Pharmaceutical Fermentation: Antibiotic Production and Processing

The widespread introduction of antibiotics into medical practice, starting in the late 1940s and early 1950s, radically transformed healthcare, raised it to a qualitatively new level, allowed saving human lives in previously hopeless situations, and became one of the most important factors leading to an increase in the life expectancy of the population of Earth by more than 20 years [1,2]. However, the widespread use of antibiotics has led to the emergence of strains of microorganisms resistant to their effects, which significantly reduces the effectiveness of treatment and leads to the deaths of millions of people [3,4]. In this regard, the search for and development of novel, effective antimicrobial drugs acting on alternative targets seems to be an urgent task [5].

Since the period of widespread use of antibiotics in the mid-20th century and up to the present time, three fundamentally different approaches have been used for the industrial production of these compounds: (i) fermentation of improved strains of microorganisms to produce natural products; (ii) in vitro modification of natural products after fermentation to produce semi-synthetic antibiotics; and (iii) chemical synthesis to produce synthetic antibiotics [6,7,8]. Among all natural products and compounds obtained by living organisms, a lot of secondary metabolites exhibit antimicrobial activity; some of them are used as antibiotics. Therefore, this Special Issue addresses the topic of obtaining secondary metabolite antimicrobial drugs (or starting substances for the creation of their highly active semi-synthetic derivatives) as a result of the fermentation of microorganism strains. Most manuscripts address not only the highly active natural products themselves but also the molecular basis of their biosynthesis as associated with biosynthetic gene clusters (BGC). This Special Issue includes both experimental articles and two reviews.

In the work of T. Antipova et al., the metabolic potential of two strains of the mold fungus Pseudogymnoascus isolated from the surface soil layer of the Kolyma Lowland (Russia, Arctic) was studied [9]. The work initially seems promising because the Arctic region is characterized by extreme living conditions and is currently poorly studied in terms of its microbiome, and microorganisms occupying extreme ecological niches may potentially possess rare or unique secondary metabolic pathways [10]. In a study of secondary metabolites in these strains, the authors discovered, for the first time for this genus of fungi, 16-membered trilactone macrolides, (+)-macrosphelides A and B. After sequencing the genomes and using the antiSMASH tool to predict secondary metabolite gene clusters, the authors found 32 BGCs for strain VKM F-4518 and 17 BGCs for strain VKM F-4519. As a result, the authors were able to identify the BGC for macrosphelides after comparing the known cluster for the biosynthesis of these compounds in Paraphaeosphaeria sporulosa [11]. The work may be continued further since macrosphelides are promising antitumor drugs [12,13], and both strains studied show high production of macrosphelide A.

The article by I. Djinni et al. is also dedicated to working with microorganisms isolated from extreme habitats. In this work, the metabolic potential of actinobacteria from an arid ecosystem was investigated [14]. The authors studied the effect of ethyl acetate extract from Streptomyces rochei CMB47, isolated from coal mine Saharan soil, on test cultures of pathogenic microorganisms, in particular, against methicillin-resistant Staphylococcus aureus (MRSA), included in the World Health Organization’s (WHO) global priority pathogens list of antibiotic-resistant bacteria [15]. The optimization of the cultivation conditions of S. rochei CMB47 allowed the authors to obtain highly active fractions with a minimum inhibitory concentration of <0.439 µg/mL against MRSA. Further research, according to the authors, will be related to establishing the structure of compounds that exhibit high activity against MRSA.

The investigation of A. Kuvarina et al. is devoted to biologically active peptides from the alkaliphilic fungus Emericellopsis alkaline [16] and continues a series of works by this group of authors devoted to this subject. Previously, the authors screened 22 strains of E. alkaline and found among them the strain E. alkalina E101 (VKM F4108; CBS 127350) with the best antifungal activity due to the high production of biologically active peptides, peptaibols [17,18], determined the structure of the first lipopeptide, emericellipsin A (EmiA) [19], and its four homologous emericellipsins (Emi B-E) [20]. In the current work, the authors optimized the fermentation conditions of E. alkaline for efficient production of emericellipsins. In particular, the authors showed that an alkaline pH of 10 is most suitable for fermentation under deep conditions for efficient production of emericellipsins for 14 days. The published work is seen as an important intermediate link in the study of these non-ribosomal peptides, emericellipsins, which exhibit high antifungal activity.

