**2. Importance of** *Streptomyces*

Streptomycetes are bacteria that produce the largest amount of commercially used antibiotics worldwide. To date, many *Streptomyces* genomes have been sequenced, and any approach that offers the possibility of increasing yields must be analyzed to increase production levels or produce more effective compounds against bacterial infections. Currently, there is great interest in the isolation of new *Streptomyces* strains from unusual environments as new sources of antibiotics [8].

Many antibiotics have precursors that act as intermediates for different metabolic pathways. Gunnarsson et al. (2004) described how central carbon metabolism is linked to producing many different antibiotics [9]; however, little has been achieved to improve the synthesis of inermediates and influence the biosynthesis of antibiotics or other commercially important compounds.

Genome sequencing of important antibiotic-producing *Streptomyces* has allowed the analysis of genes that encode the enzymes involved in carbon metabolism pathways. Gene multiplicity exists in the genomes of these organisms and up to four copies of the same gene can be found; however, the relevance of this fact has not been established. Gene multiplicity or redundancy is very common in the chromosomes of many microorganisms, especially *Streptomyces*.

## **3. Gene multiplicity in glycolytic pathway genes**

There are many databases, such as KEGG, to identify how many genes code for the same activity. It is known that nine enzymes participate in the glycolytic pathway, as shown in **Table 1**, and it was found that three genes encode phosphofructokinase, which catalyzes the phosphorylation of fructose 6-phosphate in most antibiotic-producing *Streptomyces* species. Only *S. coelicolor* and *S. venezuelae* have two genes.

The conversion of fructose 6-phosphate to fructose 1,6-bisphosphate with the concomitant hydrolysis of adenosine triphosphate represents the first irreversible step specific to glycolysis. This reaction catalyzed by phosphofructokinase (PFK; EC 2.7.1.11) is subjected to tight control, thus rendering it a critical regulatory point of the glycolytic flux [10]. The genes that encode PFK present a high multiplicity, and in these 11 antibiotic-producing *Streptomyces*, 31 proteins have been noted. The PFKs have amino acids between 341 and 345, and only *S. griseus* and *S. venezuelae* have two copies, whereas the remainder has three genes. The 31 proteins have a very high identity with some identical regions along the sequence, and at the aminoterminal end, a highly conserved domain GGDCPGLNAVIR is present; 133 residues out of 350 are fully conserved, representing 38% identity. Despite the high resemblance, the phylogenetic tree is divided into two clades, one of which is split into two subgroups, as shown in the tree, each copy is distributed in one clade, and two of them are more closely related to each other. This clade includes *S. griseus* and *S. venezuelae*, which have only two copies of the PFK-coding genes, suggesting that retention of a third gene copy has not occurred in these species (**Figure 1**). Unlike *Escherichia coli*, which has two PFKs that do not have a common ancestor, *Streptomyces* does [11].


*Actinobacteria - Diversity, Applications and Medical Aspects*


#### **Table 1.**

*Gene multiplicity in glycolytic pathway genes.*

### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

#### **Figure 1.**

*Partial multiple alignments of fosfofructokinase proteins from antibiotic producing* Streptomyces *(A) and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

*Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

The next reaction is performed using aldolase (EC 4.1.2.13), which catalyzes the conversion of fructose 1-6-diphosphate to glyceraldehyde 3-phosphate and dihydroxy-acetone phosphate. Most *Streptomyces* have two genes that code for this enzyme, except for *S. lavendulae*, which has four genes. The SLAV\_00910 and SLAV\_38480 proteins have 100% identity; therefore, are coded by duplicated genes.

Triosephosphate isomerase (EC 5.3.1.1) is an enzyme that converts dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. Of all the antibiotic-producing *Streptomyces*, only *S. coelicolor* had two proteins for this activity with an identity of 31% between the two copies. The average molecular weight of all the triose phosphate isomerases is 27 kDa with an identity between them greater than 85%, except for SCO0578.

