Actinomycetes: a Never-Ending Source of Bioactive Compounds—An Overview on Antibiotics Production

Review Actinomycetes: A Never-Ending Source of Bioactive Compounds—An Overview on Antibiotics Production

Davide De Simeis and Stefano Serra *

Consiglio Nazionale delle Ricerche (C.N.R.), Istituto di Scienze e Tecnologie Chimiche, Via Mancinelli 7, 20131 Milano, Italy; [email protected] * Correspondence: [email protected] or [email protected]; Tel.: +39-02-2399-3076

Abstract: The discovery of penicillin by Sir Alexander Fleming in 1928 provided us with access to a new class of compounds useful at fighting bacterial infections: antibiotics. Ever since, a number of studies were carried out to find new molecules with the same activity. Microorganisms belonging to Actinobacteria phylum, the Actinomycetes, were the most important sources of antibiotics. Bioactive compounds isolated from this order were also an important inspiration reservoir for pharmaceutical chemists who realized the synthesis of new molecules with antibiotic activity. According to the World Health Organization (WHO), antibiotic resistance is currently one of the biggest threats to global health, food security, and development. The world urgently needs to adopt measures to reduce this risk by finding new antibiotics and changing the way they are used. In this review, we describe the primary role of Actinomycetes in the history of antibiotics. Antibiotics produced by these microorganisms, their bioactivities, and how their chemical structures have inspired generations of scientists working in the synthesis of new drugs are described thoroughly.

Keywords: antibiotics; Actinomycetes; antibiotic resistance; natural products; chemical tailoring; chemical synthesis Citation: De Simeis, D.; Serra, S. Actinomycetes: A Never-Ending Source of Bioactive Compounds—An Overview on Antibiotics Production. 1. Introduction Antibiotics 2021, 10, 483. https:// doi.org/10.3390/antibiotics10050483 Antibiotics are a class of molecules used for the treatment and prevention of bacterial infections. It is worth nothing that, before their introduction in clinical practice, there Academic Editor: Sylvia Valdezate was no effective therapy for the treatment of different medical cases, such as pneumonia, typhoid fever, or gonorrhea [1]. Received: 25 February 2021 The first natural antibiotic isolated was penicillin, in 1928, by Fleming and co-workers. Accepted: 19 April 2021 The scientists, returning from holiday, found one of their Petri-dish containing colonies of Published: 22 April 2021 Staphylococcus contaminated with a mold, later identified as Penicillium notatum. Fleming was surprised by the fact that no bacteria were able to growth around the mold; he was, at Publisher’s Note: MDPI stays neutral the same time, perceptive enough to consider the event remarkable. with regard to jurisdictional claims in Despite Fleming’s initial observation, he dealt with chemical issues, such as stability published maps and institutional affil- and purification of the bioactive compound. After many years, in 1940, he finally left the iations. idea to isolate the antibiotic substance produced by the mold. Luckily, an Oxford team, guided by Howard Florey and Ernest Chain, managed to purify penicillin in quantities useful for clinical testing. Many firms, such as Lederle, Merck, Pfizer, Squibb, and Abbott Laboratories contributed to solve the concerns related to the scale-up of submerged fer- Copyright: © 2021 by the authors. mentation. Finally, in 1945, pharmaceutical companies started distributing and trading Licensee MDPI, Basel, Switzerland. penicillin [2]. This article is an open access article From that moment, pharmaceutical companies and universities began studies in distributed under the terms and this field, and identified several new bioactive molecules. The antibiotics era started, conditions of the Creative Commons and its “gold period” was between the 1950s and 1970s, an epoch in which the most Attribution (CC BY) license (https:// known important classes of antibiotics were discovered [1]. These bioactive compounds are creativecommons.org/licenses/by/ produced naturally from different species of fungi and bacteria, but the most attractive class 4.0/).

Antibiotics 2021, 10, 483. https://doi.org/10.3390/antibiotics10050483 https://www.mdpi.com/journal/antibiotics Antibiotics 2021, 10, 483 2 of 32

of microorganisms that are able to produce these secondary metabolites are Actinobacteria, in particular, Actinomycetes. The importance of this order is due to their abilities to produce different classes of antibiotics in terms of chemical structure and mechanisms of action. Current research suggests that Actinomycetes are also a prime resource in the finding of new natural products, thanks to their unique enzymatic sets that permit generating compounds that are potentially useful for diverse purposes. Moreover, different genera and species of Actinomycetes are able to produce the same class of antibiotics and, in few cases, the same chemical compound, indicating that the metabolic paths are strictly conserved among the order [3]. Thanks to antibiotics and the research developed in this field, many infections are now treatable, and life-quality/life expectancy are better than the past. Nature’s beauty is also found in the complexity of evolution, the engine that moves life in order to adapt itself to environmental changes. The fundamental unit of evolution is the mutation, or rather, the change, in the genetic material and the process through which the variation happens. Through mutations, bacteria become antibiotic-resistant and the infections they cause are harder to eradicate. According to WHO, at least 700,000 people die each year due to drug-resistant diseases, including 230,000 people who die from multidrug-resistant tuberculosis. Several diseases associated with the urinary and respiratory tracts, or sexually transmitted infections, are becoming untreatable. Lower respiratory infections remained the deadliest communicable disease, causing 3.0 million deaths worldwide, especially in low-income countries. Different causes have contributed to antibiotic-resistance crisis, including factors related to their overuse, inappropriate prescribing, and extensive use in agriculture and breeding [4–6]. The WHO and other organizations, such as the Centers for Disease Control and Prevention (CDC), have developed different programs for reducing this phenomenon, with the goal of potentiating the diagnosis and optimizing therapeutic regimens, in addition to adopting a prevention policy, and control tracking and prescribing practices [7]. Recently, scientists shed new light on another interesting topic: the “abuse” of antibiotics in clinics. So far, it seems that abuse of these drugs could cause resistance in commensal bacteria flora. These bacteria are regarded as potential threats. Indeed, if spread out in the environment, they could become vectors for antibiotic resistance in pathogens or could create pathologies in immunocompromised people [8]. In this context, the great diversity of the metabolites with antibiotic activity produced by the Actinomyces is an important tool to tackle the antibiotic-resistance crisis. This review guides the reader through the different antibiotics produced by microorganisms belonging to the Actinomycetes order. These natural products will be described in detail, taking into consideration their peculiarities, in terms of both bioactivity and chemical structures. Further topics that will be examined concern their industrial preparations. In particular, the variability and the complexity of their chemical structures have challenged organic chemists, inspiring researches who are “finalized” to the synthesis of these natural products or to the creation of other useful bioactive compounds.

2. Actinomycetes: Biology and Bioactive Compounds Actinomycetes are microorganisms belonging to the order of Actinomycetales, whose phylum is Actinobacteria, one of the largest taxonomic units within the bacteria domain. The phylum comprises Gram-positive bacteria with high guanine-cytosine (G + C) content in their DNA, ranging from 51% in some corynebacteria to more than 70% in Streptomyces and Frankia. An exception is the genome of the obligate pathogen Tropheryma whipplei, in which the G + C content is less than 50% [9]. The phylum is also adapted to a wide range of environments, such as soil, water (even salty), and air. Nevertheless, the predominant part of these organisms is preferentially found in alkaline soil with big amounts of organic matter [10]. Antibiotics 2021, 10, x FOR PEER REVIEW 3 of 33

Antibiotics 2021, 10, 483 3 of 32

From a morphological point of view, actinobacteria show different features. Indeed, we can recognize microorganisms with a coccoid/rod-coccoid shape-like species belong- From a morphological point of view, actinobacteria show different features. Indeed, ing to the suborder of Micrococcineae, or with a fragmenting hyphal form (microorgan- we can recognize microorganisms with a coccoid/rod-coccoid shape-like species belonging toisms the belonging suborder to of the Micrococcineae, suborder of the or withCorynebacterineae a fragmenting, hyphalsuch as formNocardia (microorganisms spp.), or with belonginga permanent to and the highly suborder differentiated of the Corynebacterineae, branched mycelium such, such as Nocardia as Streptomycesspp.), or spp. with [11]. a permanentFrom a and genetic highly point differentiated of view, genes branched involved mycelium, in the biosynthesis such as Streptomyces of antibioticsspp. [,11 and]. otherFrom metabolites a genetic belonging point of view,to the genessecondary involved metabolism, in the biosynthesis are located of in antibiotics, the genome and of otherthe microorganism metabolites belonging, and rarely to thein plasmids.secondary They metabolism, are clustered are located in very in the long genome operons, of thewhose microorganism, general assembling and rarely is schematized in plasmids. in Figure They 1. are Commonly, clustered it in is verypossible long to operons, identify whosecodifying general sequences assembling (genes) is and schematized regulatory in sequences Figure1. Commonly, (P and O) that it is possibleare able to promote identify codifyingor inhibit sequences the gene (genes)expression and regulatoryresponding sequences to specific (P environmentaland O) that are able changing to promotes. These or inhibitgenes encode the gene enzymes expression involved responding in the assembling to speciﬁc environmentaland editing of the changings. bioactive These compound, genes encodein protecting enzymes the involvedcell from inits the toxicity assembling, and facilitating and editing itsof extrusion the bioactive in the compound, environment in protecting[12,13]. the cell from its toxicity, and facilitating its extrusion in the environment [12,13].

Figure 1. Schematic view of an operon: promoter (P); operator (O); operon genegene andand terminatorterminator (T).(T). Actinomycetes It is worth noting that that the the production production of of secondary secondary metabolites metabolites in in Actinomycetes isis strictly dependent on cell morphological and physiological differentiation. The factors strictly dependent on cell morphological and physiological differentiation. The factors that influence the microorganism’s behavior is not well understood yet, but reasonably, that influence the microorganism’s behavior is not well understood yet, but reasonably, variations, such as nutrient concentration and accessibility, competitor occurrence in the variations, such as nutrient concentration and accessibility, competitor occurrence in the environment, metabolites, and cellular density, could influence the gene expression, and environment, metabolites, and cellular density, could influence the gene expression, and the enzymatic set inside the cell as a consequence [14,15]. the enzymatic set inside the cell as a consequence [14,15]. Antibiotics are always conceived as molecules able to kill other bacteria, but their Antibiotics are always conceived as molecules able to kill other bacteria, but their functions are more complicated than we thought. This simplification arose from a labo- functions are more complicated than we thought. This simplification arose from a labora- ratory “distortion” in which we considered the antibiotic producer as an isolated system. tory “distortion” in which we considered the antibiotic producer as an isolated system. Indeed, considering a composite environment, in which different microorganism pop- Indeed, considering a composite environment, in which different microorganism popula- ulations coexist, these molecules could have different roles, such as signaling agents, a tions coexist, these molecules could have different roles, such as signaling agents, a pro- promoter of the biofilm formation, or, in general, influencing the gene expression of the producermoter of the microorganism biofilm formation itself, or or of, in the general, neighbor influencing [16–18]. Interestingly,the gene expression a large numberof the proof Actinomycete-derivedducer microorganism itself antibiotics or of werethe neighbor isolated from[16–18 microorganisms]. Interestingly, thata large complete number their of wholeActinomycete lifecycles-deriv in soils—especiallyed antibiotics were biologically isolated polluted from microorganisms soils. This is further that complete proof that their the microbial-productionwhole lifecycles in soils of— antibioticespecially compounds biologically can polluted be an evolutive soils. This advantage is further that proof allows that microbialthe microbial producers-production to compete of antibiotic better compounds for natural resources. can be an evolutive advantage that allows Anothermicrobial important producers point to compete refers tobetter the uniquefor natural conditions resources. present in a deep marine environment.Another important It is presumed point that refers marine to theActinomycetes unique conditionsmay have present peculiar in a characteristics deep marine withenvironment. unique structural It is presumed elements that thatmarine have Actinomycetes not previously may been have found peculiar in terrestrial characteristicsActino- myceteswith unique, due thestructural extreme ele variationsments that in ecologicalhave not previously pressure, including been found competition in terrestrial for space, Acti- predation,nomycetes, available due the nutrients,extreme variations light, oxygen in ecological concentration, pressure, and including pressure [ 19competition,20]. for space, predation, available nutrients, light, oxygen concentration, and pressure [19,20]. 3. Mechanisms of Resistance and Antibiotics from Actinomycetes: An Overview 3. MechanismsThe antibiotic of Resistance activity of aand molecule Antibiotics is dependent from Actinomycetes on its chemical: A structuren Overview and, thus, to itsThe affinity antibiotic to a specificactivity of biological a molecule target. is dependent From a on chemical its chemical point structure of view, and there, thus, are differentto its affinity antibiotic to a specific classes biological produced target. by Actinomycetes From a chemical. Indeed, point it isof possibleview, there to identifyare dif- aminoglycosides,ferent antibiotic classes peptides, produced ansamycins, by Actinomycetes.β-lactams, tetracyclines, Indeed, it is macrolides, possible to lincosamides,identify ami- epoxides,noglycosides, and aminocoumarins.peptides, ansamycins, β-lactams, tetracyclines, macrolides, lincosamides, epoxidesIn regards, and aminocoumarins to biological activity,. a bioactive compound, useful for clinical use as antibiotic,In regards must to act biological on a specific activity, prokaryotic a bioactive target compound without, useful damage for toclinical the eukaryotic use as an- system.tibiotic, must There act are on manya specific opportunities prokaryotic intarget this without sense because damage the to the bacteria eukaryotic biology system. and morphologyThere are many are opportunities quite different in from this thosesense ofbecause the eukaryotic the bacteria cell. biology and morphology are quiteCurrently, different the from most those important of the antibiotic eukaryotic targets cell. refer to the cell wall biosynthesis, to the DNA wrapping, to the translation or transcription steps, and to some specific traits of the bacterial metabolism. Antibiotics 2021, 10, x FOR PEER REVIEW 4 of 33

Antibiotics 2021, 10, 483 Currently, the most important antibiotic targets refer to the cell wall biosynthesis,4 of to 32 the DNA wrapping, to the translation or transcription steps, and to some specific traits of the bacterial metabolism. AA relevant relevant diversity diversity between between the the prokaryotic prokaryotic an andd eukaryotic eukaryotic biological biological structure structure lies lies inin the the ribosome ribosome.. Bacterial Bacterial ribosomes ribosomes are are smaller smaller (70S) (70S) with with respect respect to to the the eukaryotic eukaryotic ones ones (80S)(80S) and and cons consistist of of two two subunits, subunits, 50S 50S and and 30S 30S (Figure (Figure 2).2 Moreover,). Moreover, the thesmall small subunit subunit 30S is30S composed is composed of rRNA of rRNA 16S, while 16S, while the eukaryotic the eukaryotic subunit subunit 40S, is 40S, formed is formed by rRNA by rRNA 18S. It 18S. is possibleIt is possible to notice to notice differences differences in the in major the major subunit subunit of the ofribosome the ribosome because because bacteria bacteria (50S) is(50S) composed is composed of rRNA of 5S rRNA and 23S 5S, andwhile 23S, in eukarya while in (60S) eukarya we find (60S) 28S, we 5.8S ﬁnd, and 28S, 5S. 5.8S,Another and im5S.portant Another difference important is in difference the protein is incontent the protein of the contentribosome. of theThe ribosome. RNA percentage The RNA in bacteriapercentage is higher in bacteria than the is higher protein than part the (60% protein rRNA part and (60% 40% rRNA peptides) and 40%, while peptides), in the eukar- while yoticin the cells, eukaryotic the ratio cells, is completely the ratio is inverted completely [21]. inverted [21].

