proteincontaining a transposase domain, respectively. In both cases anincrease in tylosin production was obtained in the wild-typestrain and in a tylosin overproducing strain (Stratigopoulos et al.,2004). Overexpression of locus dnrR1, encoding SARP regulatorDnrI, or dnrR2, encoding positive regulators DnrN and DnrOcontaining LuxR and ArsR type HTH motifs, led to a 10–100-foldincrease in the production of e-rhodomycinone and 25-foldincrease in daunorubicin (Stutzman-Engwall et al., 1992; Ottenet al., 1995). Mithramycin production by Streptomyces argillaceus

has been improved by overexpression of two different regulatorygenes in multicopy plasmids: mtrY encoding a protein with aPadR-like HTH motif (Garcıa-Bernardo et al., 2000) and mtmR

encoding a SARP family regulatory protein (Lombo et al., 1999;

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Wohlert et al., 1999). In addition, mtmR was also able to activateactinorhodin biosynthesis and complement actII-orf4 mutation inS. coelicolor JF1 (Lombo et al., 1999; Blanco et al., 2000). Othersecondary metabolite biosynthetic pathways lacking SARP activa-tors have been up-regulated using other kind of pathway-specificregulators such as pimM encoding for a PAS/LuxR regulator thatenhance pimaricin production in Streptomyces nataliensis (Antonet al., 2007) or rapH and rapG, encoding proteins with LuxR andAraC-like HTH motifs respectively, that increase rapamycinproduction in Streptomyces hygroscopicus (Kuscer et al., 2007).

SARP family proteins generally appear to function as pathway-specific regulators but, in some cases, they can work as pleiotropicregulatory proteins that control the production of multiplesecondary metabolites and morphological differentiation. Suchis the case of the afsR gene from S. coelicolor able to increaseactinorhodin and undecylprodigiosin production through itsoverexpression in S. lividans (Horinouchi et al., 1983). OthersSARP pleiotropic activators homologue to AfsR such as SsmA andAfsR-p from Streptomyces noursei and S. peucetius have beenshown to enhance nystatin and doxorubicin biosynthesis, respec-tively (Sekurova et al., 1999; Parajuli et al., 2005). In addition,overexpression of afsR-p in S. lividans, S. clavuligerus, S. griseus andS. venezuelae leads to overproduction of actinorhodin, clavulanicacid, streptomycin and pikromycin, respectively (Parajuli et al.,2005; Maharjan et al., 2008). Different pleiotropic activators havebeen shown to increase production of actinorhodin such as afsS

(Vogtli et al., 1994), the two-component regulatory system afsQ1

and afsQ2 (Ishizuka et al., 1992), abaA (Fernandez-Moreno et al.,1992) and ptpA (Umeyama et al., 1996). The last gene, pptA,encodes a phosphotyrosine protein phosphatase presumably amember of the signal transduction network that controls theproduction of actinorhodin and undecylprodigiosin (Umeyamaet al., 1996).

3.2. Down-regulation

The inactivation of pathway-specific or pleiotropic repressorscan also lead to overproduction of secondary metabolites. This isthe case of chromomycin that is overproduced when a pathway-specific transcriptional repressor cmmRII is inactivated in S. griseus

subsp. griseus (Menendez et al., 2007). A similar effect wasaccomplished in tylosin biosynthesis by disruption of tylP, genethat encode a g-butyrolactone receptor that negatively affects theproduction of the antibiotic and, in addition, influences morpho-logical differentiation in S. fradiae (Stratigopoulos et al., 2002). Anadditional pathway-specific transcriptional repressor, tylQ, ispresent in tylosin gene cluster but, in this case, its inactivationdoes not produce an increase in antibiotic production but to anearly onset of tylosin production (Stratigopoulos and Cundliffe,2002). In actinorhodin gene cluster there is also a gene, actVB-

orf10, encoding a LysR-type transcriptional regulator that has beenused for antibiotic overproduction through its inactivation inS. lividans (Martınez-Costa et al., 1999).

