**3. Metabolic pathways in the production of secondary metabolites of bacteria**

The 80–100 amino acid residues of domain T, located downstream of domain A, form a thioester bond (covalent bond) between the activated monomer and the NRPS, and this allows the peptide being synthesized to remain attached to the NRPS throughout the process of elongation. The condensation domain C (450 amino acids) is usually found after each A–T module and catalyzes the formation of peptide bonds between bound residues on two adjacent modules. In general, the number and order of modules present in an NRPS determine the length and the resulting nonribosomal peptide structure. The thioesterase domain, present only in

Polyketides are kown as natural products, having diverse functions in medical applications, and they are assembled by PKS enzymes. PKS enzymes act exactly like fatty acid synthase to generate a diverse extent of polyketides. Also, PKS enzymes start the polyketide assembly by priming the initiator molecule to the catalytic residue, and then making an extender unit for the elongation chain. On the basis of structural architecture and variation in enzymatic mechanism, PKS enzymes have been classified into three types: (1) type I PKS, (2) type II PKS,

This section describes all three types of PKS enzymes (**Table 8**). Modular PKSs include active sites, called modules; they are polypeptides used to synthesize a string of carbon. The active sites of each module are used only once during assembly of the molecule and determine the choice of units of structure and the level of reduction or dehydration for the cycle of expansion. They catalyze the length of the string of carbon, and the number of cycles of reaction is determined by the number and order of the modules in the polypeptide constituting the

These are multidomain proteins (containing several domain enzymes on the same polypeptide) that can be modular (**Figure 3**), for example, the modular systems responsible for the synthesis of macrolides (erythromycin, rapamycin, rifamycin B, etc.) in bacteria, which is

**Either modular PKS or type I Either discrete PKS or type II Either ketosynthase polyketide or type** 

**Table 8.** Classification of polyketide synthase enzymes and the functional and mechanistic differences between them.

Includes a series of modular heterodimeric enzymes. Each enzyme has a special function and use; the ACP domain transfers activated acyl-CoA substrate malonyl-CoA, an

extender unit

**III**

The homodimeric ketosynthase enzyme can carry out various biochemical reactions at a single active site; it acts in the absence of ACP or directly recognizes the acyl-CoA molecules malonyl-CoA or methylmalonyl-CoA, an extender unit

*Streptomyces* Secondary Metabolites http://dx.doi.org/10.5772/intechopen.79890 113

the last module, releases the peptide from the NRPS.

iterative (**Figure 4**) (for example, lovastatin nonaketide).

**3.2. Polyketide synthase pathways**

and (3) type III PKS.

PKS [63].

*3.2.1. Type I PKS*

Many functional enzymes organized into modules. Each module has a specific function and use; acyl carrier protein (ACP) domain activates acyl-CoA substrates malonyl-CoA or methylmalonyl-CoA or ethylmalonyl-

CoA, an extender unit

Secondary metabolic pathway reactions are formed by an individual enzyme or multienzyme complexes. Intermediate or end products of primary metabolic pathways are channeled from their systematic metabolic pathways that lead to the synthesis of secondary metabolites. There are six known pathways: the peptide pathway, the PKS pathway, the NRPS pathway, the hybrid (nonribosomal polyketide) synthetic pathway, the shikimate pathway, the β-lactam synthetic pathway, and the carbohydrate pathway. The genes encoding these synthetic pathway enzymes are generally present in chromosomal DNA and are often arranged in clusters.

### **3.1. Nonribosomal peptide synthesis pathways**

Nonribosomal peptides are peptides that are not synthesized at the level of ribosomes. One of the peculiarities of nonribosomal peptides is their small size. These peptides are not encoded by a gene, and they are not limited to the 20 basic amino acids. Indeed, the peculiarity of the NRPS system is the ability to synthesize peptides containing proteinogenic and nonproteinogenic amino acids. In many cases, these enzymes are activated in collaboration with polyketone synthases giving hybrid products. The products of these multifunctional enzymes have a broad spectrum of biological activities, and some of them have been useful for medicine, agriculture, and biological research [61].

NRPS are organized in a modular way. Each module is responsible for the incorporation of a specific monomer. The modules are subdivided into domains, and each domain catalyzes a specific reaction in the incorporation of a monomer. The number and order of modules and the type of domain present in the modules of each NRPS determine the structural variation of synthesized peptides by dictating the number, order, and choice of amino acid to incorporate during elongation. Four main areas are needed for complete synthesis (**Figure 2**). Each domain has a specific function when incorporating the monomer. Domain A, from 500 to 600 amino acid residues, is necessary for the recognition of the amino acid and its activation.