FigureFigure 2. 2. SchematicSchematic representation representation of of a a eukaryotic eukaryotic and and prokaryotic prokaryotic ribosome ribosome..

TheThe most most common common antibiotics antibiotics active active i inn bacterial bacterial protein protein synthesis synthesis are are selective selective for for the the ribosomeribosome RNA RNA,, and and they they belong belong to to different different chemical chemical classes classes,, such such as as aminoglycosides aminoglycosides,, tetracyclinestetracyclines,, and and macrol macrolidesides [22]. [22]. The The capability capability to to discriminate discriminate between between a a eukaryotic eukaryotic ribosomeribosome and and one one belonging belonging to to bacteria bacteria depends depends on on many many aspects. aspects. Nevertheless, Nevertheless, the the most most importantimportant seems seems to to be be attribute attributedd to to RNA RNA sequence. sequence. Many Many studies studies demonstrated demonstrated that that,, in in manymany sensitive sensitive bacter bacteriaia that that bec becameame resistant resistant to to the the drug drug,, the the nucleotides nucleotides responsible responsible for for thethe antibiotic antibiotic binding binding site site were were changed changed [23]. [23]. InIn regards regards toto microbialmicrobial metabolism, metabolism, there there are are specific specific biosynthetic biosynthetic pathways pathways only only iden- identifiedtified in prokaryotic in prokaryotic systems systems that couldthat could be considered be considere an interestingd an interesting target target for novel for novel drugs drugsto use to as use antibiotics. as antibiotic Ones historical. One historical example example is related is related to folic to acid folic biosynthesis. acid biosynthesis. Indeed, Indeed,folic acid folic is indispensableacid is indispensable for both for humans both humans and infecting and infecting pathogens. pathogens. However, However, humans humansobtain this obtain substance this substance as a vitamin as a vitamin in the in diet, the and diet do, and not do need not need a de a novo de novo biosynthetic biosyn- theticpathway pathway for its for synthesis. its synthesis. On the On contrary, the contrary, bacterial bacterial cell walls cell are walls impermeable are impermeable to folic acid to folicand acid its derivatives, and its derivatives because, they because lack thethey necessary lack the necessary folate receptors folate thatreceptors internalize that inter- these nalizesubstances. these substances. These considerations These considerations drove scientists drove to scientists develop drugs to develop that were drugs able that to were inter- ablefere to with interfere this process, with this and process different, and products different were products commercialized, were commercialized such as sulfadiazine, such as and sulfacetamide [24]. sulfadiazine and sulfacetamide [24]. It is worth noting that many effective drugs act via modulation of multiple essential It is worth noting that many effective drugs act via modulation of multiple essential proteins rather than a single one because the likelihood of two simultaneous mutations proteins rather than a single one because the likelihood of two simultaneous mutations occurring in two essential targets is low and limited [25]. Recently, scientists considered occurring in two essential targets is low and limited [25]. Recently, scientists considered the possibility of “cut down” virulence in pathogens, even though they are nonessential to the possibility of “cut down” virulence in pathogens, even though they are nonessential the survival. This idea was born, postulating a low-rate of mutation and a low possibility to the survival. This idea was born, postulating a low-rate of mutation and a low possibil- of developing resistance mechanisms due to little, selective pressure derived from their ity of developing resistance mechanisms due to little, selective pressure derived from their loss. Nevertheless, different studies suggested the onset of resistance mechanisms even for loss. Nevertheless, different studies suggested the onset of resistance mechanisms even these nonessential targets [26]. for these nonessential targets [26]. There are several resistance mechanisms in bacteria that could be summarized as There are several resistance mechanisms in bacteria that could be summarized as fol- follows: lows: 1. Permeability alteration; 2. Target modification/amplification; 3. Drug inactivation. Antibiotics 2021, 10, 483 5 of 32

The susceptibility of the bacterial pathogens to antibiotics is strictly related to membranes permeability. Small hydrophilic drugs, such as β-lactams and fluoroquinolones, use pore-forming porins to gain access to the cell interior, while macrolides and other larger hydrophobic drugs diffuse across the lipid bi-layers. The existence of drug-resistant strains in a large number of bacterial species is due to the modification of membrane components involved in passive diffusion and active transport (influx and efflux) in terms of presence, production levels, and structures [27]. Another important resistance mechanism involves target modification/amplification. The alteration of the antibiotic target as a result of mutation, chemical modification, substitution, or masking of key binding elements, makes the drug useless for therapeutic use. The same effect could be reached by gene duplication/amplification of the antibiotic target. Several mechanisms were suggested that could increase gene copy number; considering the duplication, it can occur following multiple mechanisms, such as non-equal homologous recombination between directly orientated repeats, RecA-independent mechanisms, and through rolling circle replication [28]. In case of target amplification, the antibiotic is still able to perform its activity, but a higher concentration is required to exploit the effect. Therefore, the clinical treatment could become incompatible due to the inherent toxicity of the drug. Drug inactivation is the last mechanism through which bacteria are able to alter the chemical structure of antibiotics, making them inactive. Different research groups have recognized a number of enzymatic inactivation processes, which involve the most important antibiotic classes. These chemical transformations comprise hydrolysis, group transfer, and redox (mainly oxidation) reactions [29–32].

3.1. β-Lactam Antibiotics The prokaryotic cell wall is composed of different proteins, lipids, and sugars, organized in particular manners depending on the species analyzed. Peptidoglycan is a particular biological structure, present predominantly in the Gram-positive bacteria wall, made from polysaccharide chains consisting of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM), cross-linked together by short peptides containing modiﬁed amino acids, such as aminopimelic acid (DAP) and native L-or D-amino acids. From a functional point of view, the bacterial cell wall is the most important structure that permits bacteria to maintain its shape and respond efﬁciently to environmental stresses, maintaining the osmotic balance [33]. In coccoid bacteria, the peptidoglycan biosynthesis starts in the cytoplasm, where NAM is linked with a pentapeptide that contains, far from the sugar moiety, two D-Alanine amino acids (D-Ala-D-Ala). The macromolecule is linked to the inner part of the cell membrane where the NAG and other amino acids are added. This intermediate is now carried out from the cell where other units of the same monomer are added via transglycosylation. Different, growing polymer strands are then cross-linked by the action of a class of enzymes called transpeptidase, able to recognize and link diverse peptidoglycan regions through the D-Ala-D-Ala moiety (Figure3). It is worth noting that the connection formed by transpeptidase is dependent on the species; some examples are well described in the literature [34,35]. β-Lactams antibiotics are molecules produced from bacterial and fungal species, with (generally) bactericidal activity. Their biological actions are correlated to the inhibition of the cell wall biosynthesis, resulting from the irreversible transpeptidase active site block due to the similarity of the antibiotic with the D-Ala-D-Ala moiety described above. From a chemical point of view, there are four main classes of natural β-lactams, all of them containing a four-membered cyclic amide (Figure4). Antibiotics 2021, 10, x FOR PEER REVIEW 6 of 33 Antibiotics 2021, 10, 483 6 of 32

Figure 3. Peptidoglycan cross-linking mediated by DD-transpeptidase. The “serpentine” attached to NAM in black Figure 3. Peptidoglycan cross-linking mediated by DD-transpeptidase. The “serpentine” attached to NAM in black repre- representssents the peptide the peptide chain. chain. The enzyme The enzyme image image is kindly is kindly taken taken by PDB by [36]. PDB [36]. Penicillin possesses a β-lactam ring, fused with a ﬁve-membered ring with a sulfur β-Lactams antibiotics are molecules produced from bacterial and fungal species, with atom; cephalosporins possess a β-lactam ring with a six-membered unsaturated ring with (generally) bactericidal activity. Their biological actions are correlated to the inhibition of Antibiotics 2021, 10, x FOR PEER REVIEWa sulfur atom, and carbapenems possess an unsaturated ﬁve-membered ring without7 of 33 the cell wall biosynthesis, resulting from the irreversible transpeptidase active site block heteroatom. Finally, monobactams only contain the β-lactam ring [37]. due to the similarity of the antibiotic with the D-Ala-D-Ala moiety described above. From a chemical point of view, there are four main classes of natural β-lactams, all of them containing a four-membered cyclic amide (Figure 4). Penicillin possesses a β-lactam ring, fused with a five-membered ring with a sulfur atom; cephalosporins possess a β-lactam ring with a six-membered unsaturated ring with a sulfur atom, and carbapenems possess an unsaturated five-membered ring without heteroatom. Finally, monobactams only contain the β-lactam ring [37].

Figure 4. Base structuralstructural nucleusnucleus of ofβ β-lactam-lactam antibiotics antibiotics and and some some representative representative examples examples of naturalof natural beta-lactam beta-lactam antibiotics antibi- producedotics produced by Actinomycetes by Actinomycetes[38–51 ].[38–51].

Resistance mechanisms that are generally recognized refer to the production of efflux pumps; modification of the antibiotic target; and synthesis of enzymes able to inactivate the antibiotic (β-lactamase, serine or Zn2+ proteases). Sometimes the latter problem could be avoided thanks to a particular class of β-lactam antibiotics called β-lactamase inhibitors. Clavulanic acid (5) and olivanic acid (6) are two natural molecules belonging to this group of antibiotics that work as suicide inhibitors of these particular enzymes. The discovery of these drugs and their natural producers had a big significance from a scientific and pharmaceutical point of view. Indeed, the finding has permitted the development of combined therapies using β-lactams and β-lactamase inhibitors, even against bacteria resistant to this antibiotic class. In nature, many compounds have these characteristics, produced by different Actinomycetes as well as fungal species. Table 1 presents natural β- lactam antibiotics and β-lactamase inhibitors produced by Actinomycetes.

Table 1. Natural β-lactam antibiotics and β-lactamase inhibitors produced by Actinomycetes.

β-Lactams Producer Microorganism 1,2 S. lipmanii; S. microflavus [38,39]; S. griseus Penicillin Penicillin N subsp. Cryophilus [46]; S. cattleya [48] S. clavuligerus [38,39]; S. griseus; S. lactamdurans [41]; S. chartreusis [42–44]; S. cinnamonensis; S. fimbriatus; S. halstedii; S. rochei; S. Cephamycins viridochromogenes [41]; S. wadayamensis; S. Cephalosporines Jumonjineneis; S. lipmanii; S. panayaensis; Amy- colatopsis lactamdurans [45]; S. cattleya [48]; S. heteromorphus [49] S. microflavus; S. lipmanii; S. clavuligerus [38– Cephalosporines 40,42]; S. hygroscopicus [43] S. cattleya [45,47]; S. griseus subsp. Cryophilus [46,50]; S. penemifaciens [48]; S. flavogriseus; S. Carbapenems Thienamycins olivaceus; S. cremeus subsp. auratilis; S. flavoviridis [49] Antibiotics 2021, 10, 483 7 of 32

Resistance mechanisms that are generally recognized refer to the production of efflux pumps; modification of the antibiotic target; and synthesis of enzymes able to inactivate the antibiotic (β-lactamase, serine or Zn2+ proteases). Sometimes the latter problem could be avoided thanks to a particular class of β-lactam antibiotics called β-lactamase inhibitors. Clavulanic acid (5) and olivanic acid (6) are two natural molecules belonging to this group of antibiotics that work as suicide inhibitors of these particular enzymes. The discovery of these drugs and their natural producers had a big significance from a scientific and pharmaceutical point of view. Indeed, the finding has permitted the development of combined therapies using β-lactams and β-lactamase inhibitors, even against bacteria resistant to this antibiotic class. In nature, many compounds have these characteristics, produced by different Actinomycetes as well as fungal species. Table1 presents natural β-lactam antibiotics and β-lactamase inhibitors produced by Actinomycetes.

Table 1. Natural β-lactam antibiotics and β-lactamase inhibitors produced by Actinomycetes.

β-Lactams Producer Microorganism 1,2 S. lipmanii; S. microflavus [38,39]; S. griseus Penicillin Penicillin N subsp. Cryophilus [46]; S. cattleya [48] S. clavuligerus [38,39]; S. griseus; S. lactamdurans [41]; S. chartreusis [42–44]; S. cinnamonensis; S. fimbriatus; S. halstedii; Cephamycins S. rochei; S. viridochromogenes [41]; Cephalosporines S. wadayamensis; S. Jumonjineneis; S. lipmanii; S. panayaensis; Amycolatopsis lactamdurans [45]; S. cattleya [48]; S. heteromorphus [49] S. microflavus; S. lipmanii; S. clavuligerus Cephalosporines [38–40,42]; S. hygroscopicus [43] S. cattleya [45,47]; S. griseus subsp. Cryophilus [46,50]; S. penemifaciens [48]; Carbapenems Thienamycins S. flavogriseus; S. olivaceus; S. cremeus subsp. auratilis; S. flavoviridis [49] Nocardia uniformis [45]; Nocardia uniformis subsp. Tsuyamanensis; Streptomyces alcalophilus Monobactam Nocardicins sp. nov. [49]; Actinosynnema mirum [48,49]; Microtetraspora caesia [51] S. clavuligerus; S. jumonjinesis; Clavulanic acid S. katsuharamanus [45] PBP inhibitors S. olivaceus [46]; Streptomyces griseus subsp. Olivanic acid cryophilus [49] 1 S. indicates the genus Streptomyces; 2 the numbers in square brackets indicate literature references.

3.2. Ansamycines Ansamycins are naturally occurring compounds produced by diverse microorganisms as secondary metabolites. Chemically, they contain aromatic moieties bridged by a polyketide chain terminating in an amide connection (Figure5). The most important natural antibiotic with this kind of structure is rifamycin B (7). Clinically, rifamycin B does not have a primary role because it has low biological activity. Nevertheless, its production is very important since it is easy to produce by fermentation and it is simple to convert in other derivatives, such as rifamycin S, an important building block for the synthesis of other drugs, such as rifampicin (8), which is widely exploited in clinical practices. Attention on this antibiotic class depends, above all, on its activity against Mycobacterium, but it is also generally important for treatment of other pathogenic Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus spp. [52]. Antibiotics 2021, 10, x FOR PEER REVIEW 8 of 33

Nocardia uniformis [45]; Nocardia uniformis subsp. Tsuyamanensis; Streptomyces alcalophilus Monobactam Nocardicins sp. nov. [49]; Actinosynnema mirum [48,49]; Microtetraspora caesia [51] S. clavuligerus; S. jumonjinesis; S. katsuha- Clavulanic acid ramanus [45] PBP inhibitors S. olivaceus [46]; Streptomyces griseus subsp. Olivanic acid cryophilus [49] 1 S. indicates the genus Streptomyces; 2 the numbers in square brackets indicate literature references.

3.2. Ansamycines Ansamycins are naturally occurring compounds produced by diverse microorganisms as secondary metabolites. Chemically, they contain aromatic moieties bridged by a polyketide chain terminating in an amide connection (Figure 5). The most important natural antibiotic with this kind of structure is rifamycin B (7). Clinically, rifamycin B does not have a primary role because it has low biological activity. Nevertheless, its production is very important since it is easy to produce by fermentation and it is simple to convert in other derivatives, such as rifamycin S, an important building block for the synthesis of other drugs, such as rifampicin (8), which is widely exploited in clinical practices. Atten- tion on this antibiotic class depends, above all, on its activity against Mycobacterium, but it is also generally important for treatment of other pathogenic Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus spp. [52]. Rifamycins are antibiotics with bactericidal activity, due to their ability to inhibit DNA-dependent RNA polymerase. The most common resistance mechanism is due to the modification of the antibiotic target through a chromosomic mutation of the DNA-dependent RNA polymerase. The mutation rate is very high when rifamycin is used in monotherapy and, for this reason, it is always associated with a second antibiotic to reduce this issue [53]. Rifamycin derivatives are produced by different microorganisms belonging to the

Antibiotics 2021Actinomycetes, 10, 483 group, such as Actinomadura formosensis, Amycolatopsis tolypomycina, Mi- 8 of 32 cromonospora sp., Amycolatopsis mediterranei, Amycolatopsis rifamycinica [38,39], Micromono- spora lacustris [54], and Salinispora arenicola [55].