The article by X. Li et al. deals with the biosynthesis of the aminoglycoside antibiotic neomycin B in Streptomyces fradiae SF-2 [21]. Neomycin was isolated from S. fradiae in the late 1940s by Selman Waksman and Hubert Lechevalier. This antibiotic exhibits good activity against Gram-negative bacteria and is partially effective against Gram-positive bacteria. Neomycin has also been shown to have potential for treating tumors [22] and SARS-CoV-2 [23]. In this regard, an urgent task is to accelerate the biosynthesis of this highly active compound. In their previous work, the authors obtained a high-yielding strain of S. fradiae SF-2 with a neomycin sulfate (NM) production level that increased to 10.849 U/mL as a result of six rounds of random mutagenesis in the Atmospheric and Room Temperature Plasma (ARTP) variant [24]. In the current article, the authors continued working with S. fradiae SF-2, carried out a comparative transcriptomic analysis, and showed that the addition of 60 mM (NH₄)₂SO₄ upregulates genes from the BGC of neomycin (neo genes) in S. fradiae SF-2. In addition, based on S. fradiae SF-2, the authors created a series of recombinant strains with overexpressed neo genes. It turned out that for the most effective genetically engineered variant, SF-NeoE, when cultivated with the addition of 60 mM (NH₄)₂SO₄, the production level of NM increased by 51.2%, to 17,399 U/mL. In conclusion, the authors note that the determination of the molecular mechanisms regulating neomycin biosynthesis, in particular the role of (NH4)2SO4, is beneficial for increasing neomycin production and applications.

In the work of H. Zhang et al., the strain Actinomadura sp. ATCC 39365 was improved to produce pentostatin [25]. Pentostatin is an important anticancer drug used to treat hairy cell leukemia and is also used in the treatment of steroid-refractory acute graft-versus-host disease, acute T cell leukemia, and chronic lymphocytic leukemia [26,27,28]. Pentastatin can be synthesized by some types of actinomycetes and filamentous fungi, but the production level in wild-type (WT) strains is insufficient for industrial purposes. For industrial production, improved high-yielding (HY) producers are used, but the level of pentostatin production in such strains is also low. In the current work, random mutagenesis using ARTP technology was performed to improve Actinomadura sp. ATCC 39365. The most active mutant, Actinomadura sp. S-15, showed an increase in pentostatin production by more than 30%, to 86 mg/L. At the same time, the upregulation of the BGC genes of pentostatin was observed; the improved producer consistently showed an increased yield of the target secondary metabolite over the observed period of time of six generations. In this regard, the authors optimized the fermentation conditions, which allowed increasing the yield of pentastatin to 152 mg/L. In this study, fermentation was performed on a small scale in a shaker. In the future, the authors plan to scale up the fermentation to obtain larger yields of pentastatin. Although at this stage, it might also be worthwhile to carry out a few more rounds of random mutagenesis, which is performed to obtain high-yield industrial producers of secondary metabolites [29]. In any case, the study presented by the team of scientists H. Zhang et al. looks like a holistic and important experiment that opens up the possibility of further work on increasing pentastatin production in Actinomadura sp. S-15.

In addition to writing the experimental article, H. Zhang et al. (with some modifications by the co-authors) contributed to this Special Issue with a review on the topic of the elucidation of pentostatin biosynthesis and its application [30]. Pentostatin is an important drug with a wide spectrum of biological and pharmacological properties, such as antibacterial, antitrypanosomal, anticancer, antiviral, herbicidal, insecticidal, and immunomodulatory effects. The review describes the history of the discovery of this nucleoside antibiotic, which has adenosine deaminase inhibitory activity [31]. The microorganisms in which the pentastatin biosynthesis pathway has been discovered, the mechanism of action of this compound, and the BGC of pentastatin are described in detail. The current knowledge on the biosynthesis of pentastatin in Streptomyces antibioticus NRRL 3238, Actinomadura sp. ATCC 39365, and Cordyceps militaris is presented in detail. A separate chapter is devoted to the use of pentastatin in the treatment of various diseases such as hairy cell leukemia [32], chronic lymphoblastic leukemia [33,34], Waldenstrom′s macroglobulinemia [35], and trypanosome inhibition [36,37]. In conclusion, the authors note that for such an important drug as pentastatin, chemical synthesis has major disadvantages compared to biosynthesis. In this regard, it is necessary to create new high-yielding producers, among other things, using metabolic engineering technologies and based on emerging knowledge about the functioning of pentastatin biosynthesis.