**Figure 2.** Minimum domains required in an NRPS [62].

The 80–100 amino acid residues of domain T, located downstream of domain A, form a thioester bond (covalent bond) between the activated monomer and the NRPS, and this allows the peptide being synthesized to remain attached to the NRPS throughout the process of elongation. The condensation domain C (450 amino acids) is usually found after each A–T module and catalyzes the formation of peptide bonds between bound residues on two adjacent modules. In general, the number and order of modules present in an NRPS determine the length and the resulting nonribosomal peptide structure. The thioesterase domain, present only in the last module, releases the peptide from the NRPS.

#### **3.2. Polyketide synthase pathways**

Polyketides are kown as natural products, having diverse functions in medical applications, and they are assembled by PKS enzymes. PKS enzymes act exactly like fatty acid synthase to generate a diverse extent of polyketides. Also, PKS enzymes start the polyketide assembly by priming the initiator molecule to the catalytic residue, and then making an extender unit for the elongation chain. On the basis of structural architecture and variation in enzymatic mechanism, PKS enzymes have been classified into three types: (1) type I PKS, (2) type II PKS, and (3) type III PKS.

This section describes all three types of PKS enzymes (**Table 8**). Modular PKSs include active sites, called modules; they are polypeptides used to synthesize a string of carbon. The active sites of each module are used only once during assembly of the molecule and determine the choice of units of structure and the level of reduction or dehydration for the cycle of expansion. They catalyze the length of the string of carbon, and the number of cycles of reaction is determined by the number and order of the modules in the polypeptide constituting the PKS [63].

#### *3.2.1. Type I PKS*

**Figure 2.** Minimum domains required in an NRPS [62].

**3.1. Nonribosomal peptide synthesis pathways**

agriculture, and biological research [61].

**3. Metabolic pathways in the production of secondary metabolites of** 

Secondary metabolic pathway reactions are formed by an individual enzyme or multienzyme complexes. Intermediate or end products of primary metabolic pathways are channeled from their systematic metabolic pathways that lead to the synthesis of secondary metabolites. There are six known pathways: the peptide pathway, the PKS pathway, the NRPS pathway, the hybrid (nonribosomal polyketide) synthetic pathway, the shikimate pathway, the β-lactam synthetic pathway, and the carbohydrate pathway. The genes encoding these synthetic pathway enzymes are generally present in chromosomal DNA and are often arranged in clusters.

Nonribosomal peptides are peptides that are not synthesized at the level of ribosomes. One of the peculiarities of nonribosomal peptides is their small size. These peptides are not encoded by a gene, and they are not limited to the 20 basic amino acids. Indeed, the peculiarity of the NRPS system is the ability to synthesize peptides containing proteinogenic and nonproteinogenic amino acids. In many cases, these enzymes are activated in collaboration with polyketone synthases giving hybrid products. The products of these multifunctional enzymes have a broad spectrum of biological activities, and some of them have been useful for medicine,

NRPS are organized in a modular way. Each module is responsible for the incorporation of a specific monomer. The modules are subdivided into domains, and each domain catalyzes a specific reaction in the incorporation of a monomer. The number and order of modules and the type of domain present in the modules of each NRPS determine the structural variation of synthesized peptides by dictating the number, order, and choice of amino acid to incorporate during elongation. Four main areas are needed for complete synthesis (**Figure 2**). Each domain has a specific function when incorporating the monomer. Domain A, from 500 to 600 amino acid residues, is necessary for the recognition of the amino acid and its activation.

**bacteria**

112 Basic Biology and Applications of Actinobacteria

These are multidomain proteins (containing several domain enzymes on the same polypeptide) that can be modular (**Figure 3**), for example, the modular systems responsible for the synthesis of macrolides (erythromycin, rapamycin, rifamycin B, etc.) in bacteria, which is iterative (**Figure 4**) (for example, lovastatin nonaketide).


**Table 8.** Classification of polyketide synthase enzymes and the functional and mechanistic differences between them.

**Figure 3.** Structure of a modular type I PKS [64]. Note: KS, ketosynthase; AT, acyl transferase; KR, ketoreductase; ACP, acyl carrier protein; TE, Thioesterase; DH, dehydrate.

proteins with a unique polypeptide chain, and are involved in the biosynthesis of flavonoid

*Streptomyces* Secondary Metabolites http://dx.doi.org/10.5772/intechopen.79890 115