FigureFigure 5. Natural 5. Natural and and synthetic synthetic ansamycins ansamycins:: rifamycin rifamycin B (natural) and and rifampicin rifampicin (semisynthetic). (semisynthetic).

3.3. Macrolides Rifamycins are antibiotics with bactericidal activity, due to their ability to inhibit DNA-dependent RNA polymerase. The most common resistance mechanism is due to The natural macrolidesthe modification class ofencloses the antibiotic a series target of compounds through a chromosomicwith 14, 15 or mutation 16 carbon of the DNA- atoms organized dependentas a macrocyclic RNA polymerase. lactone, in Thewhich mutation are often rate ispresent very high one when or two rifamycin sugar is used in moieties as “ring decorationmonotherapys”. and, The for diffe thisrences reason, among it is always these associated molecules with are a related second antibioticto their to reduce pharmacokinetic properties,this issue [53 tolerability]. , and metabolization [56]. Macrolides are generally considered bacteriostaticRifamycin agents derivatives but, in arethe produced same cases, by differentthey could microorganisms reveal bactericidal belonging to the activity. From a molecularActinomycetes pointgroup, of view, such they as Actinomadura interact with formosensis the 50S subunit, Amycolatopsis of the prokar- tolypomycina, Mi- cromonospora sp., Amycolatopsis mediterranei, Amycolatopsis rifamycinica [38,39], Micromonospora yotic ribosome, inhibiting the translocation of the ribosome. The mechanisms of resistance lacustris [54], and Salinispora arenicola [55]. are disparate and cross-resistance to other antibiotics with similar structures (lincosa- 3.3. Macrolides The natural macrolides class encloses a series of compounds with 14, 15 or 16 carbon atoms organized as a macrocyclic lactone, in which are often present one or two sugar moieties as “ring decorations”. The differences among these molecules are related to their pharmacokinetic properties, tolerability, and metabolization [56]. Macrolides are generally considered bacteriostatic agents but, in the same cases, they could reveal bactericidal activity. From a molecular point of view, they interact with the 50S subunit of the prokaryotic ribosome, inhibiting the translocation of the ribosome. The mechanisms of resistance are disparate and cross-resistance to other antibiotics with similar structures (lincosamides and streptogramins) are often associated. Generally, it is possible to observe resistances related to efflux pumps and modification of the target (70–100% of the clinical isolate). Strains able to inactivate the antibiotic are rarely found [57,58]. In nature, macrolides were isolated from different Streptomyces spp. The most common macrolides are oleandomycin, isolated from Streptomyces antibioti- cus; spiramycin, from Streptomyces ambofaciens; josamycin, from Streptomyces narbonensis var. josamyceticus; midecamycin, from Streptomyces mycarofaciens; rosaramycin, from Mi- cromonospora rosaria; and erythromycin, isolated from Aeromicrobium erythreum and Strep- tomyces erythreus [38,39,59–63]. The last mentioned compound (9), shown in Figure6, is the most important and widely-used macrolide molecule, even if the others, excluding rosaramycin, which was eliminated for its neurotoxicity, are also used as medical agents thanks their therapeutic and bioactive features. Other macrolides, such as lipiarmycin and rapamycin (10), possess larger lactone rings. Interestingly, the latter macrolide was isolated from cultures of Streptomyces hygroscopicus collected from Easter Island. This compound was named rapamycin, according to the native name of the island, Rapa Nui, and was initially used as an antifungal agent. The discovery of its immunosuppressant properties made it the drug of choice for the prevention of organ transplantation rejection. This is a further example of the great diversity of the metabolites produced by Streptomyces strains, both in terms of chemical structure and bioactivity [64]. Antibiotics 2021, 10, x FOR PEER REVIEW 9 of 33

mides and streptogramins) are often associated. Generally, it is possible to observe resistances related to efflux pumps and modification of the target (70–100% of the clinical Antibiotics 2021isolate)., 10, x FOR StrainsPEER REVIEW able to inactivate the antibiotic are rarely found [57,58]. 9 of 33

In nature, macrolides were isolated from different Streptomyces spp. The most common macrolides are oleandomycin, isolated from Streptomyces antibi- oticus; spiramycin,mides from and Streptomyces streptogramins ambofaciens) are often; josamycin, associated. from Generally, Streptomyces it is possible narbonensis to observe re- var. josamyceticussistances; midecamycin, related to from efflux Streptomyces pumps and modificationmycarofaciens of; therosaramycin, target (70– 100from% of Mi- the clinical cromonospora rosariaisolate).; and Strains erythromycin, able to inactivate isolated the from antibiotic Aeromicrobium are rarely founderythreum [57,58]. and Strep- tomyces erythreus [38,39,59In nature,–63 ]mac. Therolides last mentionedwere isolated compound from different (9), Streptomyces shown in Figurespp. 6, is the most important andThe mostwide commonly-used macrolidemacrolides aremolecule, oleandomycin, even if isolatedthe others, from excluding Streptomyces antibi- oticus; spiramycin, from Streptomyces ambofaciens; josamycin, from Streptomyces narbonensis rosaramycin, which was eliminated for its neurotoxicity, are also used as medical agents var. josamyceticus; midecamycin, from Streptomyces mycarofaciens; rosaramycin, from Mi- thanks their therapeuticcromonospora and rosariabioactive; and features. erythromycin, Other isolated macrolides, from Aeromicrobiumsuch as lipiarmycin erythreum and and Strep- rapamycin (10), tomycespossess erythreus larger lactone[38,39,59 rings.–63]. The Interestingly, last mentioned the compoundlatter macrolide (9), shown was in iso- Figure 6, is lated from culturesthe mostof Streptomyces important and hygroscopicus widely-used collected macrolide from molecule, Easter even Island. if the This others, com- excluding pound was namedrosaramycin rapamycin,, which according was eliminated to the native for its nameneurotoxicity, of the island, are also Rapa used Nui as medical, and agents was initially usedthanks as an their antifungal therapeutic agent. and Thebioactive discovery features. of Otherits immunosuppressant macrolides, such as lipiarmycin prop- and erties made it therapa drugmycin of choice (10), possess for the largerprevention lactone of rings. organ Interestingly, transplantation the latter rejection. macrolide This was iso- is a further examplelated fromof the cultures great diversityof Streptomyces of the hygroscopicus metabolites collected produced from by Easter Streptomyces Island. This com- strains, both in termspound of was chemical named structurerapamycin, and according bioactivity to the [64]. native name of the island, Rapa Nui, and was initially used as an antifungal agent. The discovery of its immunosuppressant prop-

Antibiotics 2021, 10, 483 erties made it the drug of choice for the prevention of organ transplantation rejection.9 This of 32 is a further example of the great diversity of the metabolites produced by Streptomyces strains, both in terms of chemical structure and bioactivity [64].

Figure 6. Macrolide molecules produce by Actinomycetes: erythromycin A (antibiotic), and rapamycin (immunosuppressant). Figure 6.6. Macrolide moleculesmolecules produce produce by byActinomycetes Actinomycetes: erythromycin: erythromycin A (antibiotic), A (antibiotic) and, and rapamycin rapamycin (immunosuppressant). (immunosuppressant). 3.4. Lincosamides Lincosamides3.4. are Lincosamides very similar to macrolides in terms of activity spectrum and mechanisms of action3.4., but LincosamidesLincosamides their chemical are verystructure similars are to macrolides quite different in terms (Figure of activity 7). Indeed, spectrum no and mech- macrocyclic lactoneanisms isLincosamides presen of action,t and but are the their very sugar chemicalsimilar moiety to structures macrolides is attached are in quitetoterms andifferent aminoof activity acid (Figure spectrum element.7). Indeed, and mech- no The resistance mechanismsmacrocyclicanisms of action lactoneare often, but is their presentlinked chemical andto those the structure sugar of macrolides moietys are quite is attached and different streptogramins, to an (Figure amino 7). acid inIndeed, element. no which a methylaseThemacrocyclic induces resistance lactone an mechanisms alteration is presen areont and oftenthe the 50S linked sugar subunit to moiety those of ofisthe attached macrolides ribosome. to an and aminoIn streptogramins, nature, acid element. in whichThe resistance a methylase mechanisms induces are an alterationoften linked on to the those 50S of subunit macrolides of the and ribosome. streptogramins, In nature, in lincomycin (11) was the first lincosamide isolate, but its semisynthetic chlorine derivate lincomycinwhich a methylase (11) was theinduces ﬁrst lincosamidean alteration isolate, on the but 50S its subunit semisynthetic of the chlorineribosome. derivate In nature, clin- clindamycin (12)damycinlincomycin is usually (12 () 11more is) usually was active the more first than active lincosamide lincomycin than lincomycin isolate in the, but in treatment theits semisynthetic treatment of bacterial of bacterial chlorine in- infections, derivate fections, in particularinclindamycin particular those caused those(12) is caused usually by anaerobic by more anaerobic active species species than [65]. lincomycin [65 Lincomycin]. Lincomycin in the is is treatmentavailable available offro from bacterialm different indifferent ActinomycetesActinomycetesfections, in species particularspecies,, such those such as ascaused StreptomycesStreptomyces by anaerobic lincolnensis species, Streptomyces, [65].Streptomyces Lincomycin spinosus spinosus is available, Streptomyces, from Streptomyces pseudogriseoluspseudogriseolusdifferent Actinomycetes, Streptomyces, Streptomyces species variabilis, such, , asStreptomycesStreptomyces Streptomyces variabilis variabilislincolnensis var. var., liniabilisStreptomyces liniabilis, Streptomyces, spinosus, Streptomyces vellosusvellosusStreptomyces, Micromonospora, Micromonospora pseudogriseolus halophytica halophytica, Streptomyces, and ActinomycesActinomyces variabilis, roseolusStreptomyces roseolus[38 [38,39,66,39 variabilis,66–71].–71 var.]. liniabilis, Streptomyces vellosus, Micromonospora halophytica, and Actinomyces roseolus [38,39,66–71].

Figure 7. Natural and synthetic lincosamides: lincomycin (natural) and clindamycin (semisynthetic).

3.5. Tetracyclines Tetracyclines are a class of antibiotic compounds discovered in the second half of 20th century. Chemically, they possess a naphthacene carboxamide nucleus generally substituted on carbon C2, C4, C6, and C7 of the naphthacene core. These molecules show a bacteriostatic effect inhibiting the protein synthesis through the binding with the 30S subunit of the prokaryotic ribosome. Different resistance mechanisms were identified in plasmid or transposon DNA. These genes confer to the bacteria the ability to extrude the antibiotic from the cell through efflux pump (generally in Gram-negative bacteria) or to modify structurally the ribosome in order to hinder the binding with the antibiotic (in the main part of the Gram-positive bacteria). Being that the resistance is usually located in “mobile-DNA”, the activity spectrum of tetracycline has become tighter with respect to its initial action radius. In fact, first generation tetracycline is recommended only for the treatment of intracellular pathogens, such as Brucella, Rickettsia, or Chlamydia. The first natural tetracycline approved for clinical use by the FDA was chlortetracycline (13) (1948) followed by oxytetracycline (14) (1951), tetracycline (15) (1953), and demethylchlortetracycline (16)[72,73] (Figure8). These compounds are produced by many Actinomycetes species, which are summarized in Table2 (first generation tetracyclines). Antibiotics 2021, 10, x FOR PEER REVIEW 10 of 33

Figure 7. Natural and synthetic lincosamides: lincomycin (natural) and clindamycin (semisynthetic).

3.5. Tetracyclines Tetracyclines are a class of antibiotic compounds discovered in the second half of 20th century. Chemically, they possess a naphthacene carboxamide nucleus generally substituted on carbon C2, C4, C6, and C7 of the naphthacene core. These molecules show a bacteriostatic effect inhibiting the protein synthesis through the binding with the 30S subunit of the prokaryotic ribosome. Different resistance mechanisms were identified in plasmid or transposon DNA. These genes confer to the bacteria the ability to extrude the antibiotic from the cell through efflux pump (generally in Gram-negative bacteria) or to modify structurally the ribosome in order to hinder the binding with the antibiotic (in the main part of the Gram-positive bacteria). Being that the resistance is usually located in “mobile-DNA”, the activity spectrum of tetracycline has become tighter with respect to its initial action radius. In fact, first generation tetracycline is recommended only for the treatment of intracellular pathogens, such as Brucella, Rickettsia, or Chlamydia. The first natural tetracycline approved for clinical use by the FDA was chlortetracycline (13) (1948) Antibiotics 2021, 10, 483 followed by oxytetracycline (14) (1951), tetracycline (15) (1953), and demethylchlortetra-10 of 32 cycline (16) [72,73] (Figure 8). These compounds are produced by many Actinomycetes species, which are summarized in Table 2 (first generation tetracyclines).

