The Biotechnological Potential of Secondary Metabolites from Marine Bacteria

Marine habitats are a rich source of molecules of biological interest. In particular, marine bacteria attract attention with their ability to synthesize structurally diverse classes of bioactive secondary metabolites with high biotechnological potential. The last decades were marked by numerous discoveries of biomolecules of bacterial symbionts, which have long been considered metabolites of marine animals. Many compounds isolated from marine bacteria are unique in their structure and biological activity. Their study has made a significant contribution to the discovery and production of new natural antimicrobial agents. Identifying the mechanisms and potential of this type of metabolite production in marine bacteria has become one of the noteworthy trends in modern biotechnology. This path has become not only one of the most promising approaches to the development of new antibiotics, but also a potential target for controlling the viability of pathogenic bacteria.


Introduction
Despite the significant advances in medicine, diagnostics, and treatment of infectious diseases, pathogenic microorganism still pose a serious threat to the world's human population.
Their impact is significant both in developing countries, due to the limited access to medicines there, and in developed countries, where uncontrolled administration of antibiotics has led to a wide distribution of multi-resistant bacteria. The strategy of creating new synthetic antibiotics by word combinations contained in the title, abstract, and topical catalogs: "marine bacteria and secondary metabolites", "marine bacteria and antibacterial peptides", and "marine bacteria and nonribosomal biosynthesis". The depth of search was 2007-2019.

Bacterial metabolites
Bacteria live in the environment of transmitted and received chemical signals, with signal molecules being metabolites, i.e. terminal products of cellular metabolism. The latter is a combination of two opposite but interrelated processes: energetic (catabolism) and constructive (anabolism). This is a continuous and multi-component biochemical process that occurs in every bacterial cell throughout its lifecycle [8,9,10].
Terminal products of metabolism, being small peptide molecules, are used as substrates for biochemical reactions or are utilized by microorganisms to support their life processes. This is a wide range of molecules extremely diverse in their structures and functions, with their registered number exceeding 25,000, which accounts for less than 2% of the total number of natural metabolites of microorganisms not yet available for research [9,10,11].
Depending on the functional properties and biosynthesis mechanisms, metabolites are divided into primary and secondary. Primary metabolites serve as the main energy source for providing various biochemical reactions and performing physiological functions to support life processes of bacterial cells such as growth and development. Secondary metabolites are organic compounds with a complex chemical structure and a variety of physiological functions. They are required to implement the survival strategies of bacteria in adverse conditions, acting as mediators with the external environment and means of intercellular communication (Table 1). Table 1 Key biochemical and physiological properties of primary and secondary metabolites of bacteria [8]  Since this review is focused on the bioactive properties of secondary metabolites of marine bacteria, the main emphasis will be on these complex molecules. As a rule, each bacterial species produces several antibiotics, the profile of which depends on the genus of microorganism. For instance, more than 5,000 antibiotics referred to secondary metabolites have been identified to date from the genus Actinobacteria [8], including traditional antibiotics discovered in the 1950s-1960s, as well as new antibiotics. According to forecasts, these bacteria may produce up to 150,000 different chemical antimicrobial agents [8,9]. Modern science considers secondary metabolites as a group of low-molecular-weight, structurally diverse, and complex bioactive compounds. It has been found that the active stage of synthesis of these molecules in microorganisms occurs at the end of the exponential and the beginning of the stationary phases of their growth ( Fig. 1-A). Their production is induced by depletion of nutrients and adverse habitat conditions; the genes responsible for the biosynthesis of secondary metabolites are grouped together in a small number of clusters [10,12].

Secondary metabolites of bacteria
Unlike primary metabolites, the biosynthetic pathways utilized to produce these molecules are numerous and have not been fully understood [8,10]. For biosynthesis, bacteria use multi-stage biosynthesis pathways, which involve specific enzymes or multi-enzyme complexes, being intermediate or end products of intracellular metabolism. Biosynthesis includes cascade regulations, the mechanisms of which have been studied at the transcription level [12].
Among the key pathways of biosynthesis of secondary metabolites with antibacterial activities, the best characterized are nonribosomal (with peptide synthetase as the key enzyme), β-lactam, polyketide (types I-III, with polyketide synthase as the key enzyme), ribosomalpolyketide, oligosaccharide, and shikimate pathways ( Fig. 1-B).
The significantly increased interest in obtaining new antibiotic agents derived from secondary metabolites of marine bacteria is associated with the advances in biotechnology that have been made in recent decades [12,13]. They are based on the revealed mechanism of synthesis of major microbial metabolite classes by means of polyketide synthase [14,15], nonribosomal peptide synthetase [16][17][18], which are biosynthetic pathways extensively utilized by marine microorganisms for producing antimicrobial substances.