Another *Streptomyces* gene that showed genetic redundancy was the one coding for glyceraldehyde 3-phosphate dehydrogenase. Glyceraldehyde 3P-dehydrogenase enzyme shows two classes of proteins, some with 331–335 amino acids (approx. 36.1 kDa) and others with a higher molecular weight, the majority of 481 residues (52.9 kDa), except for *S. venezuelae* with 461 amino acids (50.1 kDa). Only *S. albus* has a single small protein and the rest of the microorganisms had two or three, one large and one or two small. *S. hygroscopicus* has four paralogues, three of approximately 335 amino acids and one of 481 residues. Alignment of the amino acid sequences of glyceraldehyde 3P-dehydrogenase showed that the larger proteins (SLAV\_31635, SPRI\_6547, SVEN\_0459, CP970\_08935, SNOUR\_35775, SHJGH\_1800, SCO7040, SGR\_936, and BB341\_01000) had an extended N-terminal region of approximately 126 residues. The highly conserved regions GFGRIGR, ASCTTNA, PRVPV, and WYDNEXG were near or into the NAD-binding and C-terminal domains of the enzyme (**Figure 2**). In the phylogenetic analysis, the sequences are divided into two lineages. The first clade is formed by small-sized proteins, whereas the other included all proteins with large-size copies. Small protein clades are split into two subgroups. One is formed by a single organism with the copy with less divergence, and the other subgroup is divided into two clades, each containing a single homolog of each organism; therefore, the copies diverged to the point of being more related between species than between duplicates of the same species (**Figure 2**).

In all antibiotic-producing *Streptomyces,* the only reaction whose enzyme is encoded by one gene is 2,3-bisphosphoglycerate-dependent phosphoglycerate kinase (EC 2.7.2.3), which catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate with the generation of an ATP molecule. All proteins were 403 amino acids long and had high identity with each other. The phosphoglycerate kinase of *S. clavuligerus*, an overproducer of clavulanic acid, was detected in the proteome of this microorganism grown in tryptic soy broth, indicating that its gene was expressed in this culture medium [12].

Phosphosphoglycerate mutase (EC 5.4.2.11) performs the internal transfer of a phosphate group from the C-3 carbon to the C-2 carbon, resulting in the isomerization of 3-phosphoglycerate to 2-phosphoglycerate. Only two genes are involved in this activity in *S. griseus, S. clavuligerus, S. coelicolor*, and *S. noursei*. Most of the proteins are 252–253 amino acids in length, which are very similar (identity greater than 90%), and there are only two proteins with 511 amino acids long, SLNWT\_3230 and SCO6818 also, with 82% identity and 91.6% similarity between them. One of the two *S. noursei* mutases (SNOUR\_07945) is smaller (218 amino acid residues), with 22% identity to 253 amino acid mutases and 92% identity to histidine phosphatases of various *Streptomyces*. Then probably, this mutase is wrongly annotated in its genome.

Enolase (EC 4.2.1.11), also known as phosphopyruvate hydratase, is a metalloenzyme responsible for converting 2-phosphoglycerate to PEP. *S. coelicolor*,

**Figure 2.**

*Partial multiple alignment of glyceraldehyde 3-phosphate dehydrogenase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

*Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

*S. griseus*, *S. lavendulae*, S. *pristinaspiralis*, and *S. venezuelae* have two enolases, one with 426–428 amino acids and the other with 432–435 residues. The rest have only one enolase, which in most of these *Streptomyces* has 426 amino acids. Between the two proteins of different sizes, there is approximately 56% identity; however, between the small proteins of the different microorganisms, there is an identity greater than 90%, and between the larger ones, there is an identity greater than 83%.

Pyruvate kinase (EC 2.7.1.40) is a key enzyme involved in the last step of glycolysis that catalyzes the transfer of a phosphate group from PEP to ADP, yielding one molecule of PYR and one molecule of ATP. There are two types: type I and type II, and both enzymes show positive cooperative effects concerning PEP. The type I enzyme is activated by fructose 1,6-bisphosphate (F1,6BP) and the type II by AMP [13]. According to the amino acid sequences of PYKF and PYKA enzymes from *E. coli*, the smaller *Streptomyces* PYR kinases are type I, and the others are type II. Most proteins have 474–479 amino acids regardless of whether they are class I or class II and have greater than 60% identity between them.

