Project Description

Streptomyces is a genus of gram-positive bacteria that grow in various environments, with moderately moist soil as the main habitat, but can also be found in freshwater and marine ecosystems. They are chemoorganotrophic microorganisms, using carbon as an energy source and with a complex development of the saprophytic life cycle (Piepersberg, 1993) whose filamentous form resembles fungi, and the hyphal layers can differentiate into uninucleate spores (Ohnishi et al., 2008). This process occurs only in some bacterial genus, and these microorganisms generally have a complex and fully coordinated metabolism. Bacterial spores or endospores are important survival structures in hostile environments due to the pigment and aroma present in certain species (Chater and Chandra, 2006). They protect bacteria from external physical and chemical attacks in the form of spherical corpuscles, free or inside the microorganisms. These structures also stimulate cellular development and the production of secondary metabolites (Chi et al., 2011). As external environmental conditions improve, the spores can reproduce and multiply again.

As they are classified as gram-positive bacteria, they have a large amount of peptidoglycan in their cell wall, presenting a red or pink color when stained with saphrin or fuchsin using the Gram technique (Gregersen, 1978). These bacteria of the genus Streptomyces do not have an external membrane, being found only in gram-negative bacteria whose function is to prevent the entry of external agents. Another interesting property of Streptomyces, especially actinobacteria, is that they have the ability to produce secondary metabolites with specific biological activities such as antibacterials, antifungals, antiparasitics, antivirals, antitumors, antihypertensives, immunosuppressants and antibiotics (Omura et al., 2001; Patzer and Volkmar, 2010; Khan, 2011). For example, S. grieus, S. avermitilis, and S. tsukubensis are producers of streptomycin, avermectin, and tacrolimus, respectively. Other species can also produce these antibiotics, and they cannot be considered as a specific characteristic of the species.

These bacteria belong to the phylum Actinobacteria (Actinomycetota from 2022) and the Streptomycetes family, which consists of four genera (Hodgson, 2000), where Streptomyces is the most studied. According to a recent classification carried out by the List of Prokaryotic names with Standing in Nomenclature (LPSN), there are approximately 700 species with published and validated names. When carrying out a quick search at the National Center for Biotechnology Information (NCBI), 579 reference genomes of the genus Streptomyces were found deposited in the database. In Figure 1, it can be seen that the number of new Streptomyces species with a sequenced genome has been increasing every year. In the genome of these bacteria, there is a high proportion of guanine and cytosine with approximately 70-80% of the total of these nucleotide bases, providing greater genetic stability (Hodgson, 2000).

Bacteria can become true drug “factories” using molecular biology and genetic engineering techniques to make specific changes in bacterial DNA to increase the ability to express molecules of biotechnological interest. Many antibiotics are produced by industrial strains of bacteria that have genotypic and phenotypic characteristics appropriate for the expression of antibiotics, such as human insulin in Escherichia coli. Bacterial species of the genus Streptomyces are commercially important due to their metabolic versatility, making them an important producer of several types of antibiotics such as:

The search for antibiotics within the Streptomyces genus began with the discovery of streptomycin in 1942 and has intensified over the years. About 80% of antibiotics originate from bacteria belonging to the genus Streptomyces (Watve et al., 2001). The biotechnological potential of these bacteria still needs to be further explored, as there is little research related to the discovery of therapeutic targets against various pathogenic microorganisms.

Many bacteria that cause infectious diseases have become resistant to antibiotics known and used by the population. This acquired resistance occurs due to genetic mutations that occur in an attempt to survive by escaping the action of antibiotics. The main known mechanisms of bacterial resistance are enzymes that destroy or modify the action of antibiotics, reduced permeability of the outer membrane, overexpressed efflux systems to excrete toxic substances, and alteration, blockage, or protection of the antibiotic's target site.

Infectious diseases affect the entire world and still cause many deaths, especially in developing countries whose basic health infrastructure is still precarious. Annually, there are 17 million human deaths caused by bacterial infections. Many pathogenic bacteria manage to develop genetic mechanisms that confer resistance to antibiotics used by the population. This is a serious public health problem in the world that needs to be faced, the solution of which may lie in the discovery of new antibiotics against resistant bacteria. In Figure 2, we can see some of the antibiotics discovered and used to treat various infectious diseases.

