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

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)<sup>+</sup> 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<sup>+</sup> - 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 85kDa multidomain proteins). The greater bacterial ME complexity arises from the need for allosteric regulation owing to the noncompartmentalization 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*,

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

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.

## **6. Conclusions**

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.

#### **Acknowledgements**

This study was partially supported by grant IN214116 (DGAPA-UNAM).

#### **Conflict of interest**

The authors declare no conflict of interest.