A well-known pleiotropic repressor system related withphosphate regulation of secondary metabolite production is thetwo-component phoR–phoP system. Disruption of phoR or simul-taneous deletion of both phoR and phoP has been recently shownto increase pimaricin production in S. nataliensis (Mendes et al.,2007). Deletion of the same system in S. lividans was reported toboost actinorhodin and undecylprodigiosin production, 5- and12-fold increase, respectively (Sola-Landa et al., 2003). InS. coelicolor there is an additional pleiotropic gene, nsdA, thatnegatively affects antibiotic production and whose disruptionleads to an increase in actinorhodin, calcium-dependent antibioticand methylenomycin biosynthesis. In the nsdA mutant the levels

of the pathway-specific regulator actII-orf4 mRNA increased(Li et al., 2006). Other modifications, such as the inactivation ofpolyphosphate kinase gene ppk, have been reported to induce theexpression of actII-orf4 activating the actinorhodin pathway(Chouayekh and Virolle, 2002).

4. Engineering antibiotic resistance

Antibiotic biosynthetic gene clusters typically include one ormore genes encoding different resistance mechanisms for self-protection to overcome the toxic effects of their products. Thesesystems include enzymes to modify the antibiotic target site,antibiotic inactivating enzymes and transport systems (Cundliffe,1989; Mendez and Salas, 2001). In some cases these resistancesystems are involved not only in self-protection but also inantibiotic biosynthesis (Olano et al., 1995; Menendez et al., 2007).Consequently, it is not surprising that increased antibioticresistance has often been used to select for mutants withincreased levels of antibiotic production (Yanai et al., 2006).

4.1. Ribosome engineering

A dramatic activation of antibiotic production was observed inS. lividans and S. coelicolor containing a mutation in rpsL gene,encoding the ribosomal protein S12, which confers resistance tostreptomycin (Shima et al., 1996). This led to rationally improvethe production of actinorhodin and undecylprodigiosin byintroducing point mutations in chromosomal rpsL gene or byusing a single-copy-number plasmid to express rpsL mutantversions (Shima et al., 1996; Okamoto-Hosoya et al., 2003). Sincethen, ribosome engineering has been used as a rational approachto enhance antibiotic production in different Streptomyces spp. byconferring resistance to several antibiotics mediated by mutantribosomes (Fig. 1). The beneficial effect on antibiotic production ofmutations in rpsL gene conferring high-level resistance tostreptomycin have been confirmed by the isolation of mutantsin other Streptomyces spp. such as S. chattanoogensis, S. anti-

bioticus, S. lavendulae and S. albus producers of fredericamycin,actinomycin, formycin and salinomycin, respectively (Hosoyaet al., 1998; Tamehiro et al., 2003). Overexpression in S. coelicolor

of genes such as frr encoding a ribosome-recycling factor whoseoverproduction has been detected in streptomycin resistant rpsL

mutants, also leads to actinorhodin overproduction up to 10-fold(Hosaka et al., 2006). On the other hand, low-level resistance tostreptomycin by mutation or deletion of rsmG gene, coding for aSAM-dependent 16S rRNA methyltransferase, has been reported toraise actinorhodin production in S. coelicolor (Nishimura et al.,2007).

Apart from mutations conferring resistance to streptomycin,enhancement of antibiotic production can be also achieved bymutations conferring resistance to other antibiotics such asgentamicin, rifampin, paromomycin, geneticin, fusidic acid,thiostrepton and lincomycin. The simultaneous introduction ofseveral resistant mutations to streptomycin, gentamicin andrifampin has a cumulative effect on antibiotic production leadingto improvements in actinorhodin, salinomycin and thiazolylpep-tide GE2270 production in S. coelicolor, S. albus and Planobispora

rosea strains, respectively (Hu and Ochi, 2001; Tamehiro et al.,2003; Beltrametti et al., 2006). In addition, an increase inactinorhodin production up to 180-fold has been recentlydescribed in a S. coelicolor strain resistant to seven of the eightantibiotics mentioned above, depending on the culture mediumused (Wang et al., 2008). These mutations produce ribosomeswith aberrant protein and hyperphosphorylated guanosinenucleotide (ppGpp) synthesis activities (Okamoto-Hosoya et al.,

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2003; Wang et al., 2008). There is a positive correlation betweenppGpp and antibiotic biosynthesis since ppGpp triggers bacterialsecondary metabolism when cells enter into stationary phase(Chakraburtty and Bibb, 1997). On the other hand, in all thesemutant strains overproduction of actinorhodin correlates tohigher expression of the actII-orf4 gene probably due to theenhanced protein synthesis (Hu and Ochi, 2001; Okamoto-Hosoyaet al., 2003; Wang et al., 2008).