FigureFigure 8. 8.Natural Natural tetracycline: tetracycline: chlortetracycline chlortetracycline (13 (),13 oxytetracycline), oxytetracycline (14 (),14 tetracycline), tetracycline (15 ),(15 and), and 6-demethyltetracycline 6-demethyltetracycline (16). (16). Table 2. Natural tetracycline antibiotics produced by Actinomycetes. Table 2. Natural tetracycline antibiotics produced by Actinomycetes. Tetracycline Antibiotics Producer Microorganism 1,2 Tetracycline Antibiotics Producer Microorganism 1,2 S. aureofaciens; S. avellaneus; S. lusitanus; Tetracycline S. aureofaciens; S. avellaneus; S. lusitanus; S. Tetracycline S. viridifaciens [38,39,74,75] viridifaciens [38,39,74,75] Chlortetracycline S. lusitanus; S. aureofaciens; S. lividans 3 [38,39,74–77] S. lusitanus; S. aureofaciens; S. lividans 3 Chlortetracycline S. alboflavus; Streptomyces[38,39,74 albofaciens–77] ; Streptomyces Oxytetracycline erumpens; S. griseus; S. platensis; S. rimosus subsp. rimosus; S. alboflavusS. varsoviensis; Streptomyces[38,39 albofaciens,74] ; Strepto- myces erumpens; S. griseus; S. platensis; S. ri- 6-demethyltetracyclineOxytetracycline S. aureofaciens 3 [77] mosus subsp. rimosus; S. varsoviensis 1 S. indicates the genus Streptomyces; 2 the numbers in square brackets indicate literature references 3 genetically modified strain. [38,39,74] 6-demethyltetracycline S. aureofaciens 3 [77] 3.6.1 S. indicates Aminoglycosides the genus Streptomyces; 2 the numbers in square brackets indicate literature references 3 geneticallyAminoglycosides modified strain are. antibiotic drugs generally formed by two or three amino sugars linked with a glycosidic bond to a central aminocyclitol core. Moreover, thanks to the amino groups in the molecule, the aminoglycosides possess a polycationic character at physiological pH. These antibiotics present different issues linked to their oto- and nephro- toxicity, but also to their narrow therapeutic range. Nevertheless, they are considered fundamental and are often associated with β-lactams for the treatment of rigid infectious thanks to their capability of rapidly killing bacteria and for their post-antibiotic effects [78]. Due their cationic properties, aminoglycosides interact almost instantaneously with the anionic spots in the cell surfaces. The kinetic is dependent on concentration and the interaction is reversible. It is worth noting that other cationic species could influence this process negatively. The mechanisms through which aminoglycosides reach the cytoplasm are several, and among them, it is possible to highlight the active transport; the facilitate transport, (strictly related to the membrane potential and antibiotic concentration); and the self-promoted uptake pathway (common in Gram-negative bacteria), in which aminoglycosides alter the structure of the cross-bridged lipopolysaccharide molecules (LPS) in the outer membrane. This latter mechanism allows the generation of pores that permit a better cell-permeability to aminoglycosides [79–81]. Their most important activity consists in the alteration of the protein synthesis due to their binding on the 16S rRNA of the ribosome. The interaction with ribosomes causes an aberrant protein synthesis in which truncated proteins or proteins with an altered sequence are produced. Moreover, the aminoglycoside-binding also impairs the ribosome translocation [73,81,82]. The binding specificity of these compounds is only 10-fold higher in favor Antibiotics 2021, 10, 483 11 of 32

of the prokaryotic ribosome and this detail could explain the toxicity for the mammalian cell. Although the major actions of aminoglycosides are carried out through the ribosome impairing, the major resistance mechanisms move through the expression of speciﬁc enzymes able to modify the aminoglycoside antibiotic and by the reduction of the antibiotic permeability inside the cell. These processes are in part due to the evolutionary stability of the ribosome that remains conserved among the species, and because microorganisms possess multiple gene copies codifying for rRNA [82]. It is important to underline that a ribosomal resistance exists and was noticed for example in a clinical isolate of the E. coli strain ARS3, and other strains distributed worldwide belonging to Enterobacteriaceae, but is very rarely, with respect to the others, cited above [83,84]. There are many aminoglycosides produced by Actinomycetes in nature (Figure9), and are generally biosynthesized by streptomyces spp. (streptomycin; kanamycin; neomycin; Antibiotics 2021, 10, x FOR PEER REVIEW Micromonospora 12 of 33 tobramycin; paromomycin and spectinomycin); or by spp. (gentamicin; sisomicin; netilmicin; sagamicin; fortimicin; and dactimicin) [53].

FigureFigure 9. Selected9. Selected examples examples of of natural natural aminoglycosides: aminoglycosides: streptomycin, neomycin, neomycin, paromomycin, paromomycin, tobramycin, tobramycin, kanamycin kanamycin andand gentamycin gentamycin [38 [38,39,53,85,39,53,85–91–91].].

TheThe major major compoundscompounds used and their microbiological microbiological source sourcess are are presented presented in in TableTable 3. 3. Table 3. Natural aminoglycoside antibiotics produced by Actinomycetes. Table 3. Natural aminoglycoside antibiotics produced by Actinomycetes. Aminoglycoside Antibiotics Producer Microorganism 1,2 Aminoglycoside Antibiotics Producer Microorganism 1,2 S. griseus; S.S. bikiniensisgriseus; S.; S.bikiniensis streptomycinii; S. streptomycinii; S. ornatus; S.; humidusS. or- ; Streptomycin S. subrutilusnatus; S.; S. mashuensis humidus;; S.S. glaucescenssubrutilus; S. griseocarneusmashuensis; ; Streptomyces rimosus subsp. rimosus; S. galbus [38,39,53,85–87] Streptomycin S. glaucescens; S. griseocarneus; Streptomyces ri- S. fradiae; S. catenulae; S. chrestomyceticus; S. albogriseolus Neomycin mosus subsp. rimosus; S. galbus [38,39,53,85– [38,39,53,85,88,91] 87] StreptoalloteichusS. fradiae; S. hindustanus catenulae; S. tenebrariuschrestomyceticus; S. cremeus; S. al- TobramycinNeomycin bogriseolus[38,39,53 [38,39,53,85,88,91],88,89] Kanamycin StreptoalloteichusS. kanamyceticus hindustanus[38,39,53; S.] tenebrarius; S. Tobramycin S. rimosus var. paromomycinuscremeus; S.[38,39,53,88,89] catenulae; S. chrestomyceticus ; Paromomycym Kanamycin S. rimosusS. kanamyceticus; S. fradiae [38, 39[38,39,53],53,85] MicromonosporaS. rimosus var. purpurea paromomycinus; Micromonospora; S. catenulae pallida;; S. Gentamycin Paromomycym micromonosporachrestomyceticus echinospora; S. rimosus[38,39,53; S.,85 fradiae,90] 1 S. indicates the genus Streptomyces; 2 the numbers in square brackets indicate[38,39,53,85] literature references. Micromonospora purpurea; Micromonospora Gentamycin pallida; micromonospora echinospora [38,39,53,85,90] 1 S. indicates the genus Streptomyces; 2 the numbers in square brackets indicate literature references.

3.7. Antibiotic Peptides Antibiotic peptides is a (very interesting) class of compounds from a biosynthetic point of view. Indeed, it is possible to distinguish two groups in which these molecules could be gathered: the ribosomal peptide synthetases and the nonribosomal peptide synthetases (NRPSs). However, our interest is focused on NRPSs because the major part of antibiotics produced by Actinomycetes spp. (lipopeptide, glycopeptides, and streptogramins) is related to this mechanism [92–95]. Antibiotics 2021, 10, 483 12 of 32

3.7. Antibiotic Peptides Antibiotic peptides is a (very interesting) class of compounds from a biosynthetic point of view. Indeed, it is possible to distinguish two groups in which these molecules could be Antibiotics 2021, 10, x FOR PEER REVIEWgathered: the ribosomal peptide synthetases and the nonribosomal peptide synthetases13 of 33 (NRPSs). However, our interest is focused on NRPSs because the major part of antibiotics produced by Actinomycetes spp. (lipopeptide, glycopeptides, and streptogramins) is related to this mechanism [92–95]. NRPSNRPSss are assembled on multi multi-modular-modular enzymes enzymes that that are are able able to to generate generate a a macro- macro- cyclic structure acting as a protein protein template. template. These These multi multi-modular-modular proteins proteins are are called called non- non- ribosomal peptide synthetases (NRPSs). It It is important to underline that these particular enzenzymesymes are able toto synthetizesynthetize peptides,peptides, accepting accepting as as building building blocks, blocks not, not only only the the 20 20 classic clas- sicproteinogenic proteinogenic amino amino acids, acids, but alsobut lipids,also lipids,α-hydroxy α-hydroxy acids, acids and non-proteinogenic, and non-proteinogenic amino aminoacids. Althoughacids. Although the different the different enzymes enzymes catalyze catalyze a specific a specific synthetic synthetic step, the step, chemical the chem- and icalsequence and sequence identity identity of the product of the product is strictly is strictly dependent dependent on the orderon thein order which in which the multi- the multimodular-modular enzyme enzyme is assembled. is assembled. In each In NRPSeach NRPS module, module it is, possible it is possible to identify to identify different dif- ferentcatalytic catalytic domains domains responsible responsible for the for synthesis the synthesis of the offinal the final product produ (Figurect (Figure 10). 10). Usually, Usu- ally,there there is a domain is a domain responsible responsible for the for amino the amino acid activation acid activation (adenylation (adenylation domains, domains, (A) a (carrierA) a carrier unit impliedunit implied in the inmovement the movement of the of growingthe growing peptide peptide inthe in the different different regions regions of ofthe the NRPSs NRPSs (PCP (PCP), a), condensation a condensation domain, domain, useful useful for thefor amide-bondthe amide-bond formation formation (C), and(C), anda thioesterase a thioesterase domain domain (TE )(TE able) able to catalyze to catalyze the hydrolysisthe hydrolysis or the or macrocyclizationthe macrocyclization of the of theproduct. product. Often, Often, it isit easyis easy to to find find other other catalytic catalytic domains domains responsible responsible forfor thethe structuralstructural modificationmodification of the final final product ( M)),, such as epimerization, methylation methylation,, or cyclization. These domains are inserted in different positions of the various modules that constitute the NRPSs.NRPSs. Moreover,Moreover, other other independent independent enzyme(s) enzyme(s) could could introduce introduce further further modifications modifica- tionsusing using the rising-product the rising-product as substrate as substra [95–te98 [95]. –98].

Figure 10. 10. SchematicSchematic representation representation of ofan anNRPS NRPS enzyme. enzyme. A gene A genecodifies codifies for a module; for a module; each mod- each ulemodule is specific is specific for the for sequence the sequence and for and the for final the finalstructure structure of the of peptidic the peptidic product; product; each eachmodule module possess catalytic domains: domains: the the main main are are the the activation activation domain domain (green (green, A),, A), carrier carrier protein protein (yellow (yellow, , PCP), condensation domain (red, C), modification domain (black, E), and termination domain PCP), condensation domain (red, C), modification domain (black, E), and termination domain (light (light blue, TE). blue, TE).

The most most important important natural natural antibiotics antibiotics of of this this class, class, produced produced by bydifferent different Actinomy-Actino- cetesmycetes sppspp.,., are areglycopeptides glycopeptides,, such such as vancomycin as vancomycin (GPAs), (GPAs), lipopeptides lipopeptides,, such such as daptomy- as dapto- cinmycin (LPAs) (LPAs),, and and streptogramins streptogramins (Figure (Figure 11) 11. ).From From a achemical chemical point point of of view, view, GPAs GPAs and LPAs are molecules containing a linear or cyclic peptidic scaffold decorated with different sugars (GPAs) oror fattyfatty acids acids (LPAs) (LPAs) and and other other particular particular functional functional groups, groups such, such as chlorine,as chlorine,acetyl, acetyl or methyl, or methyl groups. groups. Conversely, Conversely, streptogramins streptogramins possess possess characteristics characteristics very close very to close to macrolides, exhibiting a macrocyclic polyunsaturated lactone structure (streptogramins A), while, in the case of streptogramins B, a cyclic hepta-or hexadepsipeptide structure is observed [94,99]. Although LPAs and GPAs are quite similar structurally, they have different mechanisms of action. GPAs are able to impair the cell wall synthesis blocking the transpeptidation and transglycosylation reactions through the binding with the D-Ala-D-Ala moieties, stabi- lized by wake–energy bonds, such as hydrophobic van der Waals contacts and hydrogen Antibiotics 2021, 10, 483 13 of 32

macrolides, exhibiting a macrocyclic polyunsaturated lactone structure (streptogramins A), while, in the case of streptogramins B, a cyclic hepta-or hexadepsipeptide structure is observed [94,99]. Although LPAs and GPAs are quite similar structurally, they have different mechanisms of action. GPAs are able to impair the cell wall synthesis blocking the transpeptidation and Antibiotics 2021, 10, x FOR PEER REVIEW 14 of 33 transglycosylation reactions through the binding with the D-Ala-D-Ala moieties, stabi- lized by wake–energy bonds, such as hydrophobic van der Waals contacts and hydrogen bonds [100]. LPAs, instead, are able to act as surfactants, binding directly to the bacterial membrane,bonds [100]. causing LPAs, ainstead, misbalance are able of to the act chemicophysical as surfactants, binding properties directly ofthe to the same bacterial biological structuremembrane [101, causing]. The resistancea misbalance developed of the chem againsticophysical GPA properties antibiotics of are the generallysame biological related to thestructure capability [101]. to The substitute resistance the developed D-Ala-D-Ala against moiety GPA antibiotics with a D-Ala-D-Lactate are generally related framework, to anthe alteration capability that to substitute could decrease the D-Ala the-D interaction-Ala moiety with with thea D antibiotic-Ala-D-Lactate up toframework, 1000-fold. In an alteration that could decrease the interaction with the antibiotic up to 1000-fold. In ad- addition, bacteria can become resistant to GPAs, increasing the thickness of the cell wall dition, bacteria can become resistant to GPAs, increasing the thickness of the cell wall (substrate iper-production) [73,102,103]. (substrate iper-production) [73,102,103]. Contrarily, streptogramins possess the same spectrum of activity and mechanism of Contrarily, streptogramins possess the same spectrum of activity and mechanism of actionaction of of macrolides, macrolides and, and the the resistanceresistance mechanisms are are often often related. related. Separately, Separately, group group A andA groupand group B streptogramins B streptogramins are are bacteriostatic, bacteriostatic, by by reversiblereversible binding to to the the 50S 50S subunit subunit of 70Sof bacterial70S bacterial ribosomes. ribosomes. Together, Together, however, however, streptogramins streptogramins from from eacheach groupgroup are syn- synergic andergic bactericidal and bactericidal [104]. [104].

FigureFigure 11. Selected 11. Selected examples examples of natural of natural peptide peptide antibiotics antibiotics produced produced by byActinomycetes Actinomycetes:: vancomycin, vancomycin, daptomycin, daptomycin, pristi- namycinpristinamycin II (streptogramin II (streptogramin A type), A and type) pristinamycin, and pristinamycin I (streptogramin I (streptogramin B type) B type) [38, 39[38,39,105,105–109–109]. ].

Antibiotics 2021, 10, x FOR PEER REVIEW 15 of 33

Regarding the activity of GPAs and LPAs against pathogens, we can assume they have a similar spectrum of action, especially against Gram-positive bacteria, even against strains The most important antibiotic peptides produced in nature by Actinomycetes are resistant to other antibiotics. summarized in Table 4. The most important antibiotic peptides produced in nature by Actinomycetes are Tablesummarized 4. Natural in peptide Table4. antibiotics produced by Actinomycetes.

Table 4. Natural peptidePeptide antibiotics Antibiotics produced by Actinomycetes. Producer Microorganism 1,2 Amycolatopsis orientalis subsp. GlycopeptidesPeptide Antibiotics vancomycin Producer Microorganism 1,2 Orientalis [38,39] Amycolatopsis orientalis subsp. Glycopeptides vancomycin StreptomycesOrientalis [38 sp.,39 KCTC12267BP] Lipopeptides daptomycin [105]; S. roseosporus [106,108]; S. Streptomyces sp. KCTC12267BP [105]; Lipopeptides daptomycin 3 S. roseosporus [106,108lividans]; S. lividans [107]3 [107] S. halstedii [38]; S. pristinaespi- S. halstedii [38]; S. pristinaespiralis; StreptograminsStreptogramins StreptograminsStreptogramins A and B A and B S.ralis virginiae; S. virginiae[109] [109] 1 2 1S.S. indicates the the genus genusStreptomyces Streptomyces; 2 the; numbersthe numbers in square in square brackets brackets indicate indicate literature literature references; references;3 S. lividans 3genetically S. lividans modiﬁed genetically strain. modified strain

3.8. Aminocumarines Aminocumarines Aminocoumarins is is an an antibiotic antibiotic class class chemically chemically formed formed by a by 3-amino a 3-amino-4,7--4,7-dihy- droxycumarindihydroxycumarin ring ringdiversely diversely decorated decorated in its indifferent its different position. position. Novobiocin Novobiocin (20) is (the20) mostis the important most important natural natural molecule molecule belonging belonging to this class to this, which class, was which approved was approved for animal for usesanimal (Figure uses (Figure 12). Indeed, 12). Indeed, Novobiocin Novobiocin,, previously previously employed employed for human for human use underuse under the namethe name albamycin albamycin,, was was withdrawn withdrawn from from the the market market by by the the FDA FDA due due to to lack lack of of safety safety or effectiveness [110]. [110].