### **4. TCA cycle**

The TCA cycle is a central metabolic pathway of all aerobic organisms and synthesizes many important precursors and molecules [14] and eight reactions are performed for complete glucose oxidation.

The first reaction is citrate synthase (CS, EC 2.3.3.1), which catalyzes the irreversible conversion of OXA and acetyl-CoA into citrate. The proteins are classified into two types: type I and type II, and are encoded by four genes in eight of the selected antibiotic-producing *Streptomyces*, three in *S. venezuelae*, two in *S. noursei,* and *S. pristinaespiralis*. Their coding genes show the greatest redundancy among all the genes involved in carbon metabolism in most *Streptomyces* species (**Table 2**). Sals\_SLNWT\_1427 (*S. albus*) and ska\_CP970\_28100 (*S. kanamyceticus*) are the largest, with 439 and 451 amino acid residues, respectively. In the amino acid sequence alignment, two groups of CSs are distinguished: small ones ranging from 363 to 395 amino acid residues and large ones ranging from 416 to 451 residues. Small or large types of either type I or II have been found, implying that type does not depend on size. As shown in **Figure 3**, a highly conserved histidine is embedded in the conserved region GPLHGXA. Another conserved amino acid is arginine in the DPR conserved amino acid sequence and aspartic or glutamic acid in the conserved NVD/E. All of these were previously reported as essential residues involved in interactions with the substrate OXA in *Streptomyces* [15]. All CSs have a common ancestor but are separated into two main lineages. One of them is split into two subgroups, which include CS classified as types 1 and 2, like the proteins of *S. lavendulae* (type 1) and *S. pristinaespiralis* (type 2) with approximately 429–433 amino acid residues. The other clade was also split into two subgroups, including proteins with 366 and 390 residues. The CSs of *S. coelicolor* encoded by *sco2736*, *sco4388* (both classified as type 2), and *sco5832* (unclassified) are grouped in one of the main clades and distributed in the two subgroups. The sequence encoded by *sco5831* is grouped into the other principal clade with another seven proteins. The % of identity among the proteins included in each subgroup is 87%–94% and similarity 91%–98%. These differences are mainly observed at the amino terminus. Taking *S. coelicolor* CSs as an example, the identity between the CSs found in each subgroup, was low, between 24.6% and 28.1%, and the similarity between 46% and 49%.

Aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), the E1 component of 2-oxoglutarate dehydrogenase (EC 1.2.4.2), and malate dehydrogenase (EC 1.1.1.37) are encoded by unique genes in all *Streptomyces,* probably because of the



#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*


**Table 2.**

*Gene multiplicity in TCA cycle genes.*

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*


#### *Actinobacteria - Diversity, Applications and Medical Aspects*

#### **Figure 3.**

*Partial multiple alignment of citrate synthase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

importance of the reactions they catalyze. The molecular weight of the aconitases of these microorganisms is 97 kDa with an identity between them greater than 92%. Isocitrate dehydrogenases have a molecular weight of 79 kDa in all these *Streptomyces*, except *S. noursei* and *S. pristinaspirales*, which also have another protein (SNOUR\_17135 and SPRI\_3067) that are smaller (45 kDa). The identity between the large proteins and between the small ones is about 85% and 77%, respectively. On the other hand, all MDHs have 329 amino acids with an identity between 87 and 93%.

The 2-oxoglutarate dehydrogenase complex is a central enzyme in aerobic metabolism that catalyzes the oxidative decarboxylation of oxoglutarate, generating

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

NADH [16]. 2-oxoglutarate dehydrogenase is composed of three subunits, E1 (EC 1.2.4.2), E2 (EC 2.3.1.61, dihydrolipoamide succinyltransferase), and E3 (EC 1.8.1.4; dihydrolipoamide dehydrogenase). As previously mentioned, there is a single gene coding for component E1 in all selected antibiotic-producing *Streptomyces*. The E2 component is encoded by one gene, except in *S. coelicolor*, which has three. *S. pristinaespiralis*, *S. albus*, *S. clavuligerus*, *S. coelicolor*, *S. griseus*, and *S. hygroscopicus* genomes have only one gene coding for E3 component, while the remaining five microorganisms have two copies with a low identity (≤ 38%) between them suggesting different origins.