The shikimate pathway groups the essential building blocks for a large assembly of aromatic metabolites and amino acids. Metabolites of the aromatic compounds present protection against ultraviolet radiation, electron transport, and signaling molecules, and also act as antibacterial agents. The shikimate pathway enzymes use specific chemical substrates, i.e. erythrose-4-phosphate and phosphoenol pyruvate (primary metabolites), to start the synthesis of aromatic building blocks. Herein, the first seven enzymes catalyze the chemical reactions in a chronological manner to produce chorismate. Two bacterial enzymes are able to transfer a complete enolpyruvoyl moiety to a metabolic pathway. 5-Enolpyruvoyl shikimate 3-phosphate synthase is considered one of the shikimate pathways. Chorismate synthase is an enzyme involved in this pathway, and its function needs the presence of a reduced cofactor, flavin mononucleotide, for its activation [69]. The Gram-positive, filamentous *Streptomyces venezuelae* (soil bacterium) and other actinomycetes gather chloramphenicol with the help of aromatic precursors. Aromatic building blocks originated from the shikimate pathway act as precursors for the phenylpropanoid unit of chloramphenicol. First, chorismic acid branches out from the shikimate pathway to produce *p*-aminophenylalanine, which could afterwards be converted into a *p*-nitrophenylserinol component by an enzymatic reaction. 4-Amino-4-deoxychorismic acid (ADC) was found as a common precursor for both para-aminobenzoic acid and PAPA: a flexible tool for identifying pleiotropic pathways using genome-wide association study summaries pathways. The genetic map reveals that pabAB genes encode enzymes for ADC biosynthesis that are clustered in a distinct region of the *S. venezuelae* chromosome. Echinosporin isolated from *Saccharopolyspora erythraea* has antibacterial and anticancer activities. This molecule has a sole tricyclic acetal-lactone structure, and the main structure does not show its biosynthetic pathway. The shikimate pathway intermediate is guided to group the echinosporin by enzy-

Cephalosporins belong to the family of β-lactam antibiotics, used for treating bacterial infections for more than 40 years. Interestingly, Gram-positive bacteria, Gram-negative bacteria,

precursors [67].

**Figure 5.** Type III PKS [68].

matic reactions [70].

**3.3. Lactam ring synthetic pathways**

#### *3.2.2. Type II PKS*

These are monofunctional protein complexes (for example, actinorhodin from *S. coelicolor*). These PKSs catalyze the formation of compounds that require aromatization and cyclization steps but no reduction or dehydration. These PKSs are involved in the biosynthesis of aromatic bacterial products such as actinorhodin, tetracenomycin, and doxorubicin [66].

#### *3.2.3. Type III PKS*

These have a single active site to catalyze the extension of the polyketide chain and cyclization without the use of an ACP (**Figure 5**). They are responsible for the synthesis of chalcones and stilbenes in plants, as well as polyhydroxy phenols in bacteria. Chalcone synthases are small

**Figure 4.** Structure of an iterative type I PKS [65].

**Figure 5.** Type III PKS [68].

proteins with a unique polypeptide chain, and are involved in the biosynthesis of flavonoid precursors [67].

The shikimate pathway groups the essential building blocks for a large assembly of aromatic metabolites and amino acids. Metabolites of the aromatic compounds present protection against ultraviolet radiation, electron transport, and signaling molecules, and also act as antibacterial agents. The shikimate pathway enzymes use specific chemical substrates, i.e. erythrose-4-phosphate and phosphoenol pyruvate (primary metabolites), to start the synthesis of aromatic building blocks. Herein, the first seven enzymes catalyze the chemical reactions in a chronological manner to produce chorismate. Two bacterial enzymes are able to transfer a complete enolpyruvoyl moiety to a metabolic pathway. 5-Enolpyruvoyl shikimate 3-phosphate synthase is considered one of the shikimate pathways. Chorismate synthase is an enzyme involved in this pathway, and its function needs the presence of a reduced cofactor, flavin mononucleotide, for its activation [69].

The Gram-positive, filamentous *Streptomyces venezuelae* (soil bacterium) and other actinomycetes gather chloramphenicol with the help of aromatic precursors. Aromatic building blocks originated from the shikimate pathway act as precursors for the phenylpropanoid unit of chloramphenicol. First, chorismic acid branches out from the shikimate pathway to produce *p*-aminophenylalanine, which could afterwards be converted into a *p*-nitrophenylserinol component by an enzymatic reaction. 4-Amino-4-deoxychorismic acid (ADC) was found as a common precursor for both para-aminobenzoic acid and PAPA: a flexible tool for identifying pleiotropic pathways using genome-wide association study summaries pathways. The genetic map reveals that pabAB genes encode enzymes for ADC biosynthesis that are clustered in a distinct region of the *S. venezuelae* chromosome. Echinosporin isolated from *Saccharopolyspora erythraea* has antibacterial and anticancer activities. This molecule has a sole tricyclic acetal-lactone structure, and the main structure does not show its biosynthetic pathway. The shikimate pathway intermediate is guided to group the echinosporin by enzymatic reactions [70].

#### **3.3. Lactam ring synthetic pathways**

**Figure 4.** Structure of an iterative type I PKS [65].

*3.2.2. Type II PKS*

acyl carrier protein; TE, Thioesterase; DH, dehydrate.