4.2. Self-resistance improvement

Increasing self-resistance levels in producing organisms hasbeen also used as an approach for increasing production yields.This strategy was used in the producers of two aminoglycosides,kanamycin and neomycin, and consisted in the introductionin the producer strains of the gene encoding an aminoglycoside60-N-acetyltransferase derived from Streptomyces kanamyceticus

that confers resistance to aminoglycoside antibiotics. This re-sulted in a substantial increase in production of these antibiotics,especially neomycin (Crameri and Davies, 1986). Similar resultswere described in several antibiotic overproducing organismssuch as S. aureofaciens, 6-demethylchlortetracycline producer,through the overexpression of a self-defense gene involved indrug efflux (Dairi et al., 1995), or the S. kanamyceticus industrialkanamycin producer strain where duplication of the entirekanamycin gene cluster, including its three self-resistance genes,leads to enhanced kanamycin resistance in addition to antibioticoverproduction (Yanai et al., 2006).

5. Engineering biosynthetic structural genes

Usually structural genes involved in the biosynthesis ofsecondary metabolites have been targeted for the generation ofnew compounds through their inactivation or deletion. Inaddition, these mutants can be used as hosts for the expressionof heterologous genes that can lead to the generation of newcompounds. There are a number of examples in the literaturewhere gene dose alteration, modification and heterologousexpression of biosynthetic structural genes have been used toboost production of secondary metabolites (Fig. 1).

5.1. Gene dose increase

S. pristinaespiralis is the producer of pristinamycin II, strepto-gramin antibiotic produced as a mixture of two compounds PIIA

and PIIB in a ratio 80:20. The complete conversion of compoundPIIB into PIIA was achieved by integration into the chromosome ofan additional copy of snaA and snaB genes coding for aheterodimeric monooxygenase catalyzing this conversion. Theduplication of snaA/snaB gene dosage did not increase the totalamount of pristinamycins but enhanced the ratio of pristinamycinPIIA leading to a better production of this compound (Sezonovet al., 1997). A similar approach has been used to boost theproduction of tetracenomycin D3, intermediate in the biosynth-esis of tetracenomycin C by Streptomyces glaucescens. In this caseoverexpression of tcmM, coding for an acyl carrier protein (ACP) ofa type II polyketide synthase (PKS), cloned into a high copy vectorleads to increase up to 30-fold the production of this biosynthesisintermediate (Decker et al., 1994).

Carbon flux from glucose can be channeled to enhance theproduction of secondary metabolites by manipulating glucosecatabolism as showed in Section 2.1 and, in addition, by alteringthe expression of structural genes specifically involved in thebiosynthesis of a particular metabolite. Among these, genes

belonging to 6DOH pathways are good candidates for this kindof manipulation. This was the case of gene sgcA1 encoding a NDP-glucose synthase, involved in the biosynthesis of 6DOH 4-deoxy-4-(dimethylamino)-5,5-dimethyl-D-ribopyranose, that has beenused to engineer the production of the enediyne antitumorantibiotic C-1027 in S. globisporus. The overexpression of sgcA1

alone resulted in a two-fold improvement for C-1027 production,and up to four-fold if cagA gene coding for C-1027 apoprotein wascoexpressed (Murrell et al., 2004). It is noteworthy that genescoding for enzymes involved in the biosynthesis of 6DOH, such assgcA1, are usually clustered together with the rest of secondarymetabolite biosynthetic genes involved in the biosynthesis of theaglycone (Salas and Mendez, 2005). However, there are a fewexamples where genes involved in the biosynthesis of 6DOH arefound outside of the biosynthetic gene cluster and sharedbetween primary and secondary metabolism. This is the case forgenes involved in the biosynthesis of L-rhamnose usually foundoutside of the cluster in all cases reported (Madduri et al., 2001;Gullon et al., 2006; Luzhetskyy et al., 2007; Ramos et al., 2008).The duplication of L-rhamnose biosynthetic genes gtt and gdh

encoding NDP-glucose synthase and NDP-glucose dehydrataserespectively, have been used for the improvement of spinosynproduction in Sac. spinosa. These enzymes are the two firstactivities responsible for channeling G1P to the biosynthesis of L-rhamnose and L-dimethyl-forosamine, 6DOHs present in spinosyn(Madduri et al., 2001).

