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Streptomycetes exhibit genetic multiplicity, like many other microorganisms, and redundancy occurs in many of the genes involved in carbon metabolism. The enzymes of the glycolytic pathway presenting the greatest multiplicity were phosphofructokinase, fructose 1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The genes that encode citrate synthase and subunits of the succinate dehydrogenase complex are the ones that show the greatest multiplicity, while in the phosphoenolpyruvate-pyruvate-oxaloacetate node, only malic enzymes and pyruvate phosphate dikinase present two copies in some Streptomyces. The extra DNA from these multiple gene copies can be more than 50 kb, and the question arises whether all of these genes are transcribed and translated. As far as we know, there is few information about the transcription of these genes in any of this Streptomyces, nor if any of the activities that are encoded by a single gene could be limiting both for growth and for the formation of precursors of the antibiotics produced by these microorganisms. Therefore, it is important to study the transcription and translation of genes involved in carbon metabolism in antibiotic-producing Streptomyces growing on various sugars.
Departamento de Biología Molecular y Biotecnología, Universidad Nacional Autómoma de México, Instituto de Investigaciones Biomédicas, México
Jonathan Alanís
Departamento de Biología Molecular y Biotecnología, Universidad Nacional Autómoma de México, Instituto de Investigaciones Biomédicas, México
Polonia Hernández
Universidad Nacional Autónoma de México, Programa Jóvenes hacia la Investigación, México
María Elena Flores*
Departamento de Biología Molecular y Biotecnología, Universidad Nacional Autómoma de México, Instituto de Investigaciones Biomédicas, México
*Address all correspondence to: mflores@iibiomedicas.unam.mx
1. Introduction
Gene multiplicity or redundancy is a characteristic of microorganisms and means there are two or more genes coding for proteins that perform the same function. Inactivation of any of these genes does not affect or has little relevance to the biological phenotype [1]. Gene redundancy has been observed in all organisms, including prokaryotes and eukaryotes, and is particularly important for actinobacteria that produce metabolites of industrial interest [2]. The most significant property of Streptomyces species is the production of several secondary metabolites (antibiotics and biologically active compounds) that can be generated at the industrial level. These metabolites are beneficial and indispensable for human and animal health [3]. Although the structures of secondary metabolites are diverse, they have been classified into at least five classes (sugar, polyketide, shikimate, amino acid, and terpene pathways) based on the precursor molecules incorporated during their biosynthesis. All precursors are derived from central primary metabolism, glycolysis (Embden–Meyerhof–Parnas EM pathway), the pentose phosphate pathway (PP), and the tricarboxylic acid (TCA) cycle. Glucose-6-phosphate, a precursor of aminoglycosides, is supplied from the early stage of the EM pathway, and acetyl-CoA and succinyl-CoA as precursors of polyketides are supplied from the final stage of the EM pathway and TCA cycle, respectively. Aromatic amino acids, as precursors of chloramphenicol, are supplied from the PP pathway, whereas other amino acids are supplied from the central metabolism to be precursors of peptide antibiotics. Acetyl-CoA, glyceraldehyde-3-phosphate, and pyruvate (PYR) are precursors of isopentenyl diphosphate and dimethylallyl diphosphate, which are the building blocks of terpenes. Malonyl-CoA, methylmalonyl-CoA, and ethylmalonyl-CoA are used as the extension units in macrolide antibiotic biosynthesis [4], and reduced cofactor (NADPH) is used during secondary metabolite biosynthesis and is generated from the PP pathway and TCA cycle. Accordingly, the dynamics of central metabolism, including the EM and PP pathways and the TCA cycle, which generate primary metabolites (as precursors for secondary metabolites) and cofactors, will influence the biosynthetic process of secondary metabolites [5].