Figure 12. The natural aminocoumarin novobiocin and the epoxide antibiotic fosfomycin.

Novobiocin is composed by a sugar residue (novobiose) and a benzoic acid derivate attached to the c coumarinoumarin ring. The The major major novobiocin novobiocin producers producers,, ActinomycetesActinomycetes, are, areStrepto- Strep- tomycesmyces spheroides spheroides, Streptomyces, Streptomyces caeruleus caeruleus,, and andStreptomyces Streptomyces niveusniveus [[38,39,111]38,39,111],, eveneven though Streptomyces spheroides spheroides couldcould be be considered considered as as a heterotypic a heterotypic synonym synonym of Streptomyces of Streptomyces niveusniveus [112].[112 Aminocoumarins]. Aminocoumarins are areable able to inhibit to inhibit DNA DNA gyrase, gyrase, an enzyme an enzyme essential essential for mak- for ingmaking accessible accessible the DNA the DNAto all the to allenzymes the enzymes implied implied in replication in replication and translation. and translation. Amino- coumarinsAminocoumarins bind with bind the with β-subunit the β-subunit of the heterotrimeric of the heterotrimeric enzyme enzyme,, partly partlycovering covering the ATP the- bindingATP-binding site of site the of thegyrase gyrase hindering hindering,, the the ATP ATP hydrolysis hydrolysis required required for for ATP ATP-dependent-dependent DNA supercoiling. Moreover, Moreover, an activity of aminocoumarins against DNA topoisomerase IV,IV, anan enzymeenzyme alsoalso involvedinvolved in in the the control control of of DNA DNA supercoiling supercoiling and and in in the the decatenation decatenation of ofthe the daughter daughter chromosomes chromosomes after after DNA DNA replication replication in bacteria,in bacteria was, was shown shown [113 [113,114].,114]. Novobiocin is a narrow narrow-spectrum-spectrum antibiotic that may be bacteriostatic or bactericidal at higher higher concentrations. concentrations. It Itis active is active mostly mostly against against Gram-positive Gram-positive bacteria, bacteria but also, but against also againsta few Gram-negative a few Gram-ne bacteria,gative bacteria including, includingNeisseria Neisseria, Bordetella, Bordetella, Brucella, Brucella, Haemophilus, Haemophilus, and, andsome some strain strain of Proteus of Proteus[115 [115].]. The The resistance resistance acquired acquired in bacteriain bacteria are are generally generally related related to tomodiﬁcation modification in thein the antibiotic antibiotic target, target, puntiform puntiform mutation mutation that that impair impair the binding the binding between be- tweenthe drug the and drug the and enzyme the enzyme [116]. [116].

3.9. Epoxides Antibiotics 2021, 10, 483 15 of 32

3.9. Epoxides The most important natural molecule belonging to this class is L-cis-1,2- epoxypropylphosphonic acid, known as fosfomycin (28). This is a polar compound in which the epoxide framework is determinant for its bactericidal activity. It is an antibiotic with a relatively broad activity spectrum because it carries out its function against most Gram+ and Gram- bacteria. It inhibits, irreversibly, the action of MurA, an enzyme involved in the ﬁrst step of the peptidoglycan synthesis. Fosfomycin is always associated with another antibiotic, because in monotherapy, it often causes the onset of resistant bacteria strains. The major resistance mechanisms are associated with a reduced uptake of fosfomycin due to mutation(s) in the structural genes codifying the transporters, an over-expression or modiﬁcation of the target enzyme MurA, or through antibiotic inactivation [117,118]. In nature, it is produced by different Actinomycetes, such as Streptomyces fradiae; Strep- tomyces viridochromogenes; and Streptomyces wedmorensis [53,119–122]. Moreover, thanks to genetic engineering, a strain of Streptomyces lividans is now able to produce fosfomycin. Indeed, Woodyer and co-workers in 2006, identify the minimal biosynthetic cluster of fosfomycin in Streptomyces fradiae, giving a new tool and the possibility to overproduce the antibiotic and create new useful analogues [123].

4. Antibiotics from Actinomycetes: From Isolation to Chemical Characterization, Total Synthesis, and Industrial Production The antibiotics produced by Actinomycetes possess the most varied chemical structures, which display a wide range of biological activities. As it happens for the main part of APIs, the structure–activity relationship (SAR) of these molecules was studied thoroughly, in order to develop new and more effective drugs. In this context, the antibiotics isolated from this class of bacteria were a wonderful source of inspiration for generations of chemists who have decided to challenge their skills. Indeed, the high structural complexity of these natural products makes it difficult to determine their chemical structures, as well as the accomplishments of their syntheses or the preparation of new synthetic analogues. It is not surprisingly that different scientists, awarded with the Nobel Prize, spent part of their careers engaging the syntheses of specific antibiotics produced by Actinomycetes [124,125]. According to their industrial manufacturing methods, antibiotics can be classified in three separate categories: natural compounds, semisynthetic antibiotics, and fully synthetic antibiotics. Natural antibiotics are obtained directly by large-scale fermentation of a given microbial strain whereas semisynthetic derivatives are compounds manufactured by chemical transformation of a natural product. Fully synthetic antibiotics are prepared exclusively through synthetic routes. Actinomyces were sources of very large numbers of natural and semisynthetic antibiotics and are producers of secondary metabolites whose chemical structures have inspired the discovery of new fully synthetic antibiotics. Each one of the above-described classes of pharmaceutical compounds include relevant drugs, whose histories started thanks to the research on Actinomyces strains. Of course, it is impossible to describe comprehensively all of the chemical efforts that have been spent to study these compounds. Therefore, we reported below only a few relevant examples of the aforementioned antibiotics whose isolations, chemical characterizations, total syntheses, and industrial productions can be regarded as milestones of the pharmacology and pharmaceutical industries.

4.1. Penicillin and Cephalosporin Large-scale production of penicillin and cephalosporin were strictly connected to two relevant industrial problems: finding a suitable, high producing strain and the development of antibiotic resistances. Since the isolation of the first penicillin (29, penicillin G, 1940), chemists have faced the problem of the synthesis of this important drug (Figure 13). Unfortunately, difficulty in assembling the high reactive beta-lactam structure delayed Antibiotics 2021, 10, x FOR PEER REVIEW 17 of 33

Antibiotics 2021 10 Antibiotics 2021, 10, x FOR PEER REVIEW, , 483 17 of 33 16 of 32

(for more than 15 years) the first synthesis of a member of this class of compounds (30, (for more thanpenicillin(for 15 years more )V, thanthe 1957) first 15 [126]. years)synthesis the of ﬁrst a member synthesis of of this a member class of ofcompounds this class of(30 compounds, (30, penicillin V, 1957)penicillin [126]. V, 1957) [126].

Figure 13. Milestone compounds in penicillin production: penicillin G, penicillin V, and 6-aminopenicillanic acid (6-APA). Figure 13. MilestoneFigure 13. compoundsMilestone compounds in penicillin in production: penicillin production: penicillin G, penicillin penicillin G, V, penicillin and 6-aminopenicillanic V, and 6-ami- acid (6-APA). nopenicillanic acid (6-APA). Consequently,Consequently, the the penicillin requirement requirement fostered fostered the quest for a biotechnological Consequently,methodmethod the of ofpenicillin its its production, production, requirement which which fosteredfinally finally lead the to quest the forselection a biotechnological of suitable high-producinghigh -producing method of its production,strains.strains. The The whichfollowing following finally development development lead to the of ofselection a a reliable reliable of fermentative fermentativesuitable high process process-producing secured secured the the large large-- strains. The followingscalescale production production development of of penicillin penicillin of a reliable G G,, which which fermentative quickly quickly foundprocess found a a securednumber number theof of medical medicallarge- applications. applications. scale productionUnlucUnluckily, of penicillinkily, many many G , bacterialwhich quickly strains strains found became became a number resistant resistant of medical to to the latter applications. antibiotic, which was Unluckily, manyphasedphased bacterial out out of of strains clinical clinical becameuse use in in the resistant the late late 196 1960s. to0s. theIn spite In latter spite of antibiotic,this of thisfact, fact,the which discovery the discovery was of a new of a class new phased out of clinicalofclass beta of -uselactam beta-lactam in the antibiotic late antibiotics, 196s0,s. the In cephalosporinsspite the cephalosporinsof this fact, (cephalosporin the discovery (cephalosporin of C, a 1955)new C, 1955)class [127] [,127 broaden], broaden the of beta-lactam pharmaceuticaltheantibiotic pharmaceuticals, the cephalosporins relevance relevance of this of(cephalosporin thisclass class of compounds. of compounds.C, 1955) In[127] addition, In, broaden addition, some the some penicillin penicillin and pharmaceuticalcephalosporinand relevance cephalosporin of antibioticsthis class antibiotics ofpossessing compounds. possessing new Inchemical new addition, chemical structures some structures penicillin were isolated wereand isolatedfrom Actino- from cephalosporin mycetesActinomycetesantibiotics (penicillin possessing(penicillin N and new Ncephamycins). chemical and cephamycins). structures These werefindings These isolated findingsindicated from indicated thatActino- only thatthe β only-lactam the mycetes (penicillinringβ-lactam isN essential and ring cephamycins). is for essential the antibiotic for These the activity antibioticfindings and indicated activity suggested and that suggestedthat only the the chemical β that-lactam the modification chemical mod- of ring is essentialtheification for ‘substituents’ the antibiotic of the ‘substituents’ linked activity to and the linked suggestedbicyclic to themoiety that bicyclic the could chemical moiety affect modification couldthe bioactivity affect theof of bioactivity these com- of the ‘substituents’pounds.these linked compounds. to the bicyclic moiety could affect the bioactivity of these compounds. TakingTaking advantage advantage of of the the already already established established process process for penicillin for penicillin G production, G production, chem- Taking advantagechemicalical efforts efforts of were the thenwere already focusedthen established focused on the on development pr theocess development for of penicillin new of semi-synthetic new G production,semi-synthetic antibiotics. antibiotics. From a chemical effortsFromchemical were a thenchemical standpoint, focused standpoint, on the the use development the of 6-aminopenicillanic use of 6-aminopenicillanic of new semi acid-synthetic (31 ,acid 6-APA) (antibiotics.31, as6-APA) the starting as the materialstarting From a chemicalmaterialfor standpoint, the syntheses for the the synthes use of these ofe 6s- aminopenicillanicof new these derivatives new derivatives was acid the (31 was most, 6- APA)the logical most as the logical choice starting choice(Figure (Figure 14). There- 14). material for theTherefore,fore, synthes thee easilys theof these easily available new available derivatives penicillin penicillin Gwas was the G used wasmost asused logical starting as choicestarting material (Figure material in 6-APA14). in 6 production.-APA pro- Therefore, the duction.Nevertheless,easily available Nevertheless, the penicillin cleavage the cleavageG of was the used phenylacetyl of the as phenylacetylstarting moiety material moiety from in the from6- latterAPA the pro- antibiotic latter antibiotic is not anis duction. Nevertheless,noteasy an transformation. easy the transformatcleavage Theof ion.the presence phenylacetyl The presence of two moiety amideof two functionalfromamide the functional latter groups antibiotic makesgroups itis makes necessary it neces- for a β not an easy transformatsaryhigh for regioselective aion. high The regioselective presence deacylation of twodeacylation reaction. amide Infunctional reaction. addition, Ingroups the addition,-lactam makes the ringit βneces--lactam is very ring sensitive is very to sary for a highsensitivehydrolysis regioselective to andhydrolysis deacylation basic reaction and basicreaction. conditions reaction In addition, leadconditions to the the lactamlead β-lactam to opening. the ring lactam is very opening. sensitive to hydrolysis and basic reaction conditions lead to the lactam opening.

FigureFigure 14. 14. TheThe industrial industrial processes processes to to 6 6-aminopenicillanic-aminopenicillanic acid acid (6 (6-APA).-APA). Figure 14. The industrial processes to 6-aminopenicillanic acid (6-APA). To overcome these problems, an ingenious and reliable cleavage procedure was developed in the late 1970s [128]. Accordingly, the carboxylate functional group of the penicillin G salt is protected as trimethylsilyl ester, whereas the secondary amide bond is Antibiotics 2021, 10, x FOR PEER REVIEW 18 of 33

To overcome these problems, an ingenious and reliable cleavage procedure was developed in the late 1970s [128]. Accordingly, the carboxylate functional group of the penicillin G salt is protected as trimethylsilyl ester, whereas the secondary amide bond is Antibiotics 2021, 10transformed, 483 into an imine chloride, through reaction with phosphorus trichloride at low 17 of 32 temperatures (−40 °C). The obtained intermediate (32) is treated with an alcohol (usually butanol) to give derivative (33), whose quenching with water leads to the final cleavage of the phenylacetictransformed moiety intoas the an iminecorresponding chloride, through alcohol reaction-ester. The with 6 phosphorus-APA is obtained trichloride in at low ◦ high overall yieldtemperatures and the above (−40 -describedC). The obtained process intermediate was largely (32 emplo) is treatedyed within the an industry alcohol (usually for about 20 yearsbutanol) after toits give discovery. derivative (33), whose quenching with water leads to the final cleavage of the phenylacetic moiety as the corresponding alcohol-ester. The 6-APA is obtained in high The chemical methods for producing 6-APA are environmentally burdensome as overall yield and the above-described process was largely employed in the industry for they require hazardousabout 20 years chemicals after its and discovery. generate a considerable amount of waste. There- fore, the developmentThe of chemical new enzymatic methods forprocesses producing has 6-APA received are environmentally increasing attention burdensome [129]. as they More specifically,require the improvement hazardous chemicals of the and technology generate ain considerable recombinant amount protein of waste. production Therefore, the made new penicillindevelopment G acylases of new affordable enzymatic, which processes possess has receivedenhanced increasing efficiency attention and stabil- [129]. More ity. As a result specifically,of the considerabl the improvemente efforts spent of the in technology the optimization in recombinant of the protein enzymatic production pro- made cess, 6-APA is currentlynew penicillin produced G acylases through affordable, hydrolysis which possessof penicillin enhanced G salt efficiency, using and water stability. as As a reagent and immobilizedresult of the penicillin considerable G effortsacylase spent as a incatalyst the optimization [130,131]. of the enzymatic process, 6-APA is currently produced through hydrolysis of penicillin G salt, using water as reagent and The gained large-scale availability of the 6-APA secured the synthetic access to the immobilized penicillin G acylase as a catalyst [130,131]. main part of the semisyntheticThe gained large-scaleantibiotics availability possessing of the the β 6-APA-lactam secured nucleus the of synthetic penicillin. access to the Similarly, the discoverymain part of of the the semisyntheticcephalosporins antibiotics produced possessing by Acremonium the β-lactam and nucleusStreptomyce of penicillin.s genera fosteredSimilarly, a new quest the discovery for a suitable of the synthetic cephalosporins building produced block by forAcremonium the preparationand Streptomyces of semisynthetic cephalosporinsgenera fostered.a Unfortunately, new quest for a in suitable those years, synthetic the building structural block complexity for the preparation of the natural cephalosporinsof semisynthetic and cephalosporins. the lack of high Unfortunately, producer strains in those did years, not thelead structural to any out- complexity standing fermentativeof the natural process cephalosporins that could support and the lackthe ofsynthesis high producer of all strainsthe semisynthetic did not lead to any cephalosporinsoutstanding. Consequently, fermentative a completely process different that could preparative support the synthesisapproach of was all the investi- semisynthetic gated. More specifically,cephalosporins. the research Consequently, focused a completelyon chemical different reactions preparative that could approach transform was investi- gated. More specifically, the research focused on chemical reactions that could transform the easily availablethe easily penicillin available G into penicillin new derivatives, G into new possessing derivatives, the possessing cephalosporin the cephalosporin β-lac- β- tam nucleus [132].lactam In nucleusthis context, [132]. two In this new context, chemical two rearrangement new chemical rearrangement reactions were reactions spe- were cially developedspecially (Figure developed 15). Penicillin (Figure sulfoxides15). Penicillin (34 sulfoxides), easily (available34), easily byavailable oxidation by oxidation of of penicillin, are thepenicillin, starting are compound the startings compoundsfor both transformations. for both transformations.