Other primary metabolism precursors such as G3P, lysine or2-oxoglutarate can be channeled to enhance the production ofseveral secondary metabolites by using specific biosyntheticstructural genes (Fig. 2). Overexpression or integration into thechromosome of clavaminate synthase gene cas2, resulted in up tofive-fold increase in clavulanic acid production in S. clavuligerus.An additional improvement, up to 23-fold, was achieved by thesimultaneous integration into the chromosome of pathway-specific activator ccaR and the clavaminate synthase gene cas2

(Hung et al., 2007). In addition, the duplication of pah2 gene,encoding a proclavaminate amidino hydrolase, has been recentlyreported to improve clavulanic acid production (Song et al., 2008).By expressing lat gene, encoding a lysine e-aminotransferase, in ahigh-copy-number plasmid in S. clavuligerus, cephamycin Cproduction was greatly enhanced (Malmberg et al., 1993, 1995).A similar approach has been used in order to increase nikkomycinX production in S. ansochromogenes by the overexpression of sanU

and sanV genes coding for the two subunits of a glutamate mutaseinvolved in the conversion of glutamate into nikkomycin pre-cursor 3-methylaspartate (Li et al., 2005). Duplication of sanO

gene coding for a non-ribosomal peptide synthetase (NRPS)responsible for the formation of the 4-formyl-4-imidazolin-2-one moiety present in nikkomycin X leads to an equivalentincrement in antibiotic production (Wang and Tan, 2004).

5.2. Gene inactivation or deletion

A different approach for improving metabolite production is toremove genes coding for activities that transform the metaboliteinto a different one. Such is the case of dnrX and dnrH genesidentified in S. peucetius and involved in the transformation ofdaunorubicin and doxorubicin into their polyglycosylated formsknown as baumycins. Inactivation of dnrX and dnrH representimprovements of 3- and 8.5-fold in the biosynthesis of doxor-ubicin and daunorubicin, respectively (Lomovskaya et al., 1998;Scotti and Hutchinson, 1996). In addition, disruption of dnrU,involved in the transformation of daunorubicin into 1,3-dihydro-daunorubicin, leads to a similar improvement of doxorubicinproduction (Lomovskaya et al., 1999). Production of doxorubicin

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was further improved by disruption of several genes in the samestrain, which lead to increases up to seven-fold in the dnrU/dnrX

double mutant and to 26-fold in the dnrU/dnrX/dnrH triplemutant. An additional 1.3–2.8-fold raising was obtained byoverexpressing dnrV and doxA genes, involved in late steps duringdoxorubicin biosynthesis, in the double or triple mutants(Lomovskaya et al., 1999). Following a similar approach, over-expressing dnmT, involved in the biosynthesis of daunorubicindeoxysugar L-daunosamine, in the dnrH mutant led to animprovement in daunorubicin production (Scotti and Hutchinson,1996).

Channeling of carbon flux by modification of amino acid-related precursor supplies has been applied to increase clavulanicacid production in S. clavuligerus, strain that also producescephamycin C and other clavams. Production of cephamycin Ccan be abolished by inactivation of lat gene coding for a lysine e-aminotransferase. This inactivation leads in turn to an increase inclavulanic acid production (Paradkar et al., 2001). A similar effectcan be achieved if production of non-clavulanic acid clavams isdepleted by inactivation of cvm1 (Mosher et al., 1999). Bothapproaches, inactivation of lat and cvm1 applied at the sameorganism (Fig. 2), in this case an industrial strain of S. clavuligerus,resulted in a significant improvement of clavulanic acid produc-tion, several orders of magnitude higher than in the wild-typestrain (Paradkar et al., 2001).