My own review contains information related to the use of fungal strains for the industrial production of antibiotics [38]. Improved strains of mold fungi are one of the most important sources for obtaining antibiotics, since the compounds obtained as a result of their fermentation, according to various sources, occupy approximately half of the modern antibiotic market [39,40,41,42]. The most important fungal antibiotics are beta-lactams, such as penicillins, obtained from the fermentation products of Penicillium chrysogenum, and cephalosporins, obtained from the fermentation products of Acremonium chrysogenum [43,44,45]. The review also examines antibiotics from fungi of other classes, such as fusidanes, griseofulvin, pleuromutilins, echinocandins, enfumafungins, and enniatins, and their current status and prospects for use are considered. Highly active fungal compounds in clinical trials stage I–III are also described; their targets and mechanisms of action are discussed. A separate chapter is devoted to methods for obtaining highly active fungal strains for the industrial production of antibiotics, and classical and genetically engineered approaches are compared. The creation of improved producers is necessary because natural isolates produce an insufficient amount of the target secondary metabolites for industrial production, up to several tens of mg per liter of culture fluid. Industrial strains produce 100–1000 times more antibiotics. For example, for P. chrysogenum, the production was 87,650 mg per liter of penicillin G [46], and for A. chrysogenum, the production of 35,770 cephalosporin C was obtained [47]. The review collects numerous data on the improvement of fungal strains for the production of secondary metabolites and antibiotics, in particular; it is shown that, starting in the 1950s and up to the present, the most effective are classical strain improvement (CSI) methods associated with multi-round random mutagenesis and screening. And there is no magic bullet, in the terminology of Paul Ehrlich, that would allow, as a result of some direct genetic engineering event, to transform a wild-type (WT) strain into a high-yielding (HY) producer of a secondary metabolite. In order to understand which molecular events are selected after random mutagenesis to create an HY strain from a low-activity natural isolate, the final chapter of the review analyzes the available multi-omics data comparing the genomes, transcriptomes, and proteomes of WT strains with their HY progeny strains. As a result, a series of universal events is identified that are characteristic of the currently studied fungal producers of secondary metabolites improved by classical methods. This set of events includes: (i) upregulation of the target biosynthetic gene cluster (BGC), which may or may not be accompanied by an increase in gene dosage (during duplication); (ii) changes at the level of global regulation of secondary metabolism, including mutations in regulators such as LaeA or velvet complex proteins; (iii) disruption in alternative BGCs; (iv) redistribution of cellular energy flows for the needs of the target secondary metabolism; (v) changes in response to stress on the background of high-yield production; and (vi) redistribution of primary metabolism flows for the efficient production of precursors of the target metabolism. In conclusion, the prospects for the further use of fungi as biofactories for the production of antibiotics and sources for obtaining new drugs are considered.

This Special Issue, devoted to pharmaceutical fermentation in the context of “antibiotic production and processing”, brings together both experimental articles and reviews devoted to highly active secondary metabolites of microorganisms. The ultimate practical goal of such research is to create an industrial strain for the production of a drug that is in demand on the market. From this point of view, these works are at various stages of implementation. The investigation of I. Djinni et al. seems to be at the initial stage, where the presence of highly active (<0.439 µg/mL against MRSA) metabolites was shown for Streptomyces rochei CMB47, and what these compounds are remains to be studied [14]. In the work of T. Antipova et al., the mold fungus Pseudogymnoascus was first found to produce macrosphelides, promising antitumor drugs; these strains can potentially be improved to obtain these compounds [9]. The work by Kuvarina et al. is the next logical step, as the authors previously discovered biologically active peptides from the alkaliphilic fungus Emericellopsis alkaline and determined their structure and in the current work, they optimized the fermentation process [16]. In the work of H. Zhang et al., the collection strain Actinomadura sp. ATCC 39365 was improved by the classical method, and the fermentation process was optimized [25]. In the article by X. Li et al., the authors introduced an effective genetic engineering modification into the previously improved producer Streptomyces fradiae SF-2 and optimized fermentation, which led to an increase in NM production by more than 50% [21]. The described experimental works demonstrate the diversity of modern tools for obtaining improved producers of secondary metabolites in microorganisms, starting with the determination of BGC in silico and ending with the combination of classical and genetic engineering approaches. All experimental work presented in this Special Issue fills gaps in knowledge about the secondary metabolism of the studied microorganisms and provides prerequisites for future research.

The review by H. Zhang et al. summarizes the knowledge on pentostatin biosynthesis in microorganisms [30]. One of the most significant achievements in this field is the determination of pentostatin BGC for several actinomycetes and fungi, which is described in detail in the review. The authors emphasize the need to combine classical strain improvement methods with metabolic engineering to create highly efficient pentostatin producers. However, in order to effectively use metabolic engineering, it is necessary to know the fundamental molecular events that occur when creating an HY strain from a natural low-activity isolate. Another review in this Special Issue attempts to classify such changes [38]. It is also stated that some side mutations that arise in programs for improving various fungal producers of secondary metabolites may also be of a universal nature. For example, in phylogenetically distant representatives of the classes Sordariomycetes (A. chrysogenum) and Eurotiomycetes (Aspergillus terreus), as a result of CSI programs, the content of intracellular polyamines increased, and resistance to inhibitors of the key enzyme of polyamine biosynthesis, ornithine decarboxylase (EC 4.1.1.17, ODC), appeared [48,49]. Such knowledge may be useful since exogenous administration of polyamines results in increased production of target secondary metabolites in the highly active strains studied [50,51,52].

I thank all the authors for participating in this Special Issue; I consider the published articles interesting and useful for readers.