Figure 15.FigureThe transformation 15. The transformation of penicillin of penicillin sulfoxides sulfoxides through Morin through rearrangement Morin rearrangement (upper pathway) (upper or path- through Kukolja rearrangement.way) or through Kukolja rearrangement.

The acid-promotedThe acid-promoted stereospecific stereospeciﬁc transformation transformation of penicillin of penicillinsulfoxides sulfoxides into corre- into corresponding desacetoxycephalosporinssponding desacetoxycephalosporins (36) or (37 ()36 is) orgenerally (37) is generally known known as the as Morin the Morin rear- rearrange- ment [133]. This reaction is performed by heating sulfoxide (34) in presence of an acid rangement [133]. This reaction is performed by heating sulfoxide (34) in presence of an catalyst. The process involves a number of steps comprising of activation of the sulfoxide acid catalyst. Theby theprocess acid leadinginvolves to a the number generation of steps of the comprising azetidinone of sulfenic activation acid (35of), the protonation sul- of foxide by the acidsulfenic leading acid to and the cyclization generation with of thethe adjacentazetidinoneπ-bond. sulf Theenic preferential acid (35), protona- formation of one tion of sulfenic ofacid the and two cyclization isomers (36) with or (37 the) is controlledadjacent π by-bond. the experimental The preferential conditions formation used. of one of the two isomersIn the same(36) or contest, (37) is Kukolja controlled discovered by the the experimental oxidative ring conditions expansion used. of sulfoxide 34 yielding 3-methylenecepham sulfoxides (39)[134]. The reaction is based on the transformation of sulfoxide (34) into sulﬁnyl halide (38), by means of halogenating agents. The Antibiotics 2021, 10, x FOR PEER REVIEW 19 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 19 of 33

In the same contest, Kukolja discovered the oxidative ring expansion of sulfoxide 34 Antibiotics 2021, 10, 483 18 of 32 yielding 3-methylenecepham sulfoxides (39) [134]. The reaction is based on the transfor- In the same contest, Kukolja discovered the oxidative ring expansion of sulfoxide 34 mation of sulfoxide (34) into sulfinyl halide (38), by means of halogenating agents. The yielding 3-methylenecepham sulfoxides (39) [134]. The reaction is based on the transfor- following cyclizationmation of (of38 sulfoxide) by Lewis (34 )acids into sulfinyland the halide reductio (38)n, byof meanthe obtaineds of halogenating sulfoxide agents. The following cyclization of (38) by Lewis acids and the reduction of the obtained sulfoxide (39) afford 3-methylenecephamsfollowing cyclization of type of ( (3840) )by. Lewis acids and the reduction of the obtained sulfoxide (39) afford 3-methylenecephams of type (40). Overall, both( 39Morin) afford and 3-methylenecephams Kukolja reactions ofwere type widely (40). employed for the industrial Overall, both Morin and Kukolja reactions were widely employed for the industrial synthesis of the cephalosporinsOverall, both [135,136]. Morin and As veryKukolja relevant reactions examples, were widely we report employed the indus-for the industrial synthesis of the cephalosporins [135,136]. As very relevant examples, we report the in- synthesis of the cephalosporins [135,136]. As very relevant examples, we report the industrial processes fordustrial the synthesis processes of 7 for-aminodeacetoxycephalosporanic the synthesis of 7-aminodeacetoxycephalosporanic acid (7-ADCA) acidand (7-ADCA) trial processes for the synthesis of 7-aminodeacetoxycephalosporanic acid (7-ADCA) and cefaclor. and cefaclor. Morin rearrangementcefaclor.Morin is rearrangement the key step in is thethe key transformation step in the transformation of penicillin of G penicillin sulfoxide G sulfoxide (41) into the cephalosporanic(41) intoMorin the rearrangement cephalosporanicacid derivative is the acid (42 key) derivative, whose step in hydrolysisthe (42) transformation, whose gives hydrolysis 7 of-ADCA penicillin gives (43 7-ADCA ),G sulfoxide (43), the most important(the41 building) mostinto the important blockcephalosporanic for building cephalosporin block acid derivative for cephalosporin synthesis (42) ,(Figure whose synthesis hydrolysis 16). (Figure gives 16). 7-ADCA (43), the most important building block for cephalosporin synthesis (Figure 16).

Figure 16.Figure The industrial 16. TheFigure industrial synthesis 16. The synthesis industrial of 7-aminodeacetoxycephalosporanic of synthesis 7-aminodeacetoxycephalosporanic of 7-aminodeacetoxycephalosporanic acid acid (7- (7-ADCA).ADCA) acid. (7-ADCA).

Similarly, penicillinSimilarly, V (30 ) penicillin is converted V (30 )into isis convertedconverted the corresponding intointo thethe correspondingcorresponding protected sulfoxide protectedprotected sulfoxidesulfoxide (44),), which is transformed into 3 3-methylenecepham-methylenecepham sulfoxides (45) by the Kukolja process (44), which is transformed into 3-methylenecepham sulfoxides (45) by the Kukolja process (Figure 17).17). The The production production of of the the latter latter pharmaceutical pharmaceutical intermediate intermediate was was exploited exploited in (Figure 17). The productiondifferentin different industrial industrialof the platterrocesses processes, pharmaceutical, affording affording semi semi-synthetic-intermediatesynthetic cephalosporin was cephalosporin exploited antibiotic. antibiotic.in A repre- A different industrialsentativerepresentative processes example, affording example is the cefaclor issemi the-synthetic cefaclor synthesis, synthesis, cephalosporin developed developed by theantibiotic. Eli by Lilly the A EliCompany. repre- Lilly Company. Accord- sentative exampleingly,Accordingly, is the enol cefaclor (46 enol) is synthesis, prepared (46) is prepared by developed ozonolysis by ozonolysis by of thecompound Eli of compoundLilly (45 Company.). (45). Accord- ingly, enol (46) is prepared by ozonolysis of compound (45).

Figure 17. The key synthetic steps of the synthesis of cefaclor®® by Eli Lilly Company. PG = protecting group. Reagents and conditions: (a) NCS, toluene, reflux; (b) SnCl4, CH2Cl2, rt; (c) O3, CH2Cl2, −70 °C; (d◦) (PhO)3PCl2, CH2Cl2, −15 °C then ◦iBuOH; conditions: (a) NCS, toluene, reflux; (b) SnCl4, CH2Cl2, rt; (c)O3, CH2Cl2, −70 C; (d) (PhO)3PCl2, CH2 Cl2, −15 C then (e) 2-amino-2-phenylacetylchloride; deprotection. Figure 17. The key syntheticiBuOH; steps (e) 2-amino-2-phenylacetylchloride; of the synthesis of cefaclor® by deprotection. Eli Lilly Company. PG = protecting group. Reagents and conditions: (a) NCS, toluene, reflux; (b) SnCl4, CH2Cl2, rt; (c) O3, CH2Cl2, −70 °C; (d) (PhO)3PCl2, CH2Cl2, −15 °C then iBuOH; Hence, the use of the triphenyl phosphite-chlorine complex as a halogenating and (e) 2-amino-2-phenylacetylchloride; deprotection. Hence, the use of the triphenyl phosphite-chlorine complex as a halogenating and reducing reagent allows the one pot transformation of of three three different functional groups. groups. The halogenation halogenation of of the the enol enol group group affor affordsds the the corresponding corresponding vinyl vinyl chloride chloride whereas whereas the Hence, the use of the triphenyl phosphite-chlorine complex as a halogenating and sulfoxidethe sulfoxide group group is reduced is reduced to sulfide. to sulfide. Finally, Finally, the theacyl acyl side side chain chain is cleaved is cleaved through through the reducing reagent formationtheallows formation the of one the of correspondingpot the transformation corresponding chloro chloro-imine, -ofimine, three which different which is hydrolyzed functional is hydrolyzed by groups. addition by addition of isobu- of The halogenation tanol.isobutanol.of the The enol resulting Thegroup resulting aminoaffords deriv amino the ativecorresponding derivative (47) is treated (47) vinyl is with treated chloride phenylglycine with phenylglycinewhereas chloride the followed chloride sulfoxide group isbyfollowed reduced the deprotection by to the sulfide. deprotection of theFinally, carboxyl of thethe functional carboxylacyl side functional groupchain to is afford groupcleaved tocefaclor affordthrough (48 cefaclor). the (48). formation of the corresponding chloro-imine, which is hydrolyzed by addition of isobutanol. The resulting4.2. amino Tetracyclines derivative (47) is treated with phenylglycine chloride followed by the deprotection ofThe the history carboxyl of functional tetracyclines group started to in afford the early cefaclor 1940s,1940s (, 48 with). the identificationidentification of the chlortetracycline ( (1313)) from a strain of Streptomyces isolated from soil [[72].72]. Since the new 4.2. Tetracyclines antibiotics and the Streptomyces colonies were gold-colored, the names aureomycin and Streptomyces aureofaciens were assigned to the tetracycline, and to the new bacteria species, The history of tetracyclines started in the early 1940s, with the identification of the chlortetracycline (13) from a strain of Streptomyces isolated from soil [72]. Since the new Antibiotics 2021, 10, x FOR PEER REVIEW 20 of 33

Antibiotics 2021, 10, 483 19 of 32 antibiotics and the Streptomyces colonies were gold-colored, the names aureomycin and Streptomyces aureofaciens were assigned to the tetracycline, and to the new bacteria species, respectively. The The first first pharmacological pharmacological studies studies revealed revealed that that aureomycin aureomycin is a potent is a potent broad- broadspectrum-spectrum antibacterial antibacterial agent, andagent in, 1948,and in the 1948 drug, the was drug approved was approved for clinical for use, clinical although use, althoughits exact chemicalits exact structurechemical hadstruct yeture to had be determined.yet to be determined. Within ten Within years, ten three years more, three nat- moreural tetracyclines—terramycin natural tetracyclines—terramycin (14), achromycin (14), achromycin (15), and (15 declomycin), and declomycin (16)—entered (16)— theen- teredpharmaceutical the pharmaceutical market. However, market. the However, increasing the incidence increasing of incidencebacterial resistance of bacterial to this re- sistancegroup of to antibiotics this group led of antibiotics to a decline led in to their a decline use for in their human use medicine. for human As medicine. a result, As the a result,need for the the need development for the development of new active of new tetracyclines active tetracyclines stimulated stimulated the researchers, the research- in their esynthesesrs, in their and synthes synthetices and modifications. synthetic modification R.B Woodward,s. R.B Woodward who was awarded, who was the awarded Nobel Prize the Nobelin Chemistry Prize in in Chemistry 1965, collaborated in 1965, with collaborated Pfizer to determinewith Pfizer the to structuredetermine of the terramycin structure and of terramycinto study the and total to synthesis study the of total tetracyclines. synthesis of Because tetracyclines. of the complicated Because of the stereochemistry complicated stereochemistryand substitution, and Woodward substitution, once Woodward described these oncecompounds described these as a “diabolicalcompounds concatena- as a “dia- bolicaltion of concatenatio reactive groupings”.n of reactive Moreover, groupings”. the many Moreover substituents, the many in the substituents tetracyclines in the render tet- racyclinesthese drugs render very sensitivethese drugs to acidic, very sensitive alkaline, to and acidic, reducing alkaline, reagents; and reducing thus, making reagents their; thusstereospecific, making their syntheses stereospecific an outstanding synthese challenge.s an outstanding As exemplified challenge. with As exemplified aureomycin with and aureomyciterramycinn degradation and terramycin (Figure degradation 18), basic and(Figure acidic 18), environments basic and acidic can triggerenvironment their degra-s can triggerdation [their137]. degradation In the presence [137]. of In bases, the presence the tetracyclines of bases, canthe tetracyclines isomerize to can isotetracyclines. isomerize to isotetracyclines.Chlortetracycline Chlortetracycline (13) is particularly (13 labile,) is particularly and forms isochlortetracycline labile, and forms isochlortet (49) even whenracy- warmedcline (49) ateven pH when 7.5. In warmed strong acidic at pH conditions, 7.5. In strong tetracyclines acidic conditions, containing tetracyclines a hydroxyl contain- group ingat C-6 a hydroxyl readily eliminate group at waterC-6 readily with concomitant eliminate water aromatization with concomitant of the ring. aromatization In these con- of theditions, ring. oxytetracyclineIn these conditions, (14) formedoxytetracycline an epimeric (14) mixtureformed an of epimeric the apo-oxytetracycline mixture of the apo (50),- whereasoxytetracycline the epimerization (50), whereas at C-4 the occurs epimerization readily atat pHC-4 values occurs between readily 2 at and pH 6, values affording be- tweencompound 2 and ( 516, ).affording compound (51).

Figure 18. The chemicalchemical sensitivity of naturalnatural tetracyclinestetracyclines exempliﬁedexemplified with aureomycin and terramycinterramycin degradation in basic and acidic environments,environments, respectively.respectively. TetracyclineTetracyclinenumbering numberingis isreported reported for for aureomycin. aureomycin.