Antimicrobial substances of marine microorganisms
Microorganisms from terrestrial ecosystems and their metabolites have always been a source of many biologically active compounds applied in medicine, pharmaceutical industry, and agriculture. After The history of study of secondary metabolites from marine bacteria is an example of the joint efforts and achievements of microbiologists, chemists, biochemists, molecular biologists, and geneticists. The discovery of the phenomenon of unusual peptides synthesized in microorganisms independently of ribosomes and RNA was followed by a long series of findings and evidence of extremely diverse natural bacterial metabolites exhibiting antibiotic and antitumor activities ( Table 2).

Secondary metabolites of marine bacteria are products nonribosomal synthesis
During their life cycle, marine microorganisms actively synthesize secondary metabolites which are low-molecular-weight peptides. They represent specific protein fragments that, in addition to being sources of nitrogen and amino acids, perform numerous biological functions [5,7,11,53]. These substances were derived from algae, marine bacteria, and fungi. The anti-infection activity of marine peptides has been shown to depend on their structural properties, amino acid composition and sequence, as well as on the habitat conditions for producer bacteria [7,53,54] ( Table 2).
The major part of marine bacteria are exposed to extreme conditions of high pressure, salinity, low temperature, and lack of sunlight. These factors caused them to develop the unique properties and the ability to biosynthesize substances with unusual characteristics, different from their terrestrial counterparts. To date, biological properties of only a small number of these peptides have been studied, but the proportion of described substances increases each year, attracting increasingly more attention of researchers [11,[54][55][56].
Most marine bacteria and other microorganisms use numerous gene clusters for metabolite biosynthesis [7,56]. Studies of genome sequences have shown that a significant part of them is responsible for the biosynthesis of secondary metabolites. For example, among marine microorganisms, isolates of the genus Bacillus are referred to as phylogenetically heterogeneous groups of marine bacteria. They need much nutrients and space, and, in order to compete with other bacteria, they synthesize a significant amount of secondary metabolites with pronounced antimicrobial activity, encoded by genes constituting up to 8% of the genome [54,57].
To date, dozens of metabolites, which are peptides consisting of 20-40 amino acids and used for inter-and intraspecific competition, have been isolated from various marine microorganisms [5,7,54]. In recent years, much of the attention of researchers has been focused on secondary metabolites of marine bacteria, which are products of nonribosomal biosynthesis considered a new class of natural antimicrobial agents that can potentially become an alternative to traditional antibiotics [54,56,59,60].
The capability of nonribosomal peptide synthesis is widely distributed among bacteria.
In the second half of the 20th century, this biosynthesis mechanism was simulated in a laboratory to obtain antimicrobial peptides produced by marine isolates of Bacillus sp., in which the pharmacologically-induced inhibition of ribosomes or RNA removal did not prevent protein synthesis [64].
It has been established that the ATP-dependent synthesis of nonribosomal peptides (NRP) occurs by means of peptide synthetase, an enzyme complex independent of messenger RNA, transmitting the genetic information from DNA to ribosomes, where the amino acid sequence of protein products of gene expression is determined [61,63,65]. mechanisms and the diversity of pharmacological properties of NRPs. To date, several pathways of nonribosomal synthesis of peptides have been characterized from both terrestrial (human commensals and pathogens) and marine species of bacteria, which are enough comprehensively considered in recent reviews [59,60,64].
It should be noted that, from the evolutionary aspect, the understanding of nonribosomal biosynthesis mechanisms has evolved from the erroneous view of peptide synthetase as a precursor of ribosomes, as well as from the discovery of the "thiotemplate" mechanism [62,65] and its revision in connection with the advent of the modern modular-domain "multiple carrier model" [58,63,64]. In the present review, we consider only some of the antimicrobial peptide substances that are products of this biosynthesis pathway, being components of secondary metabolites of marine bacteria.

Cyclic lipopeptides (cLPs)
Cyclic lipopeptides (cLPs) are common metabolites synthesized by various bacterial genera and are of interest as substances having various biological activities (Fig. 2). Lipopeptides of marine bacteria consist of a short cyclic oligopeptide (backbone) bound to fatty acids (tail) and show strong antibacterial activity against common human, animal, and plant pathogens, due to which these metabolites have attracted attention as potential natural antibiotic agents (Table 2).
Tauromamid is a relatively new nonribosomally biosynthesized antibiotic [30,31] (Table   2), belonging to the group of cyclic lipopeptides (like daptomycin, the first permitted antibiotic of this class). It is produced by the marine bacterial isolate of Brevibacillus lateosporus PNG276, inhabiting the Gulf of Papua [31]. Tauromamid has a strong and selective inhibitory action on the Gram-positive pathogen Enterococcus sp., as well as exhibits unexpressed activity against the methicillin-resistant Staphylococcus aureus (MRSA; MIC = 200 µg/mL) and Candida albicans (MIC = 50 µg/mL) [30,31].
Halobacillin and methylhalobacillin are two cyclic lipopeptides isolated from bacteria inhabiting deep-sea sediment in the Gulf of California, Mexico [32]. Halobacillin is also one of the most effective known biosurfactants [4]. This antibiotic inhibits the growth of human colon tumor cells (HCT-116) at IC50 0.98 µg/mL and exhibits analogous, but lower than surfactin (a known surfactant antibiotic isolated from terrestrial strains of Bacillus subtilis), antimicrobial activity against S. aureus, Proteus vulgaris, and Enterococcus faecalis [4,32] ( Table 2).
The wide distribution of cLPs among secondary metabolites of marine bacteria is evidenced by the fact that they make up the major part of products of marine isolates from Bacillus sp., one of the most common inhabitants of the World Ocean [3].