114 Basic Biology and Applications of Actinobacteria

*3.2.3. Type III PKS*

These are monofunctional protein complexes (for example, actinorhodin from *S. coelicolor*). These PKSs catalyze the formation of compounds that require aromatization and cyclization steps but no reduction or dehydration. These PKSs are involved in the biosynthesis of aro-

**Figure 3.** Structure of a modular type I PKS [64]. Note: KS, ketosynthase; AT, acyl transferase; KR, ketoreductase; ACP,

These have a single active site to catalyze the extension of the polyketide chain and cyclization without the use of an ACP (**Figure 5**). They are responsible for the synthesis of chalcones and stilbenes in plants, as well as polyhydroxy phenols in bacteria. Chalcone synthases are small

matic bacterial products such as actinorhodin, tetracenomycin, and doxorubicin [66].

Cephalosporins belong to the family of β-lactam antibiotics, used for treating bacterial infections for more than 40 years. Interestingly, Gram-positive bacteria, Gram-negative bacteria, and fungi are the major sources of β-lactam antibiotics. The Gram-positive *Streptomyces clavuligerus* is able to produce both clavulanic acid and cephamycin, since the Gram-negative bacterium *Lysobacter lactamgenus* produces cephabacins. Two hypotheses have been put forward for β-lactam biosynthesis: (1) horizontal gene transfer (HGT) from bacteria to fungi and (2) vertical descent (originated from a common ancestor). Bioinformatics, genetic designs, and sequence identity are more beneficial in HGT.

**Conflict of interest**

**Author details**

and Rebecca Pogni4

Ben Bella, Oran, Algeria

University, M'sila, Algeria

**References**

2006.05.008

Temouchent, Temouchent, Algeria

Mohammed Harir1,2\*, Hamdi Bendif2

The authors declare that no conflicting interest exists.

\*Address all correspondence to: mohamedharir31@gmail.com

Society for Microbiology. 2007;**2**(3):125-131

, Miloud Bellahcene3

1 Biology of Microorganisms and Biotechnology Laboratory, University of Oran 1 Ahmed

3 Department of Natural and Life Sciences, Institute of Sciences, University Center of Ain

4 Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy

[1] Baltz R. Antimicrobials from actinomycetes: Back to the future. Microbe - American

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[4] Claessen D, de Jong W, Dijkhuizen L, Wösten HAB. Regulation of Streptomyces development: Reach for the sky! Trends in Microbiology. 2006;**14**(7):313-319. DOI: 10.1016/j.tim.

[5] Granozzi C, Billetta R, Passantino R, Sollazzo M, Puglia AM. A breakdown in macromolecular synthesis preceding differentiation in *Streptomyces coelicolor* A3 (2). Journal of General

[6] Zitouni A. Taxonomic study and antagonistic properties of Nocardiopsis and Saccharothrix isolated from Saharan soil and production of new antibiotics by Saccharothrix sp. 103 p.

*octospinosus*. PLoS One. 2011;**6**(8):e22028. DOI: 10.1371/journal.pone.0022028

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2 Department of Natural and Life Sciences, Faculty of Sciences, Mohamed Boudiaf

, Zohra Fortas1

*Streptomyces* Secondary Metabolites http://dx.doi.org/10.5772/intechopen.79890 117

The production of β-lactam antibiotic occurs through three different steps: prebiosynthetic steps, intermediate formation steps, and late steps (also known as decorating steps) [71–76]. The biosynthesis of building blocks for β-lactam consist of L-α-aminoadipic acid, L-cysteine, and L-valine. L-α-Aminoadipic acid is not a proteinogenic amino acid formed from L-lysine. The actinomycete lysine 6-aminotransferase converts L-lysine into L-α-aminoadipic acid.

The two starting enzyme reactions are omnipresent in fungi and cephalosporin biosynthesis. D-(L-Aminoadipyl)-L-cysteinyl-D-valine synthase is the first enzyme, using all three amino acids gathered into a tripeptide through condensation reaction. This enzyme is NRPS encoded by the acvA (pcbAB) gene. The next step is the synthesis of a bicyclic ring (a four-member β-ring is fused with a five-member thiazolidine ring) through an oxidative reaction, catalyzed by isopencillin N-synthase, and results in the formation of isopenicillin N. Cephalosporin– cephamycin biosynthesis is the development of the five-member thiazolidine ring into a six-member dihydrothiazine ring. Several enzymes consecutively contribute to this ring conversion. β--Lactam biosynthesis is synthesized by a gene, which is usually clustered in the DNA of all reproducing bacteria. Bacterial species capable of producing β--lactam antibiotics exhibit an ecological benefit. In contrast, β-lactam–producing bacteria show low sensitivity to β-lactams on their own, or they have evolved to inactivate β-lactam antibiotics by β-lactamase enzymes.
