Novel bioactive natural products from bacteria via bioprospecting,genome mining and metabolic engineering

For over seven decades, bacteria served as a valuable source of bioactive natural products some of which were eventually developed into drugs to treat infections, cancer and immune system-related diseases. Traditionally, novel compounds produced by bacteria were discovered via conventional bioprospecting based on isolation of potential producers and screening their extracts in a variety of bioassays. Over time, most of the natural products identifiable by this approach were discovered, and the pipeline for new drugs based on bacterially produced metabolites started to run dry. This mini-review highlights recent developments in bacterial bioprospecting for novel compounds that are based on several out-of-the-box approaches, including the following: (i) targeting bacterial species previously unknown to produce any bioactive natural products, (ii) exploring non-traditional environmental niches and methods for isolation of bacteria and (iii) various types of ‘genome mining’ aimed at unravelling genetic potential of bacteria to produce secondary metabolites. All these approaches have already yielded a number of novel bioactive compounds and, if used wisely, will soon revitalize drug discovery pipeline based on bacterial natural products.     

Bioactive substances produced by marine isolates of Pseudomonas

Pseudomonas is a genus of non-fermentative gram-negative Gammaproteobacteria found both on land and in the water. Many terrestrial isolates of this genus have been studied extensively. While many produce bioactive substances, enzymes, and biosurfactants, other Pseudomonas isolates are used for biological control of plant diseases and bioremediation. In contrast, only a few marine isolates of this genus have been described that produce novel bioactive substances. The chemical structures of the bioactive substances from marine Pseudomonas are diverse, including pyroles, pseudopeptide pyrrolidinedione, phloroglucinol, phenazine, benzaldehyde, quinoline, quinolone, phenanthren, phthalate, andrimid, moiramides, zafrin and bushrin. Some of these bioactive compounds are antimicrobial agents, and dibutyl phthalate and di-(2-ethylhexyl) phthalate have been reported to be cathepsin B inhibitors. In addition to being heterogeneous in terms of their structures, the antibacterial substances produced by Pseudomonas also have diverse mechanisms of action: some affect the bacterial cell membrane, causing bacterial cell lysis, whereas others act as acetyl-CoA carboxylase and nitrous oxide synthesis inhibitors. Marine Pseudomonas spp. have been isolated from a wide range of marine environments and are a potential untapped source for medically relevant bioactive substances.     

Antifouling activities expressed by marine surface associated Pseudoalteromonas species

Members of the marine bacterial genus Pseudoalteromonas have been found in association with living surfaces and are suggested to produce bioactive compounds against settlement of algal spores, invertebrate larvae, bacteria and fungi. To determine the extent by which these antifouling activities and the production of bioactive compounds are distributed amongst the members of the genus Pseudoalteromonas, 10 different Pseudoalteromonas species mostly derived from different host organisms were tested in a broad range of biofouling bioassays. These assays included the settlement of larvae of two ubiquitous invertebrates Hydroides elegans and Balanus amphitrite as well as the settlement of spores of the common fouling algae Ulva lactuca and Polysiphonia sp. The growth of bacteria and fungi, which are the initial fouling organisms on marine surfaces, was also assayed in the presence of each of the 10 Pseudoalteromonas species. It was found that most members of this genus produced a variety of bioactive compounds. The broadest range of inhibitory activities was expressed by Pseudoalteromonas tunicata which inhibited all target fouling organisms. Only two species, Pseudoalteromonas haloplanktis and Pseudoalteromonas nigrifaciens, displayed negligible activity in the bioassays. These were also the only two non-pigmented species tested in this study which indicates a correlation between production of bioactive compounds and expression of pigment. Three members, P. tunicata, Pseudoalteromonas citrea and Pseudoalteromonas rubra, were demonstrated to express autoinhibitory activity. It is suggested that most Pseudoalteromonas species are efficient producers of antifouling agents and that the production of inhibitory compounds by surface associated Pseudoalteromonas species may aid the host against colonisation of its surface.     

Exploration of antimicrobial potential in LAB by genomics

A tremendous flow of information has been created through various genome sequencing projects worldwide. So far, 128 bacterial genome sequences have been completed and 391 are under way. Many of these bacteria, including several lactic acid bacteria (LAB), are used in the production and preservation of food and feed. The major antimicrobial and biopreservative substance produced by LAB is organic acid; however, some LAB produce additional antimicrobial compounds. Among these, the bacteriocins have demonstrated great potential as food preservatives. Additionally, antimicrobial compounds different from the bacteriocins have recently been identified, of which several display strong antifungal activity. The information obtained from genomics and related technologies will have great impact on the future identification and development of new antimicrobial agents. Developments will include the identification of pathways for the production of antimicrobials and genome mining for new antimicrobial peptides.     