One little-studied area is the carbon metabolism in these organisms. Few studies have examined the presence of genes that participate in the glycolytic pathway, TCA cycle, or phosphoenolpyruvate-pyruvate-oxaloacetate (PEP-PYR-OXA) node. In general, microorganisms metabolize glucose through the glycolysis and hexose monophosphate pathways [6]. Many of the intermediates in these metabolic pathways are used to synthesize other essential bacterial compounds (amino acids, polysaccharides, nucleic acids, lipids, fatty acids, and antibiotics).
The EMP pathway consists of nine reactions, in which the final product is pyruvate. The first reaction consists of the isomerization of glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. Another phosphate with adenosine triphosphate (ATP) as the donor is incorporated into fructose 6-phosphate by phosphofructokinase. The next step is the cleavage of fructose 1,6-diphosphate by aldolase, generating dihydroxyacetone phosphate and glyceraldehyde 3-phosphate, which can be interconverted by triose phosphate isomerase. Glyceraldehyde 3-phosphate is oxidized to 1,3-diphosphoglycerate by glyceraldehyde 3-phosphate dehydrogenase to generate NADH. The phosphoglycerate kinase-catalyzed reaction generates an ATP molecule, an example of substrate-level phosphorylation. The 3-phosphoglycerate is converted to 2-phosphoglycerate by phosphoglycerate mutase and is subsequently dehydrated by enolase. Phosphoenolpyruvate (PEP) is used to generate another molecule of ATP and PYR through a reaction catalyzed by pyruvate kinase. Then, four ATP molecules are generated, and two high-energy phosphate bonds are used; thus, the net gain is two ATPs per oxidized glucose molecule [6].
The TCA cycle is one of the most important metabolic pathways, not only as part of catabolism but also as an important intermediate for amino acid biosynthesis and synthesizing secondary metabolites. Generally, the citric acid cycle is the main oxidation pathway for carbon chains of carbohydrates, fatty acids, and many amino acids to CO2 and water. At each turn of the cycle, two molecules of CO2 are released. Most of the energy generated during oxidation is stored as NADH, FADH, or ATP (or GTP).
Because the intermediates of the TCA cycle are used as precursors in other pathways, they are replenished through anaplerotic reactions. Under normal conditions, the reactions that take intermediates in the cycle and those that replace them are kept in dynamic equilibrium; therefore, their concentrations remain constant. The most common anaplerotic reactions are those in which PYR or PEP are converted to Oxaloacetic acid (OXA) or malate. For example, this first reaction can be mediated by PEP carboxylase in some plants, yeasts, and bacteria or by the malic enzyme (ME), which is widely distributed in prokaryotes and eukaryotes [7].
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.
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.
Streptomyces lavendulae (SLAV), Streptomyces albus DSM 41398 (SALS), Streptomyces clavuligerus F613-1 (SCLF), Streptomyces pristinaespiralis HCCB 10218 (SPRI), Streptomyces kanamyceticus ATCC 12853 (SKA), Streptomyces noursei ATCC 11455 (SNOUR), Streptomyces hygroscopicus subps. jinggagensis TL01 (SHJGH), Streptomyces coelicolor A3(2) (SCO), Streptomyces avermitilis MA-4680 (SAVERM), Streptomyces griseus subsp. griseus NBRC 13350 (SGR), Streptomyces venezuelae ATCC 10712 (SVEN). The number of amino acids of each encoded protein is shown in parentheses.
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 amino-terminal 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].
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-diphospho-glycerate 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, 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.
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 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 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 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+ or NADP+ 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%.
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.
Streptomyces lavendulae (SLAV), Streptomyces albus DSM 41398 (SALS), Streptomyces clavuligerus F613-1 (SCLF), Streptomyces pristinaespiralis HCCB 10218 (SPRI), Streptomyces kanamyceticus ATCC 12853 (SKA), Streptomyces noursei ATCC 11455 (SNOUR), Streptomyces hygroscopicus subps. jinggagensis TL01 (SHJGH), Streptomyces coelicolor A3(2) (SCO), Streptomyces avermitilis MA-4680 (SAVERM), Streptomyces griseus subsp. griseus NBRC 13350 (SGR), Streptomyces venezuelae ATCC 10712 (SVEN). The number of amino acids of each encoded protein is shown in parentheses.