Polyketides / lipoamides
Polyketides are extremely large classes of secondary metabolites that contain acylcoenzyme A and constitute the basis of many pharmaceutical, agrochemical, and veterinary drugs. The biosynthesis of these metabolites occurs with the involvement of multimodular megasynthases known as polyketide synthases [14,15].

Mechanisms of antimicrobial action of antibacterial peptides
In recent years, a large group of secondary metabolites of different types, structures, and mechanisms of antibacterial action has been isolated from a number of other marine bacteria [3-5, 7, 8]. Unlike those from terrestrial ecosystems, producers of antimicrobial peptides were mainly strains of Gram-positive marine bacteria [7, 9,  Most of the isolated antimicrobial metabolites are capable of rapidly killing a wide range of microbes. Large-sized antimicrobial proteins (>100 amino acids) are often lytic proteins binding nutrients [68,71] or destroying specific cell patterns [71][72][73], causing DNA degradation [73,74], and inhibiting intracellular synthesis of peptidoglycan [75][76][77] and specific proteins by disrupting the structure or function of microbial cell membranes [7,54,78,79] (Fig. 3). In recent years, two accessible databases have been created for storing and querying information on almost two hundred antibacterial peptides: BACTIBASE [80] and BAGEL [81,82]. Moreover, members of this metabolite class are mentioned in other relevant databases, such as APD3 [83], ANTIMIC [84], CyBase [85], or StraPep [86], the use of which in the mode suggests the antibacterial activity of the substances obtained.
Despite the isolation and study of secondary metabolites from marine bacteria is currently at the initial stage, the obtained results show them as the most promising agents to control infectious diseases of fish. This is especially important in the light of the current trend of increasing proportion of marine aquaculture in the global seafood production and its increasing role in world's fisheries [87][88][89].

Conclusions and prospects to the future
Marine bacteria are an extremely rich source of structurally diverse classes of proteinbased secondary metabolites. In recent years, significant progress has been made in our understanding of the complex mechanisms of their nonribosomal biosynthesis. These natural metabolic byproducts of marine bacteria have a wide range of antimicrobial activities, low rate of elimination from the organism, high specificity to cell targets, and reduced risk of undesirable side effects. Due to these properties, they are already considered as a source of effective biologically active therapeutic agents that can become an alternative to traditional antimicrobial drugs.
Modern biotechnologies of invention and development of novel antibiotics having a medical value are based on application of natural strategies of nonribosomal peptide synthesis [4,9,10]. Revealing the mechanisms and potential of this type of metabolite production in terrestrial and marine bacteria is not only of fundamental, but also of great practical importance.
In human pathogens, this biosynthetic pathway probably causes the emergence of microbial isolates with multidrug resistance. Under these conditions, the activity of peptide synthetase and clusters of the genes responsible for nonribosomal synthesis become a new target for the strategy of treatment of infections caused by drug-resistant forms of bacteria [3,5,91,92].
As regards marine bacteria and their secondary metabolites, the targeted control of biosynthesis mechanisms by using the metabolic engineering methods to create hybrid peptides or obtain hybrid peptide synthetases by disrupting the target gene of nonribosomal synthesis is now one of the noteworthy trends in modern biotechnology. This pathway becomes not only one of the most promising approaches to the development of novel antibiotics, but also a potential target for controlling the exocrine activity of pathogenic bacteria and, consequently, their viability [5,[93][94][95].
The range of active antimicrobial agents derived from marine bacteria, discovered and tested in recent years, indicate a high pharmacological potential of their secondary metabolites, and the study of these peptides is expected to be an interesting and fruitful activity in the coming years.

Conflict of interests.
The authors declare that they have no conflict of interest.
Funding. The study was supported by the Far Eastern Branch, Russian Academy of Sciences, within the framework of the "Far East" Integrated Program for Basic Research, project no. 18-5-099.