Antibacterial activity of resin based coatings containing marine microbial extracts

Bioactive agents produced by marine surface‐associated bacteria have been incorporated into experimental antifouling coatings. It was found that coating formulation can affect the action spectra of bacterial extracts involved. Little work has been done on the effect of permeability on the leaching of bioactive compounds from antifouling coatings. The formulations with 1.5% and 1.0% alginate produced significantly larger zones (p < 0.05) than with 0.5% alginate. Test formulations continued to leach active compounds after continuous immersion in seawater for 120 h. The bacterial strains used to produce the active compounds were identified by biochemical tests and/or the analysis of the DNA from a 500 bp section of the gene coding for the 16S rRNA subunit. Strains used were tentatively identified as Planococcus citreus, seven Bacillus strains (including three Bacillus pumilus and three Bacillus lautus strains), Cytophaga fucicola, Cytophaga uliginosa and a Pseudoalteromonas spp.     

植物内生菌及其活性代谢产物

植物内生茵是一种新的微生物资源,具有潜在的应用价值。近年来,从植物内生茵中寻找新的生物活性物质的研究方兴未艾。对近年来从植物内生茵中发现的抗肿瘤、抗茵、抗病毒、杀虫、免疫抑制、抗氧化、降糖等活性化合物及其相应的产生茵的研究作一简要综述。     

Construction of Saccharomyces cerevisiae strain FAV20 useful in detection of immunosuppressants produced by soil actinomycetes

The screening of microbial natural products continues to represent an important route to the discovery of novel chemicals for development of new therapeutic agents. The aim of this work was to develop an efficient method for the detection of immunosuppressive compounds produced by soil actinomycetes. Mutant strain of Saccharomyces cerevisiae, named FAV20, sensitive to FK506 was constructed by disrupting VMA22 gene using the selectable marker kanMX4 which allowed detection of integration events. Actinomycetes were isolated from different soil samples and in a newly developed test with S. cerevisiae FAV20, six strains have been identified that produce bioactive compounds with the same mechanism of action as FK506. S. cerevisiae FAV20 can be easily used as a test strain in drug screening programs based on inhibition of the calcineurin phosphatase dependent signaling pathway in the cell.     

天然活性先导化合物生物转化研究进展

天然活性先导化合物生物转化是利用生物催化剂(如：酶、微生物、动植物细胞)将加入到生物反应系统中的天然活性先导化合物进行特异性的分子结构修饰以获得高效、低毒新化合物的方法。该方法可以有效地提高已知的天然活性先导化合物的活性、降低毒副作用、改善水溶性和生物利用度,也可以用来生产具有重要应用价值的微量天然活性先导化合物,同时可用于药物代谢机制的研究。国内外学者已经针对甾体、醌类、黄酮类、萜类等化合物开展了天然活性先导化合物生物转化研究,筛选出一批有重要应用价值的生物转化反应类型,但针对天然活性先导化合物生物转化的机制、生物转化过程工程以及生物转化产物活性等方面的研究较少。将现代生命科学技术(如：生物催化剂的定向改造、高通量筛选、组合生物转化、非水相生物转化)引入天然活性先导化合物生物转化研究中,必将推进天然活性先导化合物的快速发展。     

Heterogeneity in Bacterial Specialized Metabolism

Specialized metabolites (SMs) like typical antibiotics, signaling molecules or other bioactive compounds of bacterial origin (sometimes also used in human therapy) are often complex natural products that are costly for the cell to make. Several bacterial taxa are known to produce multiple SM classes in parallel and therefore a division of labor within a clonal population of bacteria might be beneficial. In this review, examples of SM of gram-negative and gram-positive bacterial taxa that are produced by different cell types are presented, and the possibility that such a heterogeneity is more widespread in SM biosynthesis is discussed. In addition, tools to study SM production at the single cell level are presented.     

Quorum sensing inhibitors: An overview

Excessive and indiscriminate use of antibiotics to treat bacterial infections has lead to the emergence of multiple drug resistant strains. Most infectious diseases are caused by bacteria which proliferate within quorum sensing (QS) mediated biofilms. Efforts to disrupt biofilms have enabled the identification of bioactive molecules produced by prokaryotes and eukaryotes. These molecules act primarily by quenching the QS system. The phenomenon is also termed as quorum quenching (QQ). In addition, synthetic compounds have also been found to be effective in QQ. This review focuses primarily on natural and synthetic quorum sensing inhibitors (QSIs) with the potential for treating bacterial infections. It has been opined that the most versatile prokaryotes to produce QSI are likely to be those, which are generally regarded as safe. Among the eukaryotes, certain legumes and traditional medicinal plants are likely to act as QSIs. Such findings are likely to lead to efficient treatments with much lower doses of drugs especially antibiotics than required at present.     

Gene clusters for beta-lactam antibiotics and control of their expression: why have clusters evolved, and from where did they originate?