Multiple alignments of the amino acid sequences of the E2 subunit of 2 oxoglutarate dehydrogenase showed close resemblance, with many highly conserved amino acids within the conserved domains, such as the YDHR region, which is part of the 2-oxoacid dehydrogenase acyltransferase catalytic domain. The proteins encoded by *sco7123* and *sco1268* of *S. coelicolor* were smaller than the rest, with sequences of 372 and 417 amino acids, respectively. In contrast, the one encoded by *sco5281* had 1272 residues, similar to the proteins of the rest of the *Streptomyces* studied. This characteristic was reflected in the phylogenetic tree, where the former two proteins were separated from the others in one clade. Large proteins were grouped in the second clade, with SCO5281 being the less related group member (**Figure 4**).

There is an alternative way to perform the synthesis of 2-oxoglutarate via 2 oxoglutarate ferredoxin oxidoreductase (EC 1.2.7.11) formed by two subunits called a and b. *S. avermitilis, S. griseus*, *S. kanamyceticus*, and *S. noursei* have only one copy of this pair of genes, whereas *S. albus* has two genes for subunit b and one for a, and *S. clavuligerus* and *S. noursei* have two for subunit a and one for b. In contrast, *S. coelicolor* has two copies of each, generated by gene duplication because the proteins have 99% identity. *S. lavendulae* also has two copies of each gene, with an identity of 38.4%.

The next reaction is catalyzed by succinyl-CoA synthetase (EC 6.2.1.5), which is also composed of two protein subunits: α and β. *S. albus*, *S. clavuligerus*, *S. griseus*, *S. lavendulae*, *S. noursei*, and *S. venezuelae* have a single copy of the genes that code for each subunit. Two copies are present in the genomes of *S. avermitilis*, *S. coelicolor*, *S. pristinaespiralis, S. hygroscopicus, and S. kanamyceticus*; a subunit is smaller (290–299 residues) than b (376–394), one of the copies being around 376 amino acids and the other larger in all cases. In the first four microorganisms, the genes for the a and b subunits are physically together for both pairs of genes, whereas in the latter, they are separated by five genes in both cases (CP970\_05375/CP970\_5370; CP970\_16770/ CP970\_16765). The identity and similarity between the two proteins subunits a or b in each of the *Streptomyces* range from 57.9% to 78.6% and 74.8% to 86.2%, respectively.

Succinate dehydrogenase (SDH; EC 1.3.5.1, EC 1.3.5.4), which catalyzes the oxidation of succinic acid to generate fumaric acid is a four-subunit multimeric enzyme (iron-sulfur protein, flavoprotein subunit, hydrophobic membrane anchor protein, and cytochrome b-556 subunit). The number of genes that code for each subunit varies, and even the same genome *Streptomyces* can have different number of genes for each subunit. As shown in **Table 2**, the *S. albus* genome contains two genes for the SDH flavoprotein subunit, two for the iron-sulfur protein, one for the SDH hydrophobic membrane anchor protein, and two for the SDH cytochrome b-556 subunit. *S. avermitilis* has three genes for flavoprotein subunit, while *S. coelicolor* has four. On the other hand, all of these microorganisms have one copy of the gene coding the hydrophobic membrane anchor protein and two for the cytochrome b-556 subunit.

The genes that code for iron-sulfur protein have great redundancy, finding three copies in all antibiotic-producing *Streptomyces* except *S. albus*, which has two. In total, 28 proteins were found that have very similar molecular weights, however, according to the identities between them, three groups can be distinguished. The

#### **Figure 4.**

*Multiple alignment of 2-oxoglutarate dehydrogenase component E2 (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

#### *Multiplicity in the Genes of Carbon Metabolism in Antibiotic-Producing Streptomycetes DOI: http://dx.doi.org/10.5772/intechopen.106525*