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 proteins are very similar; they have 909 and 921 amino acids in and identity and similarity between 87.3–90.2% and 90.5–94.5%, respectively.
PEP carboxykinase, an enzyme with the opposite action to the previous ones, decarboxylates OXA to form PEP. This enzyme could be considered gluconeogenic; however, it is also part of the PEP-PYR-OXA node for distributing the carbon flux between the different central pathways of metabolism [19]. Its molecular weight is approximately 67 kDa, some of which are a few amino acids long. In the same way as the previous enzymes, Streptomyces carboxykinases have an identity greater than 84%.
MEs catalyzes the oxidative decarboxylation of l-malate to produce PYR and CO2, coupled with the reduction of NAD(P)+ cofactors (EC 1.1.1.38, EC 1.1.1.40). In general, NAD+- dependent MEs function to provide PYR for the TCA cycle, whereas NADP+- dependent MEs function to generate NADPH for anabolic reactions. Bacterial ME isoforms are comparatively understudied and collectively demonstrate greater structural and functional diversity (ranging from “minimal” 40 kDa subunits to much larger 85 kDa multidomain proteins). The greater bacterial ME complexity arises from the need for allosteric regulation owing to the non-compartmentalization of the bacterial cell [20].
The genes coding for MEs in these antibiotic-producing Streptomyces are those that present multiplicity. All these microorganisms have two MEs, one dependent on NAD+ and the other on NADP+, with molecular weights of approximately 48 kDa and 42 kDa, respectively. Three proteins (SAVERM_1514, SAVERM_3870, and SHJGH_4528) from S. avermitilis and S. hygroscopicus have higher molecular weight, with 570 and 583 amino acid residues, and less than 25% identity with the NAD- and NADP-dependent MEs. Its molecular weight is approximately 61–63 kDa, however, BLAST analysis showed that these enzymes are common in many other Streptomyces.
Pyruvate phosphate dikinase (EC 2.7.11.32) converts PEP, inorganic pyrophosphate, and AMP into PYR, inorganic phosphate, and ATP. This protein is a gluconeogenic and anaplerotic enzyme and, in some bacteria, it plays other important roles. This protein is associated with virulence in Brucella ovis, and in Mycobacterium tuberculosis, it is indispensable for its growth as a part of the node [21, 22]. The gene encoding pyruvate phosphate dikinase in these antibiotic-producing Streptomyces species is present in all of them, in some cases with two copies, for example, S. coelicolor, S. clavuligerus, S. griseus, S. hygroscopicus, and S. lavendulae, with molecular weights ranging from 98 to 102 kDa. One of the S. clavuligerus proteins is smaller (61.7 KDa), with 25.9% identity to the amino acid sequence of the protein encoded by SCO0208; however, as in the previous case, BLAST analysis found many other different Streptomyces species that have this enzyme of different size. The expression of the gene that encodes pyruvate phosphate dikinase has been little studied, however, Llamas et al. (2020) reported that sco0208 which codes for this enzyme in S. coelicolor, was maximum at 36 h of growth in minimal medium with casamino acids as carbon and nitrogen sources.
Finally, the genes that encode all enzymes of the glycolytic pathway, the TCA cycle, and the PEP-PYR-OXA node are distributed throughout the genome and represent up to 50 kb that might not be needed. Although many genes are found in the core, others are also found in the arms, and the question arises as to whether all genes are expressed and translated. For example, four genes that encode CS are transcribed to generate functional proteins? Viollier et al. (2001) reported that the deletion of the citA gene of S. coelicolor (sco2736), which codes for one of the four CS, generated glutamate auxotrophy, indicating that it is the main enzyme in this microorganism for the condensation reaction to form six-carbon citrate [23]. Similarly, Takahashi and Flores (unpublished results) found that in S. coelicolor grown on glucose as a carbon source, sco2736 mRNA levels were higher than sco5832, whereas sco4388 and sco5831 mRNA levels were very low, confirming that one of the four CS is predominant under these growth conditions. In Saccharopolyspora erythraea, another actinobacterium, strong relative transcription of gltA-2 was observed only during the early exponential phase and declined thereafter, whereas the other two genes citA and citA4 exhibited relatively low transcript levels during the early exponential phase, and transcription was gradually increased and reached a maximum level during the early stationary phase [24]. The expression of different genes for the same activity likely occurs under different growth conditions or carbon sources. If this is the case, genetic multiplicity allows Streptomyces to adapt and be robust, which may drive the expansion of primary metabolic capability [2].