Due to the above above-described-described synthetic problems, the first first enantioselective synthesis of a natural tetracyclinetetracycline waswas onlyonly reported reported in in 2000 2000 [138 [138],], almost almost 60 60 years years after after the the isolation isolation of ofaureomycin, aureomycin, the the first first known known tetracycline. tetracycline On. On the the contrary, contrary, the the large-scale large-scale availability availability of ofnatural natural tetracyclines tetracyclines by fermentationby fermentation fostered fostered the studythe study on the on preparationthe preparation of semisynthetic of semisyn- thetictetracyclines. tetracyclines. Some Some chemical chemical transformations, transformations, suitable suitable for industrial for industrial production, production, were werespecially specially developed developed for these for these antibiotics antibiotics [72,125 [72,125,137,139],137,139]. For example,. For example, hydrogenolysis hydrogenol- of ysisthe benzylicof the benzylic hydroxyl hydroxyl groups groups at C-6 at provided C-6 provided a reliable a reliable entry intoentry a into series a series of acid-stable of acid- s6-deoxytetracyclinetable 6-deoxytetracycline while while the same the same reaction reaction allowed allowed removing removing the chloride the chloride functional func- tionalgroup group from the from C-7 the position C-7 position (Figure 19(Figure). As a19). result, As a hydrogenation result, hydrogenation of the aureomycin of the aureo- and mycindemethylchlortetracycline and demethylchlortetracycline gave tetracycline gave tetracycline and sancycline and sancycline (52). The obtained(52). The syntheticobtained synthetictetracyclines tetracyclines showed minor showed toxicity minor and toxicity higher and chemical higher stability. chemical Moreover, stability. theseMoreover, structural modifications increase the reactivity of the positions 7 and 9 towards electrophilic substitution. Accordingly, the nitration reaction is the key step in the synthetic processes for the preparation of relevant antibiotics, such as minocycline (53) and tigecycline (54). Antibiotics 2021, 10, x FOR PEER REVIEW 21 of 33

Antibiotics 2021, 10, 483 20 of 32 these structural modifications increase the reactivity of the positions 7 and 9 towards electrophilic substitution. Accordingly, the nitration reaction is the key step in the synthetic processes for the preparation of relevant antibiotics, such as minocycline (53) and tigecy- Anothercline (54). clever Another procedure clever procedure for chemical for modiﬁcation chemical modification of the position of the 6 isposition illustrated 6 is illus- with thetrated preparation with the preparation of methacycline of methacycline (57) and doxycycline (57) and doxycycline (58) from oxytetracycline.(58) from oxytetracy- The benzyliccline. The hydroxyl benzylic group hydroxyl of the group latter of natural the latter antibiotic natural is antibiotic not easily is cleaved not easily through cleaved hy- drogenationthrough hydrogenation because it is because quaternary it is andquaternary sterically and hindered. sterically Therefore, hindered. the Therefore, addition the of chloride,addition of by chloride means of, by NCS means treatment of NCS allows treatme thent formationallows the offormation the pentacyclic of the pentacyclic derivative (derivative55). This compound(55). This compound is treated withis treated hydroﬂuoric with hydrofluoric acid, and acid the, resulting and the resulting elimination elimi- of onenation water of one molecule, water molecule affords,the affords key intermediatethe key intermediate (56). The (56 latter). The compoundlatter compound possesses pos- ansesses exomethylenic an exomethylenic double double bond instead bond instead of the quaternaryof the quaternary hydroxyl hydroxyl group. group. The reductive The re- removalductive removal of the chloride of the functionalchloride functional group at positiongroup at 11a position gives methacycline11a gives methacycline (57), whereas (57 a), furtherwhereas reductive a further stepreductive (by hydrogenation) step (by hydrogenation) affords doxycycline affords doxycycline (58) commercialized (58) commercial- under ® theized name under Vibramycin the name V.ibramycin® .

Figure 19.19. The synthetic approaches toto thethe mostmost relevantrelevant semisyntheticsemisynthetic tetracyclines.tetracyclines.

Overall, thethe above-describedabove-described chemical chemical processes processes provide provide the the main main part part of the of semisyn-the sem- theticisynthetic tetracycline tetracycline on theon the market. market. However, However, the the transformation transformation of of natural natural tetracyclinetetracycline allows a limitedlimited numbernumber ofof modiﬁcationsmodifications ofof theirtheir chemicalchemical frameworks.frameworks. Otherwise,Otherwise, dede novo synthesissynthesis wouldwould give give access access to to a largera larger number number of newof new and and more more different different derivatives. derivatives.As mentioned previously, these drugs are a formidable synthetic challenge, and only in 2005As didmentioned the Myers previously group report, these adrugs versatile are a and formidable enantioselective synthetic syntheticchallenge route, and only to a diversein 2005 rangedid the of Myers 6-deoxytetracycline group report antibioticsa versatile [and140, 141enantioselective]. The new process synthetic targeted route not to aa singlediverse compound, range of 6- butdeoxytetracycline a group of structures antibiotics with the[140,141]. D ring Th ase a new site of process structural targeted variability not a (Figure 20). This strategy alternatively involves the parallel preparation of the left fragment (D ring, eventually substituted with a further E ring) and the right fragments (A and B ring) followed by their efﬁcient union, with concomitant formation of the central C ring. The key Antibiotics 2021, 10, x FOR PEER REVIEW 22 of 33

Antibiotics 2021, 10, x FOR PEER REVIEW 22 of 33

single compound, but a group of structures with the D ring as a site of structural variabil- Antibiotics 2021, 10ity, 483 (Figure 20).single This strategycompound alternatively, but a group involves of structures the withparallel the Dpreparation ring as a site of of the structural left frag- variabil-21 of 32 ment (D ring, eventuallyity (Figure 20). substituted This strategy with alternatively a further E involves ring) and the the parallel right preparation fragments of(A the and left frag- B ring) followedment by (Dtheir ring, efficient eventually union, substituted with concomitant with a further formation E ring) and of thethe right central fragments C ring. (A and B ring) followed by their efficient union, with concomitant formation of the central C ring. The key couplingcoupling reaction reaction for merging for merging the thetwo two moieties moieties involves involves a a highly highly stereocontrolledstereocontrolled Michael– The key coupling reaction for merging the two moieties involves a highly stereocontrolled Michael–ClaisenClaisen cycliza cyclizationtion reaction. reaction. Accordingly, Accordingly, a a carbanion intermediate intermediate of typeof type (59) ( undergoes59) Michael–Claisen cyclization reaction. Accordingly, a carbanion intermediate of type (59) undergoes a conjugatea conjugate addition addition to to the the α,α, β--unsaturatedunsaturated ketone ketone (60 (),60 followed), followed by ring by ring closure clo- reaction undergoes a conjugate addition to the α, β-unsaturated ketone (60), followed by ring closure reaction toto afford afford the the whole tetracyclinetetracycline framework framework (61 ().61 Overall,). Overall, two two carbon-carbon carbon-carbon bonds, and sure reaction to afford the whole tetracycline framework (61). Overall, two carbon-carbon two stereogenic centers are created in only one experimental step. bonds, and twobonds stereogenic, and two centers stereogenic are createdcenters are in onlycreated one in experimentalonly one experimental step. step.

Figure 20. The key steps of the Myers approach to fully synthetic tetracyclines. Figure 20. FigureThe key 20. stepsThe key of the steps Myers of the approach Myers approach to fully to synthetic fully synthetic tetracyclines. tetracyclines. The extraordinary flexibility of the method has now made it possible to identify new The extraordinaryThe extraordinary flexibility of flexibilitythe method of the has method now made has now it possible made it possibleto identify to identify new new tetracyclinetetracycline derivatives derivatives with with activity activity against against a a wide wide range range of of antibiotic antibiotic-resistant-resistant bacteria. bacteria. tetracycline derivatives with activity against a wide range of antibiotic-resistant bacteria. InIn this this context, context, Tetraphase Pharmaceuticals has recently developed eravacycline [142], [142], a In this context,syntheticsynthetic Tetraphase halogenated halogenated Pharmaceuticals tetracycline tetracycline has closely closely recently related developed to tig tigecycline.ecycline eravacycline. Eravacycline [142], has a shown synthetic halogenatedpotentpotent broad broad-spectrum tetracycline-spectrum closely activity relatedagainst a to wide wide tigecycline variety variety of of. Eravacyclinemicroorganisms, microorganisms, has including including shown many many potent broad-spectrumGGram-positiveram-positive activity organisms, organisms against, such asuch wide as as methicillin-resistant variety methicillin of microorganisms,-resistantS. aureusS. aureus, and including, and vancomycin vancomycin many resistant re- Gram-positivesistantEnterococcus organisms Enterococcus faecalis, such and faecalis as Enterococcus methicillin and Enterococcus- faeciumresistant; Gram-negativefaecium S. aureus; Gram, and- organisms,negative vancomycin organisms such as Escherichia re-, such as sistant EnterococcusEscherichiacoli, and faecalis anaerobic coli , andand speciesanaerobicEnterococcus of microorganisms,species faecium of microorganisms; Gram such-negative as Bacteroides, such organisms as .Bacteroides This fluorocycline, such. This as fluoro- was Escherichia colicycline,compared and anaerobic was to compared ertapenem species to and ertapenem of meropenemmicroorganisms and meropenem for the, such treatment foras Bacteroidesthe of complicatedtreatment. This of intra-abdominalcomplicated fluoro- in- cycline was comparedtrainfections-abdominal to and ertapenem infections levofloxacin and and for meropenemlevofloxacin the treatment forfor ofthe the complicated treatment treatment of urinaryof complicated complicated tract infections. urinary in- tract Er- infections.avacycline Eravacycline was granted was fast granted track designation fast track designation by the FDA by and the isFDA currently and is currently in phase IIIin tra-abdominal infections and levofloxacin for the treatment of complicated urinary tract phaseclinical III trials clinical as a trials broad-spectrum as a broad- antibiotic.spectrum antibiotic. This new, fullyThis syntheticnew, fully antibiotic synthetic is antibiotic currently infections. Eravacycline was granted fast track designation by the FDA and is currently in isproduced currently according produced to accordingthe Myers syntheticto the Myers protocol synthetic (Figure protocol 21). The (Figure key building 21). The blocks key phase III clinicalbuildingare trials the fluoro-benzoate blocksas a broad are the-spectrum fluoro (62) and-benzoate antibiotic. the α, (β62-unsaturated) Thisand thenew, α, βfully ketone-unsaturated synthetic (63), whose ketone antibiotic condensation (63), whose is currently producedcondensationaffords tetracycline according affords derivative tetracycline to the Myers (64 derivative). Finally, synthetic ( deprotection64) . Finally, protocol deprotection reactions (Figure and 21). reactions functional The key and group func- building blockstionaltransformation are groupthe fluoro transformation give-benzoate eravacycline give(62) eravacyclineand (65). the α, β (-65unsaturated). ketone (63), whose condensation affords tetracycline derivative (64). Finally, deprotection reactions and functional group transformation give eravacycline (65).

Figure 21. EravacyclineEravacycline synthesis according to the Myers process process..

4.3.4.3. Lipiarmycins Lipiarmycins Actinomycetes are producers of different antibiotics, whose chemical structures range Figure 21. Eravacyclinefrom relatively synthesis small accordingβ-lactam to derivativesthe Myers process to the larger. macrolide derivatives. All of these substances have high structural complexity in common, which represents an exceptional 4.3. Lipiarmycins Antibiotics 2021, 10, x FOR PEER REVIEW 23 of 33

Actinomycetes are producers of different antibiotics, whose chemical structures range from relatively small β-lactam derivatives to the larger macrolide derivatives. All of these substances have high structural complexity in common, which represents an exceptional challenge concerning both structure determination and synthesis. Of course, a higher molecular complexity is linked to tricky analytical procedures and synthetic processes. The histories of the most important antibiotics are very similar to each other. Usually, their studies started many years ago, with the isolation from a given Actinomyces strain followed by the struggle for the determination of their chemical structures. Meanwhile, the study of their total syntheses helped to confirm the structure assignation and laid the foundations for the preparation of new semi-synthetic or fully synthetic analogues. Alt- hough, with respect to their specific features, the histories of a number of macrolide antibiotics, such as erythromycin, vancomycin, rifamycin, daptomycin, and their semisynthetic analogues have followed the above-described path. In order to illustrate a very relevant example, we singled out the less known antibiotic lipiarmycin A3 (Figure 22). The reason for our choice lies in the long and intriguing history of this natural product. Indeed, this macrolide returned to the fore as demonstrated by the great number of scientific studies that were published in very recent years [143–145]. Antibiotics 2021, 10, 483 22 of 32 Lipiarmycin was first isolated in the early seventies by the Lepetit group (Italy), during a screening for antibiotics produced by strains of the genus Actinoplanes. A new Acti- noplanes specie [146] was identified from a soil specimen collected in locality Decca in In- diachallenge and, accordingly concerning, the both bacter structureium was determination named Actinoplanes and synthesis. deccanensis Of. This course, microorgan- a higher ismmolecular produce complexityd a substance is linked with to strong tricky activity analytical against procedures Gram- andpositive synthetic bacteria. processes. The new antibioticThe histories was isolated of the on most February important 29, 1972 antibiotics, and was are named very similarlipiarmycin to each (from other. leap Usually, year). theirAlbeit studies, its started chemical many physical years ago,properties with the and isolation its biological from activity a given [147,148] Actinomyces were strain thor- oughlyfollowed described by the struggle within a for few the years determination later, this material of their was chemical recognized structures. to be a Meanwhile, mixture of twothe studyrelated of products their total in syntheses1987, when helped the first to comprehensive confirm the structure study [149] assignation on the chemical and laid structurethe foundations determination for the preparationof lipiarmycin of was new published semi-synthetic. On the or basis fully of synthetic chemical analogues. degrada- Although,tions and NMR with respect studies, to the their antibiotics specific features, extracted the from histories Actinoplanes of a number deccanensis of macrolide, named lipiarmycinantibiotics, such A3 ( as66) erythromycin, and lipiarmycin vancomycin, A4 (67), were rifamycin, characterized daptomycin, by a common and their 18 semisyn--mem- beredthetic analoguesmacro lactone have attached followed to the two above-described glycosyl moieties, path. namely 2-O-methyl-4-O-homodi- chloroIn-orsellinate order to illustrate-β-D-rhamnose a very relevant and 4- example,O-isobutyrate we singled-5-methyl out- theβ-rhamnose less known (Figure antibiotic 22). Furtherlipiarmycin studies A3 (Figure[150] revealed 22). The that reason the for same our choicestrain was lies inalso the able long to and produce intriguing two historyminor compounds,of this natural lipiarmycin product. Indeed, B3 (68) this and macrolide B4 (69), respectively returned to,the which fore differ as demonstrateded from the bycorre- the spondinggreat number A3 and of scientific A4 by the studies position that of were the isobutyric published ester in very on recentthe rhamnose years [143 moiety–145]..

Figure 22. The chemical structures of lipiarmycins.lipiarmycins.