#### **Figure 5.**

*Partial multiple alignment of malate dehydrogenase proteins (A) in antibiotic producing* Streptomyces *and derived bootstrapped tree (B). slx,* Streptomyces lavendulae*, sals,* Streptomyces albus *DSM 41398, sclf,* Streptomyces clavuligerus *F613-1, spri,* Streptomyces pristinaespiralis *HCCB 10218, ska,* Streptomyces kanamyceticus *ATCC 12853, snr,* Streptomyces noursei *ATCC 11455, sho,* Streptomyces hygroscopicus *subps.* jinggagensis *TL01, sco,* Streptomyces coelicolor *A3(2), sma,* Streptomyces avermitilis *MA-4680, sgr,* Streptomyces griseus *subsp.* griseus *NBRC 13350, sve,* Streptomyces venezuelae *ATCC 10712.*

first includes proteins of 246–249 amino acids and that have an identity between them greater than 88% and is one of the copies in all these microorganisms. In another group are those with 252 amino acids and identities greater than 94%. The last group includes proteins with 256–267 amino acids with identities greater than 85%. All these data indicate that although the subunits have very similar molecular weights, they are actually not so similar, suggesting that they had a common ancestor but that they have diverged a lot over time.

It has been found that there are three different flavoprotein proteins, some with 649–652 amino acids with identities greater than 90%, another group of proteins with 584 amino acids and identities between 91 and 93%, and the last group with 633–667 residues and identities greater than 80%. The flavoproteins SCO7109 and SHJGH\_5964 do not resemble each other or the proteins of the previous groups.

The hydrophobic membrane anchor proteins are smaller than the previous ones, around 17 kDa, and present identities between 77% and 82%, while there are two types of cytochrome b subunit, one of around 223–243 amino acids with lower identities than the previous ones, between 58% and 71%. The second type includes proteins of smaller size and identity between 74% and89%.

The hydration reaction of fumarate to generate malate is catalyzed by fumarate hydratase (EC 4.2.1.2; fumarase). There are two classes of proteins: fumarase classes I and II. Most antibiotic-producing *Streptomyces* have a single gene for fumarase Class I, while only seven of these *Streptomyces* have fumarase class II (**Table 2**). The class I proteins are larger (60.2 kDa) than the class II proteins (50.3 kDa). The identity among class I fumarases and class II is 74% and 75%, respectively.

The final TCA cycle reaction is catalyzed by malate dehydrogenase, which catalyzes the reversible conversion of malate to OXA using NAD<sup>+</sup> or NADP<sup>+</sup> as the coenzyme [17], which is only encoded by a single gene in all microorganisms that produce different antibiotics. The multiple sequence alignment of this enzyme showed a high degree of conservation between them, distributed in two main clades from a common ancestor, which was then subdivided into eight subclades (**Figure 5**). The identity is higher than 90%.

#### **5. PEP-PYR-OXA node**

The PEP-PYR-OXA node is a major branch of central carbon metabolism and acts as a connection point between glycolysis, gluconeogenesis, and the TCA cycle [7]. A large variety of enzymes involved in the node have been reported, such as PEP carboxylase (EC 4.1.1.31), pyruvate carboxylase (EC 6.4.1.1), PEP carboxykinase (EC 4.1.1.32), malic enzymes (EC 1.1.1.38), pyruvate kinase (EC 2.7.1.40), and pyruvate phosphate dikinase (EC 2.7.9.1). These enzymes are indispensable for distributing PEP, PYR, and OXA in *Streptomyces*. The enzymes involved in this node vary among microorganisms, and their activity depends on the culture conditions [2, 18].

This anaplerotic pathway does not present multiplicity in any of the genes that encode the enzymes participating in the PEP-PYR-OXA node, except for malic enzymes and pyruvate phosphate dikinase as shown in **Table 3**. PYR carboxylase is an enzyme that is present only in *S. albus*, *S. coelicolor*, *S. hygroscopicus*, and *S. pristinaspiralis*, with only one copy of the gene. The molecular weight is 121 kDa and with 77%–88% identity and 88%–95% similarity among all proteins.

PEP carboxylase, an enzyme that catalyzes the carboxylation of PEP to OXA, is encoded by a single gene and is present in all antibiotic-producing *Streptomyces*. The


**Table 3.** *Gene multiplicity in PEP-PYR-OXA*

 *node.*