In the 1960s, many strains of the Streptomyces genus were isolated by the Federal University of Pernambuco (UFC), located in the state of Pernambuco, northeastern Brazil. The research carried out at UFC aimed to identify strains with potential antitumor capacity. Of the isolated strains, S. olindensis DAUFPE 5622 was isolated from soil, this species is a producer of a secondary metabolite called cosmomycin D (Furlan et al., 2004). This metabolite demonstrated antitumor and antimicrobial properties with strong inhibition power against gram-positive bacteria.

Cosmomycin D is an aromatic polyketide compound from the anthracycline family, which appears in the form of a red powder that is poorly soluble in water and soluble in alcohols (Lima et al., 1969; Pamboukin, 2003; Inoue, 2006). Polyketide compounds are secondary metabolites produced by bacteria, fungi, and plants (Cragg and Newman, 2013) whose structural diversity is classified into three classes – peptide, macrocyclic lactones, and quinone derivatives (Leeper et al., 2000). Anthracyclines are highly effective chemotherapeutic agents that destroy tumor cells and are used in the treatment of some types of breast cancer. In Figure 3, there is the chemical structure of cosmomycin produced by S. olindensis DAUFPE 5622. This compound has a molecular weight of 1189 g/mol and a molecular formula of C60H59O22N2, presenting one of the most complex glycosylation models found in anthracyclines (Miyamoto et al., 2002).

The genome of S. olindensis DAUFPE 5622 has been completely sequenced and is available at NCBI – ID 32003 (Garrido et al., 2006; Rojas et al., 2014). It has a size of 9.4 Mb, 71.5% GC, 19 rRNAs, 67 tRNAs, 9 other RNAs, 8,260 genes, 152 pseudogenes and 8,013 encoded proteins. With the genome sequencing of S. olindensis it was possible to identify the genes responsible for the biosynthesis of cosmomycin D, which involve between 34 and 40 genes (Garrido, 2005; Metsä Ketelä et al., 2008).

Molecular regulation of antibiotic biosynthesis in Streptomyces typically depends on a specific activating gene that is likely within the cryptic gene cluster (Liu et al., 2013). For example, Streptomyces Antibiotic Regulatory Proteins (SARPs) are a family of paralogous proteins involved in the biosynthesis of secondary metabolites such as antibiotics. Liu and colleagues (2013) listed SARPs in several Streptomyces species through biosynthetic pathways as Types I and II polyketides and Nonpolyketides. Some pathways may present more than one SARP forming parts of regulatory steps of gene clusters (Bate et al., 1999; Sun et al., 2003).

Only a small portion of metabolic biosynthesis pathways are expressed under usual bacterial cultivation conditions in the laboratory (Baltz, 2016). The metabolic pathways of cryptic genes can be activated by different cultivation media and incubation temperatures, the addition of substances, and the induction of resistance to antimicrobials (Bode et al., 2002; Tanaka et al., 2013; Ochi et al., 2014).

The St(r)ep by St(r)ep project proposes an innovative solution for the scientific community and industry with the aim of optimizing a certain metabolic pathway for the production of a secondary metabolite, from a gene cluster described in the literature for Streptomyces. These bacteria are responsible for the production of 80% of the antibiotics used clinically to treat various infectious diseases. They have sufficient cellular “machinery” to produce several secondary metabolites. These molecules are produced under certain conditions in which bacteria are found. Most of the genes responsible for the production of metabolites are silenced in cellular metabolism, this prevents Streptomyces from having the same impact and biotechnological interest as other microorganisms such as E. coli. Through synthetic biology and metabolic engineering, we can reverse this process by making Streptomyces more efficient and competitive, transforming them into bacterial antibiotic biofactories. As a proof of concept, S. olindenses DAUFPE 5622 was used, whose genome has been completely sequenced and produces an interesting secondary metabolite called cosmomycin D.