Deletion of biosynthetic genes can lead occasionally to raisesecondary metabolite production. Such is the case of nysF

inactivation that increases nystatin production in S. noursei. nysF

encodes a putative 40-phosphopantetheinyl transferase that issupposed to be involved in post-translational modification of theACP domains in nystatin type I PKS, but it was shown to negativelyregulate nystatin production (Volokhan et al., 2005). In the case ofnanchangmycin production by Streptomyces nanchangensis, thiscan be improved by selective deletion of other PKS-containingclusters found in the same organism. Eight of these clusters werefound in S. nanchangensis and the inactivation of only one of themled to a three-fold increase in nanchangmycin yield. This deletionprobably affects precursor supply for the rest of PKSs (Sun et al.,2002).

5.3. Gene modification

Doramectin (also named CHC-B1) is an antihelmintic polyke-tide compound produced by a S. avermitilis mutant strain, whichlacks a branched-chain a-keto acid dehydrogenase activity codedby bkdF. This strain produces two related compounds, doramectinand CHC-B2. The conversion of CHC-B2 into doramectin is aprocess that remains to be clarified but it involves in some wayaveC, a gene with unknown function. Improvement of productionratios for doramectin was achieved by generating several modifiedversions of aveC by site-directed mutagenesis and error-pronePCR, screening for the best variants and chromosomal insertion ofthe refined gene in the producer strain where the wild-type aveC

gene was previously inactivated (Stutzman-Engwall et al., 2003).Further enhancements of doramectin production were accom-plished by aveC semi-synthetic DNA shuffling and chromosomalinsertion of the best versions (Stutzman-Engwall et al., 2005).

5.4. Expression of heterologous genes

In S. spiramyceticus F21 the production of shengjimycin (alsonamed 400-isovalerylspiramycin) was improved by inserting in itschromosome a copy of the 400-O-acyltransferase gene, ist, from S.

mycarofaciens 1748 (Guangdong et al., 2001) (Fig. 3). On the otherhand, productivity of erythromycin has been improved in a Sac.

erythraea industrial strain by a chromosomally integrated copy ofa bacterial hemoglobin gene vhb, originally isolated fromVitreoscilla spp. (Brunker et al., 1998; Minas et al., 1998). Thissystem was previously used for the enhancement of cephalospor-in C in the fungus Acremonium chrysogenum (DeModena et al.,1993). Improvement of erythromycin production in that strainmay be the consequence of an increase in erythromycinbiosynthetic flux as a result of the increased activity of anoxygen-dependent step in erythromycin synthesis, most likelyhydroxylation steps (Brunker et al., 1998). However, it is uncertainhow the presence of the oxygen-binding heme protein affectserythromycin production (Minas et al., 1998).

6. Genome shuffling

Genome shuffling has been described and demonstratedas a new method for rapid enhancement of secondary metaboliteproduction. Using this approach six- to eight-fold increase intylosin production in S. fradiae were obtained by two rounds ofgenome shuffling over a population of classically improvedstrains. Similar titers were obtained by classical improvementmethods but along 20 rounds of mutagenesis and screening(Zhang et al., 2002). Following the same method Streptomyces spp.U121 has been engineered for the overproduction of (2S,3R)-hydroxycitric acid by three rounds of genome shuffling afterone round of mutagenesis using nitrosoguanidine (Hida et al.,2007).

7. Heterologous expression of entire gene clusters

Advances in developing DNA manipulation tools and theimprovement in genome sequencing technologies have provedfruitful for the isolation of many gene clusters involved in naturalproducts biosynthesis. However, in some cases production titersof the encoded compound are low, and there are no genetic toolsoptimized for metabolic engineering in the particular producerstrain. The consequence of these problems is an increased interestfor transferring secondary metabolite pathways to new hostswhere genetic tools are available or they have been previouslyengineered for the heterologous production of bioactive com-pounds.