InLipiarmycin the meantime, was first two isolated research in thegroups early from seventies Japan by United the Lepetit States group independently (Italy), during re- porteda screening the for isolation antibiotics of clostomicins, produced by strainsfrom Micromonospora of the genus Actinoplanes echinospora. A subsp. new Actinoplanes armeniaca [151],specie and [146 tiacumicins] was identified from from Dactylospor a soil specimenangium aurantiacum collected in subsp. locality hamdenensis Decca in India [152], and, respectively.accordingly, Based the bacterium on NMR wasstudies, named clostomicinActinoplanes B1 and deccanensis tiacumicin. This B microorganismwere recognized pro- as duced a substance with strong activity against Gram-positive bacteria. The new antibiotic was isolated on February 29, 1972, and was named lipiarmycin (from leap year). Albeit, its chemical physical properties and its biological activity [147,148] were thoroughly described within a few years later, this material was recognized to be a mixture of two related products in 1987, when the first comprehensive study [149] on the chemical structure determination of lipiarmycin was published. On the basis of chemical degra- dations and NMR studies, the antibiotics extracted from Actinoplanes deccanensis, named lipiarmycin A3 (66) and lipiarmycin A4 (67), were characterized by a common 18-membered macro lactone attached to two glycosyl moieties, namely 2-O-methyl-4-O-homodichloro- orsellinate-β-D-rhamnose and 4-O-isobutyrate-5-methyl-β-rhamnose (Figure 22). Further studies [150] revealed that the same strain was also able to produce two minor compounds, lipiarmycin B3 (68) and B4 (69), respectively, which differed from the corresponding A3 and A4 by the position of the isobutyric ester on the rhamnose moiety. In the meantime, two research groups from Japan United States independently reported the isolation of clostomicins, from Micromonospora echinospora subsp. armeniaca [151], and tiacumicins from Dactylosporangium aurantiacum subsp. hamdenensis [152], respectively. Based on NMR studies, clostomicin B1 and tiacumicin B were recognized as identical to lipiarmycin A3. In the same years (The late 80s), a very large number of effective semisynthetic antibiotics with broad activities were developed and the studies on this small class of compounds were interrupted for more than a decade. A renewed interest in lipiarmycin A3/tiacumicin B started in the late 1990s, when Op- timer Pharmaceuticals began the commercial development of tiacumicin for the treatment of Clostridium difficile [153,154]. This Gram-positive bacterium is an important nosocomial Antibiotics 2021, 10, x FOR PEER REVIEW 24 of 33

identical to lipiarmycin A3. In the same years (The late 80s), a very large number of effective semisynthetic antibiotics with broad activities were developed and the studies on this small class of compounds were interrupted for more than a decade. A renewed interest in lipiarmycin A3/tiacumicin B started in the late 1990s, when Optimer Pharmaceuticals began the commercial development of tiacumicin for the treat- Antibiotics 2021, 10, 483 ment of Clostridium difficile [153,154]. This Gram-positive bacterium is an important 23noso- of 32 comial pathogen frequently diagnosed in infectious hospital-acquired diarrhea; it is regarded as a major public health concern. Tiacumicin B (Fidaxomicin) was then approved bypathogen the FDA frequently in 2011; it diagnosed became the in infectiousfirst-line therapy hospital-acquired for the cure diarrhea; of Clostridium it is regarded difficileas in- a fections.major public health concern. Tiacumicin B (Fidaxomicin) was then approved by the FDA in 2011;Increasing it became interest the first-line in tiacumicin therapy B forinduced the cure more of Clostridium detailed structural difficile infections. studies, culmi- natingIncreasing in the determination interest in tiacumicin of its structure B induced by single more crystal detailed X-ray structural analysis studies,[155]. The culmi- ste- reonating structure in the 66 determination, possessing the of its(18 structureS,19R) absolute by single configuration, crystal X-ray was analysis thus assessed [155]. Thefor tiacumicinstereo structure B. In 66this, possessing context, the the (19 (18SS)-,19diastereoisomerR) absolute configuration, of 66 was prepared was thus by assessed synthesis for andtiacumicin the spectroscopic B. In this context, analyses the of (19 theS)-diastereoisomer latter compound of were66 was not prepared compared by to synthesis those of and ti- acumicinthe spectroscopic B, but appeared analyses ofidentical the latter to compound those reported were notfor lipiarmycin compared to A4 those (67 of). tiacumicinHowever, theB,but NMR appeared spectra identicalof the semi to- thosesynthetic reported and of for the lipiarmycin tiacumicin A4B (of (67 unambiguously). However, the deter- NMR minedspectra configuration) of the semi-synthetic were acquired and of thein different tiacumicin solvents, B (of unambiguously thus inhibiting determined the evaluation con- offiguration) the subtle were spectroscopic acquired in differences different solvents, between thus the inhibitingtwo. Following the evaluation the same of reasoning, the subtle lipiarmycinspectroscopic A4 differences was assigned between the (19 theS two.) configuration Following thewithout same reasoning,any further lipiarmycin confirmatory A4 analysis.was assigned the (19S) configuration without any further confirmatory analysis. AccordingAccording to to the the pharmaceutical pharmaceutical regulations regulations adopted adopted by by the the majority majority of of the the nations, nations, eveneven a a little little difference difference in in the chemical structure structuress of two pharmaceutical products imply thethe need need to to present present a a new new common common techni technicalcal document document (CTD) (CTD) for for the the new new substance. substance. Therefore,Therefore, the determination of the lipiarmycin A3 and tiacumicin B structures and/orand/or the the demonstrationdemonstration of of their their co co-identity-identity became became a a relevant relevant issue issue for for the the pharma pharma industry industry [143]. [143]. InIn addition, addition, recent recent studies studies [156] [156] demonstrated that lipiarmycin possess possesseded good activity againstagainst multidrug multidrug-resistant-resistant strainsstrains ofofMycobacterium Mycobacterium tuberculosis tuberculosis, expanding, expanding its its pharmaceu- pharma- ceuticaltical relevance. relevance. ForFor these these reasons, reasons, in in the the last last few few years, years, a a number number of of research research groups groups have published studiesstudies on on the the determination determination of the lipiarmycin lipiarmycin/tiacumicin/tiacumicin structure structure,, as well as on its its total total synthesis.synthesis. More More specifically, specifically, the the chemical chemical degradation degradation of of samples samples of of tiacumicin tiacumicin B B from DactylosporangiumDactylosporangium aurantiacum and lipiarmycin A3 from from ActinoplanesActinoplanes allowed the the isolat isolationion ofof the the same same derivative derivative 7171 (Figure(Figure 23),23), which which proved proved identical identical to to a a synthetic synthetic sample sample of of 7171 andand unambiguously unambiguously demonstrated demonstrated that lipiarmycin lipiarmycin A3 and tiacumicin tiacumicin B possess possess the the same same (18(18SS,19,19RR)) absolute absolute configuration configuration (2014) [157]. [157].

Figure 23. The chemical degradationdegradation ofof lipiarmycinlipiarmycin A3 A3 and and tiacumicin tiacumicin B B allows allows assigning assigning (18 (18S,19S,19R)R absolute) absolute conﬁguration. configura- ◦ ◦ tion.Reagents Reagents and conditions:and conditions: (a) KOH(a) KOH aq.; aq.; (b) Ac2O,(b) Ac2O, Py; Py; (c) ( O3,c) O MeOH/CH2Cl2,3, MeOH/CH2Cl2, −−7878 °C;C; (d ()d NaBH) NaBH4,4, from from –78− °C78 toC rt; to (e rt;) chromatographic(e) chromatographic separation; separation; (f) Ac (f)2 Ac2O,O, Py, Py,DMAP DMAP..

OneOne year year later later (2015) (2015),, three three different groups independently reported reported [158 [158––160160]] the enantioselectiveenantioselective synthesis synthesis of of the the tiacumicin tiacumicin B B aglycon aglycon and and of the putative lipiarmycin aglyconaglycon,, followed followed by by the the first first total total synthesis synthesis of the fidaxomicin fidaxomicin [161]. [161]. The The latter latter synthetic synthetic compound proved identical to a commercial sample of tiacumicin B from Dactylosporangium aurantiacum, thus confirming the structure assigned. Finally, a study on the comparative X-ray analysis of lipiarmycin A3 and tiacumicin B was published in 2017, providing the definitive proof that these pharmaceutical compounds are identical in all respects and confirmed the previous assigned chemical structure of lipiarmycin A3 [162]. Although the debate on the lipiarmycin structural determination is now closed, the studies on the synthesis of this antibiotic have increased, fostered by renewed interest for the preparation of synthetic derivatives. Accordingly, two new syntheses of tiacumicin B Antibiotics 2021, 10, x FOR PEER REVIEW 25 of 33

compound proved identical to a commercial sample of tiacumicin B from Dactylosporan- gium aurantiacum, thus confirming the structure assigned. Finally, a study on the comparative X-ray analysis of lipiarmycin A3 and tiacumicin B was published in 2017, providing the definitive proof that these pharmaceutical compounds are identical in all respects and confirmed the previous assigned chemical structure of lipiarmycin A3 [162]. Although the debate on the lipiarmycin structural determination is now closed, the studies on the synthesis of this antibiotic have increased, fostered by renewed interest for the preparation of synthetic derivatives. Accordingly, two new syntheses of tiacumicin B aglycone [163,164], the total synthesis of tiacumicin A [165], the total synthesis of tiacumicin B [166], and two reviews on tiacumicin B [144,145] were reported between 2017 and Antibiotics 2021, 10, 483 24 of 32 2020. Although the synthetic strategies are very similar each other, the order of assembling the fragments change between the different approaches (Figure 24). For example, the final ringaglycone closure [163 of,164 the], thelactonic total synthesisring was ofbuilt tiacumicin-up either A [165applying], the total a ring synthesis closing of metathesis tiacumicin betweenB[166], andC(5) two and reviews C(6) or on through tiacumicin Yamaguchi B [144,145 macrolactonization.] were reported between The Suzuki, 2017 andStille, 2020. or the alleneAlthough–alkyne the coupling synthetic were strategies employed are very for similarthe formation each other, of the the carbon order- ofcarbon assembling single bondthe fragments between changeC(4) and between C(5) and the between different C(14) approaches and C(15). (Figure In addition, 24). For the example, carbon– thecarbon ﬁnal singlering closure bond between of the lactonic C(11) and ring C(12) was built-upwas formed either through applying aldol a ringbased closing reactions metathesis or [2,3] thebetween Wittig C(5) rearrangement and C(6) or, throughwhereas Yamaguchi the carbon– macrolactonization.carbon double bond The between Suzuki, C(15) Stille, and or C(16)the allene–alkyne was obtained coupling by Horner were–Wadsworth employed– forEmmons the formation reaction. of the carbon-carbon single bondOver betweenall, none C(4) of and the C(5) reported and between synthetic C(14) approaches and C(15). we Inre addition,designed thein order carbon–carbon to secure ansingle industrial bond between process C(11)for lipiarmycin and C(12) waspreparation. formed through Nevertheless aldol, based they represent reactions ora relevant [2,3] the advanceWittig rearrangement, in the study of whereas this antibiotic, the carbon–carbon providing new double chemical bond insights between for C(15) the anddesign C(16) of newwas obtainedsemisynthetic by Horner–Wadsworth–Emmons derivatives [167,168]. reaction.

Figure 24. The key synthetic steps employed in the synthesis of lipiarmycins. The The reaction reaction used used to build the indicated carboncarbon–carbon–carbon single bonds, carbon carbon–carbon–carbon double bonds, macrolactone bond bond,, and glycosidic bonds are are highlighted highlighted with blue, red, pink pink,, and green color, respectively.

5. ActinomycetesOverall, none Are of thestill reported Considered synthetic a Source approaches of Bioactive were Compounds designed in order to secure an industrialIt seems that process regarding for lipiarmycin antibiotics preparation. all has been Nevertheless, already discovered they represent. Despite athis relevant com- monadvance perception, in the study Actinomycetes of this antibiotic, remain providing the most newimportant chemical source insights of genetic for the variability design of innew different semisynthetic ecosystems. derivatives Molecular [167 biology,168]. and genetics give us new equipment that permit better sensitivity and accuracy in finding new specific microbial molecular targets and 5. Actinomycetes Are still Considered a Source of Bioactive Compounds bioactive compounds. Screening techniques become more sophisticated and precise and are basedIt seems essentially that regarding on (i) the antibiotics use of antisense all has RNA; been already(ii) new discovered.adjuvant-molecules; Despite this(iii) newcommon natural perception, antibioticActinomycetes-resistant bactremaineria; (iv) the new most cultivation important methods; source of and genetic (v) activation variability ofin silent different operons ecosystems. responding Molecular to the biology secondary and metabolism genetics give [169]. us new equipment that permit betterThe sensitivity antisense and RNA accuracy is a particular in finding small new single specific-stranded microbial RNA able molecular to pair targetswith a spe- and cificbioactive mRNA compounds. of the cell, causing Screening itstechniques degradation become and, consequently, more sophisticated the missed and precisetranslation and ofare the based cell essentiallygenetic heritage. on (i) the Thanks use of to antisense this knowledge, RNA; (ii) we new are adjuvant-molecules; now able to build (iii)specific new natural antibiotic-resistant bacteria; (iv) new cultivation methods; and (v) activation of silent operons responding to the secondary metabolism [169]. The antisense RNA is a particular small single-stranded RNA able to pair with a specific mRNA of the cell, causing its degradation and, consequently, the missed translation of the cell genetic heritage. Thanks to this knowledge, we are now able to build specific vectors carrying the information to synthetize these small antisense RNAs inside the cell in order to impair the expression of a specific target gene. This technique permits us to choose new possible target(s) for the therapy, observing altered bacterial phenotypes in terms of morphological changes, impaired growth, and toxicity. Moreover, we are able to validate the mechanism of action of a molecule against the presumed target, enhancing our sensibility in screening protocols [170]. It is possible to try to restore the sensitivity to a drug using an adjuvant molecule. This goal could be reached creating a screening system, seeding a specific antibiotic resistant strain in a plate containing the same antibiotic Antibiotics 2021, 10, 483 25 of 32

at which it is resistant. At this point, a molecule or an extract of a fermentative broth could be added, evaluating the restored antibiotic toxicity. Finally, it is possible to study the target and understand the mechanism of action. Natural antibiotic-resistant bacteria help our new drug research because we start from the premise that natural resistant bacteria could be produce the same antibiotic at which it is resistant (first filter). A second cut-off, based on the known antibiotic open reading frames (ORFs), is then applied, enriching our microbial library in probable new drug producers [171]. Our cultivation procedures are not suitable to permit the growth of a big portion of the prokaryotic world [172]. Enhancing cultivation technologies and comprehending the molecular biology of bacteria could give access to a major number of possibilities to study the biodiversity and the enzymatic tools in the environment [173,174]. This is possible, for example, using strong selective factors, i.e., higher salinity, different temperatures, alkaline or acid growth media (collections of novel or poorly represented strains). Moreover, it is possible to recreate the microenvironment in an in vitro scale, to understand the “cross-talk molecules” implied in the relationship among the bacterial populations. Another relevant aspect concerns the syntheses of new semisynthetic antibiotics. Actinomycetes are producers of bioactive compounds belonging to each one of the most representative classes of antibiotics. These APIs are currently produced through well- established, high volume fermentation processes. Therefore, the pharmaceutical industry can take advantage of these useful building blocks for the creation of new APIs. The development of antibiotic-resistant bacteria leads to the need for new antibiotics and the chemical modification of the known ones is still the most successful approach adopted to this end. To date, the main part of the antibiotics used in clinical practice are semisynthetic derivatives of compounds obtained through Actinomycetes strain fermentation. Conse- quently, understanding the mechanisms of antibiotic resistance can also help design the chemical modification of a given API in order to obtain a new compound with higher biological activity. On the other hand, chemical research will provide the synthetic and technological tools for the industrial production of new antibiotics.

6. Conclusions Bacteria evolutionary success depends on their capability to adapt themselves quickly to an environmental stress, thanks to the fine-tuning of their mutational rates [175]. The mutation could establish (or not) in the population, based on its fitness advantage and, of course, a general evolutionary advantage. Obviously, the mutational rate depends on the growth speed of the population: the faster they replicate, the more mutational events could occur. Moreover, the role of the toxin–antitoxin system in bacterial persistence was under- lined. The activation of these loci permits bacteria to arrest their growth under different stress pressures, such as starvation, non-optimal pH, temperature, high population density, or antibiotic presence in the environment [176,177]. Whatever the mechanism is, the result is the temporary inhibition of the essential cellular processes, such as DNA replication, transcription, and translation. The most important antibiotics perform their actions against active and proliferating cells and they have no effect on these quiescent cells. It seems clear that their capability to adapt to a stress is a big issue to overcome. The discovery of new useful, clinical drugs, is slow, for sure, and particular attention must be kept using antibiotics. Researches of new microbial targets and new high-performance drugs must go forward. Actinomycetes, with their extraordinary metabolisms and their enzymatic and genomic potential, could help us in this aim, providing bioactive compounds never observed or unique structural moieties that chemists could modify in order to obtain other useful compounds. Moreover, the study of their biology and metabolism could assist not only medicine, but also agriculture, cosmetics, laundry, pharmaceutics, and bioremediation [178]. It also important that different scientific fields share their experience, knowledge, technologies, and expertise to improve health and wellness. Antibiotics 2021, 10, 483 26 of 32

“Somewhere something incredible is waiting to be known” (Carl Sagan, astronomer).

Author Contributions: The authors contributed equally to this work. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Data Availability Statement: No new data were created or analyzed in this study. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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