As mentioned before, antibiotics are synthesized from precursors that are intermediates from carbon metabolism. By providing an overview of the gene multiplicity and determining which enzymes are encoded by a single gene, it will be possible to design strategies aimed at the sufficient biosynthesis of precursors for the metabolism of microorganisms to satisfy the demand for these same compounds to form antibiotics.
On the other hand, the growth of Streptomyces could be limited by insufficient synthesis of some of the enzymes involved in glucose metabolism. For example, the E1 component of 2-oxoglutarate dehydrogenase is encoded by a single gene in Streptomyces, whereas gene multiplicity exists in the other two components to form a complex that performs the conversion reaction of 2-oxoglutarate to succinate. In addition, this protein is a part of the PYR dehydrogenase complex, therefore, the question arises as to whether the expression of this protein could limit the growth of glucose as a carbon source. To date, no evidence has been reported indicating that it may or maybe not limiting.
Genetic multiplicity is present in all antibiotic-producing Streptomyces included here. The number of base pairs representing the redundant DNA was approximately 50 kb. The enzymes of the glycolytic pathway presenting the greatest multiplicity were phosphofructokinase, fructose 1,6-bisphosphate aldolase, glyceraldehyde 3 phosphate dehydrogenase and PYR kinase. The TCA cycle enzymes with the most gene copies were CS, both subunits of succinyl-CoA synthetase, the iron-sulfur protein subunit, flavoprotein subunit, and cytochrome b-556 subunit of succinate dehydrogenase, and fumarase. The MEs genes of the PEP-PYR-OXA node were the only ones that presented multiplicity. More research is required on the transcription of these genes and also on translation in order to establish their importance in these microorganisms.
1.Nowak MA, Boerlijst MC, Cooke J, Smith JM. Evolution of genetic redundancy. Nature. 1997;388:167-171
2.Schniete JK, Cruz-Morales P, Selem-Mojica N, Fernández-Martínez LT, Hunter IS, Barona-Gómez F, et al. Expanding primary metabolism helps generate the metabolic robustness to facilitate antibiotic biosynthesis in. MBio. 2018;9
3.Pye CR, Bertin MJ, Lokey RS, Gerwick WH, Linington RG. Retrospective analysis of natural products provides insights for future discovery trends. Proceedings of the National Academy of Sciences. 2017;114:5601
4.van Keulen GS, Dijkhuizen J. Central carbon metabolic pathways in Streptomyces. In: P D Streptomyces: Molecular Biology and Biotechnology. Norfolk, UK: Caister Academic Press; 2011. pp. 105-124
5.Doi S, Komatsu M, Ikeda H. Modifications to central carbon metabolism in an engineered Streptomyces host to enhance secondary metabolite production. Journal of Bioscience and Bioengineering. 2020;130:563-570
6.Kim BH, Gadd GM. Glycolysis. In: Kim BH, Gadd GM, editors. Prokaryotic Metabolism and Physiology. 2nd ed. Cambridge: Cambridge University Press; 2019. pp. 58-79
7.Llamas-Ramírez R, Takahashi-Iñiguez T, Flores ME. The phosphoenolpyruvate-pyruvate-oxaloacetate node genes and enzymes in Streptomyces coelicolor M-145. International Microbiology. 2020;23:429-439
8.Quinn GA, Banat AM, Abdelhameed AM, Banat IM. Streptomyces from traditional medicine: Sources of new innovations in antibiotic discovery. Journal of Medical Microbiology. 2020;69:1040-1048