7.1. Plasmids for heterologous expression

Heterologous production of secondary metabolites in S.

coelicolor can be significantly enhanced by using specific plasmidsdesigned for that purpose. This is the case of plasmid pSMALL thatwas obtained by co-integration of plasmids pJRJ2 and SCP2@ bothderived from plasmid SCP2*. The utilization of pSMALL for theproduction of 15-methyl-6-deoxyerythronolide B (15-methyl-6-dEB) resulted in four-fold increase in both S. coelicolor and S.

lividans, probably due to increased gene dosage and higherplasmid stability. This production can be further enhanced until25-fold if plasmid pBOOST is used for co-integration instead ofSCP2@ (Hu et al., 2003). Other plasmids have been developed forits use in the erythromycin producer Sac. erythraea and otheractinomycetes. Such is the case of pCJR24, integrative vectorcontaining the pathway-specific activator actII-orf4 and promotersfrom actI and actIII, that lead to increments in erythromycinproduction up to five-fold compared to wild-type strain afterplacing the entire PKS under the control of the actI promoter(Rowe et al., 1998).

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7.2. Using Streptomyces hosts

In some cases heterologous expression has being used as agenetic tool for the identification of a particular gene cluster thatresulted in the improvement of the production yields. This wasthe case of the anthracycline tetracenomycin C produced by S.

glaucescens and the indolocarbazole rebeccamycin produced byLechevaleria (formerly Saccharothrix) aerocolonigenes. Tetraceno-mycin C cluster was identified by its expression in S. lividans,which resulted in the overproduction of pigmented intermediatesof the biosynthetic pathway (Motamedi and Hutchinson, 1987),while rebeccamycin gene cluster was identified through theexpression of L. aerocolonigenes DNA containing cosmids into S.

albus, which led to production levels several folds higher thatthose of the original strain (Sanchez et al., 2002).

In other cases, a set of genes was heterologously expressed. Anexample is the production of megalomycin in Sac. erythraea.Megalomycin was originally produced by Micromonospora mega-

lomicea and it is structurally very similar to erythromycin butdiffers in the presence of an additional deoxysugar, megosamine,attached at the C-6 hydroxyl group of the aglycone. Since theproduction of megalomycin in M. megalomicea is quite poor, theexpression of the megosamine pathway in Sac. erythraea wasattempted. This led to a significant increment in megalomycinproduction and opened the possibility for further improvementboth in production and in new compound development (Volche-gursky et al., 2000). In a similar way, production of the tylosinaglycone tylactone and its glycosylated derivative desosaminyltylactone was achieved by expressing the tylosin PKS genes into aS. venezuelae strain where the pikromycin PKS genes werepreviously deleted to avoid competition for the acyl-CoA pre-cursors (Jung et al., 2006). Furthermore, 2.7- and 17.1-foldincrements in production of these compounds were accomplishedby introducing an additional copy of the pikromycin pathway-specific positive regulator gene pikD (Jung et al., 2008).

Expression of different secondary metabolite gene clusters inheterologous hosts usually is accompanied by approaches pre-viously described in this review such as overexpression of genesinvolved in 6DOH metabolism, fatty acid precursors supply orpathway-specific regulatory genes. In the case of the aminocou-marins novobiocin and clorobiocin produced by S. spheroides andS. roseochromogenes, respectively, the entire gene clusters wereexpressed independently in S. coelicolor M512 leading to equiva-lent production titers of the respective compounds as in theoriginal strains (Eustaquio et al., 2005a). However, in theheterologous host these pathways can be more easily engineeredthan in their wild-type strains. Metabolic engineering of thesepathways in S. coelicolor has led to enhance novobiocin productionby introducing the regulatory gene novG in a multicopy plasmidleading to a three-fold overproduction (Eustaquio et al., 2005b)and to increase 8–26-fold the production of novclobiocin 122 bythe introduction of genes involved in the biosynthesis ofL-rhamnose from the elloramycin pathway (Freitag et al., 2006a).Production of novclobiocin 122 was attempted before inS. coelicolor expressing the novobiocin gene cluster, by inactivationof methyltransferase gene cloU, but only small amounts of thedesired L-rhamnoside derivative were obtained (Freitag et al.,2006b).

A good example on how to engineer fatty acid precursor supplyin order to enhance the production of a secondary metabolite in aheterologous host is the production of 6-deoxy-erythronolide B(6-dEB) in S. coelicolor. This compound is produced frompropionyl-CoA and methylmalonyl-CoA. Production titers of thiscompound were increased four-fold by the heterologous expres-sion of genes matB and matC genes from Rhizobium trifolii,involved in the generation of malonyl-CoA and methylmalonyl-

CoA from exogenous malonate and methylmalonate, in a S.

coelicolor strain expressing erythromycin PKS from Sac. erythraea

(Lombo et al., 2001).

7.3. Using industrially optimized strains

A further increase in production can be achieved by expressionof the newly engineered pathways into industrially optimizedstrains. Using this approach, high erythromycin titers wereobtained in the industrial Sac. erythraea K41-135 strain afterexpressing a genetically modified PKS. This is possible because ithas been demonstrated that the overproduction phenotype is dueto mutations in non-PKS genes (Rodrıguez et al., 2003).

7.4. Using other microorganisms as hosts

In the production of secondary metabolites not only importantis the final yield but also the fermentation process. Heterologousexpression of gene clusters in Escherichia coli to overproducedesired compounds would first reduce fermentation times andwould allow industrial scale fermentations since there are well-established scalable protocols. In addition, the genetic tools forengineering E. coli strains are highly developed. Following thesecriteria during the last years important efforts have been made forthe production of erythromycin in E. coli. These efforts started byexpressing erythromycin PKS in an E. coli strain geneticallymodified to facilitate (i) post-translational PKS modifications byexpressing sfp phosphopantetheinyl transferase gene from Bacillus

subtilis and (ii) methylmalonyl-CoA production by expressing PCCgenes (pccA and pccB) from S. coelicolor. The activity of thebiotinylated subunit PccA was enhanced by overexpression of E.

coli birA biotin ligase gene. The resultant strain was able toproduce 6-dEB at levels equivalent to a Sac. erythaea industrialstrain (Pfeifer et al., 2001). Further metabolic engineering of thatstrain such as the expression of metK (Fig. 2) from S. spectabilis

encoding a SAM synthetase, led to a two-fold improvement in6-dEB production (Wang et al., 2007). All accumulated knowledgehas led to the final production of erythromycin C and D in E. coli byexpression of erythromycin PKS and 17 additional genes frommegosamine pathway coding for enzymes involved in 6DOHbiosynthesis and other tailoring modifications (Peiru et al., 2005).Similar approaches are underway for the production of polyke-tides in other heterologous hosts such as Saccharomyces cerevisiae,where different pathways, propionyl-CoA ‘‘dependent and inde-pendent’’ for the production of methylmalonyl-CoA have beenintroduced (Mutka et al., 2006).

In addition to polyketides, other secondary metabolites such asnon-ribosomal peptides are produced in E. coli. In these cases, thestrain also has to be engineered by introducing sfp gene for post-translational modification of the NRPS. In that way antitumorechinomycin and its intermediate triostin A were produced inE. coli by expressing the entire gene cluster isolated fromStreptomyces lasaliensis (Watanabe et al., 2006).

8. Conclusions

The future success of the pharmaceutical industry depends onthe identification or development of new compounds with novelactivities or directed to more specific targets. The rapidly growingamount of secondary metabolite gene clusters identified andcharacterized provides new genetic tools for the generation ofnovel compounds by combinatorial biosynthesis. In addition,these clusters contain elements that can be used to increasethe production yields. As shown in this review, significant

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improvement of secondary metabolite production has beenobtained during recent years applying different methods ofgenetic and metabolic engineering both of wild-type producerorganisms and heterologous host after expression of biosyntheticgene clusters. These genetic methods include the deletion andoverexpression of genes directed to up- or down-regulate theexpression of secondary metabolite gene clusters, or to overridebottlenecks in the biosynthesis of these compounds. Particularattention has been drawn to alter carbon flux by redirecting thecentral primary metabolic networks toward specific parts of themetabolism. Following these approaches, further improvementsin the production of the compounds reviewed here and new onesshould be expected in the future, with special attention to theefforts in the development of heterologous hosts for the expres-sion of secondary metabolites.

Acknowledgments

Research in the authors’ laboratory has been supported bygrants from the Spanish Ministry of Education and Science(BFU2006-00404 to J.A.S. and BIO2005-04115 to C.M.), the RedTematica de Investigacion Cooperativa de Centros de Cancer toJ.A.S. (Ministry of Health, Spain; ISCIII-RETIC RD06/0020/0026)and from the UE FP6 (Integrated Project no. 005224). We thankObra Social Cajastur for financial support to Carlos Olano andFelipe Lombo.

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