9.Gunnarsson N, Eliasson A, Nielsen J. Control of fluxes towards antibiotics and the role of primary metabolism in production of antibiotics. Advances in Biochemical Engineering/Biotechnology. 2004;88:137-178
10.Webb BA, Forouhar F, Szu FE, Seetharaman J, Tong L, Barber DL. Structures of human phosphofructokinase-1 and atomic basis of cancer-associated mutations. Nature. 2015;523:111-114
11.Cabrera R, Baez M, Pereira HM, Caniuguir A, Garratt RC, Babul J. The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: Kinetic and structural analysis of the allosteric ATP inhibition. The Journal of Biological Chemistry. 2011;286:5774-5783
12.Ünsaldı E, Kurt-Kızıldoğan A, Voigt B, Becher D, Özcengiz G. Proteome-wide alterations in an industrial clavulanic acid producing strain of. Synthetic and Systems Biotechnology. 2017;2:39-48
13.Muñoz ME, Ponce E. Pyruvate kinase: Current status of regulatory and functional properties. Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology. 2003;135:197-218
14.Ferraris DM, Spallek R, Oehlmann W, Singh M, Rizzi M. Structures of citrate synthase and malate dehydrogenase of Mycobacterium tuberculosis. Proteins. 2015;83:389-394
15.Ge Y, Cao Z, Song P, Zhu G. Identification and characterization of a novel citrate synthase from Streptomyces diastaticus No. 7 strain M1033. Biotechnology and Applied Biochemistry. 2015;62:300-308
16.Ambrus A, Nemeria NS, Torocsik B, Tretter L, Nilsson M, Jordan F, et al. Formation of reactive oxygen species by human and bacterial pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes reconstituted from recombinant components. Free Radical Biology and Medicine. 2015;89:642-650
17.Ge Y-D, Cao Z-Y, Wang Z-D, Chen L-L, Zhu Y-M, Zhu G-P. Identification and biochemical characterization of a thermostable malate dehydrogenase from the mesophile Streptomyces coelicolorA3(2). Bioscience, Biotechnology, and Biochemistry. 2014;74:2194-2201
18.Sauer U, Eikmanns BJ. The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiology Reviews. 2005;29:765-794
19.Papagianni M. Recent advances in engineering the central carbon metabolism of industrially important bacteria. Microbial Cell Factories. 2012;11:50
20.Harding CJ, Cadby IT, Moynihan PJ, Lovering AL. A rotary mechanism for allostery in bacterial hybrid malic enzymes. Nature Communications. 2021;12:1228
21.Basu P, Sandhu N, Bhatt A, Singh A, Balhana R, Gobe I, et al. The anaplerotic node is essential for the intracellular survival of. The Journal of Biological Chemistry. 2018;293:5695-5704
22.Vizcaíno N, Pérez-Etayo L, Conde-Álvarez R, Iriarte M, Moriyón I, Zúñiga-Ripa A. Disruption of pyruvate phosphate dikinase in Brucella ovis PA CO. Veterinary Research. 2020;51:101
23.Viollier PH, Minas W, Dale GE, Folcher M, Thompson CJ. Role of acid metabolism in Streptomyces coelicolor morphological differentiation and antibiotic biosynthesis. Journal of Bacteriology. 2001;183:3184-3192
24.Liao CH, Yao L, Ye B-C, Ye BC. Three genes encoding citrate synthases in Saccharopolyspora erythraea are regulated by the global nutrient-sensing regulators GlnR, DasR, and CRP. Molecular Microbiology. 94:1065-1084
Written By
Toshiko Takahashi, Jonathan Alanís, Polonia Hernández and María Elena Flores
Submitted: 22 February 2022Reviewed: 13 July 2022Published: 12 August 2022
Open access peer-reviewed chapter