While beta-lactam compounds were discovered in filamentous fungi, actinomycetes and gram-negative bacteria are also known to produce different types of beta-lactams. All beta-lactam compounds contain a four-membered beta-lactam ring. The structure of their second ring allows these compounds to be classified into penicillins, cephalosporins, clavams, carbapenens or monobactams. Most beta-lactams inhibits bacterial cell wall biosynthesis but others behave as beta-lactamase inhibitors (e.g., clavulanic acid) and even as antifungal agents (e.g., some clavams). Due to the nature of the second ring in beta-lactam molecules, the precursors and biosynthetic pathways of clavams, carbapenems and monobactams differ from those of penicillins and cephalosporins. These last two groups, including cephamycins and cephabacins, are formed from three precursor amino acids that are linked into the alpha-aminoadipyl-L-cysteinyl-D-valine tripeptide. The first two steps of their biosynthetic pathways are common. The intermediates of these pathways, the characteristics of the enzymes involved, the lack of introns in the genes and bioinformatic analysis suggest that all of them should have evolved from an ancestral gene cluster of bacterial origin, which was surely transferred horizontally in the soil from producer to non-producer microorganisms. The receptor strains acquired fragments of the original bacterial cluster and occasionally inserted new genes into the clusters, which once modified, acquired new functions and gave rise to the final compounds that we know. When the order of genes in the Streptomyces genome is analyzed, the antibiotic gene clusters are highlighted as gene islands in the genome. Nonetheless, the assemblage of the ancestral beta-lactam gene cluster remains a matter of speculation.     

Marine actinomycetes: An ongoing source of novel bioactive metabolites

Actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications and hold a prominent position due to their diversity and proven ability to produce novel bioactive compounds. There are more than 22,000 known microbial secondary metabolites, 70% of which are produced by actinomycetes, 20% from fungi, 7% from Bacillus spp. and 1–2% by other bacteria. Among the actinomycetes, streptomycetes group are considered economically important because out of the approximately more than 10,000 known antibiotics, 50–55% are produced by this genus. The ecological role of actinomycetes in the marine ecosystem is largely neglected and various assumptions meant there was little incentive to isolate marine strains for search and discovery of new drugs. The search for and discovery of rare and new actinomycetes is of significant interest to drug discovery due to a growing need for the development of new and potent therapeutic agents. Modern molecular technologies are adding strength to the target-directed search for detection and isolation of bioactive actinomycetes, and continued development of improved cultivation methods and molecular technologies for accessing the marine environment promises to provide access to this significant new source of chemical diversity with novel/rare actinomycetes including new species of previously reported actinomycetes.     

Impact of incubation period on biodegradation of petroleum hydrocarbons from refinery wastewater in Kuantan,Malaysia by indigenous bacteria

This work studied the biodegradation of petroleum hydrocarbons (PHCs) extracted from refinery wastewater to produce industrially important by-products at different incubation periods. Two out of 13 bacterial isolates, KRD2 and KRA4 were isolated. Dichloromethane was used to extract the PHC, and gas chromatography-mass spectrometry (GC-MS) analysis revealed that the refinery wastewater PHC was successfully biodegraded using the selected bacterial isolates within 15 days of incubation. Both KRD2 and KRA4 isolates degraded all 13 initially extracted PHC compounds within 5 days, except C13BD and C9BD, which produced 6 and 4 compounds as secondary metabolites with peak area percentages of 1.58, 1.38, 0.85, 29.94, 7.59, and 11.16% and 3.55, 2.88, 52.31, and 6.14%, respectively. These metabolites have been reported in industrial and medical applications. After 10 days, only 6 and 8 compounds were degraded by both isolates, respectively, and C11PAD compound was produced, as well as C5PAD, C7PAD, and C13PAD. After 15 days, it was clear that all the initial PHC compounds have been completely degraded by both isolates. Metabolites C5PAD, C6PAD, C8PAD, and C13PAD were produced by KRD2, and metabolites C5PAD, C6PAD, C8PAD, and C9PAD were produced by KRA4 at different peak areas. The alignment revealed that the KRA4 isolate was included in the genus Chryseobacterium gambrini, while KRD2 isolate was successfully identified as Mycobacterium confluentis using the Biolog microbial identification system. The incubation period evidently affected biodegradation process by indigenous degraders. These effective bacteria were shown to be of great potential for further application in biodegradation technology of PHC contaminated refinery wastewater to produce industrially important by-products.     

Identification of novel inhibitors of fibroblast growth factor (FGF-2) binding to heparin and endothelial cell survival from a structurally diverse carbohybrid library

Inhibitors of FGF-2 binding to a heparin-albumin conjugate were identified by ELISA from a library of glucuronic acid derivatives. These compounds were also inhibitors of endothelial cell survival that is dependant on FGF-2 and heparin or heparan sulfate proteoglycans. The results indicate that these bioactive compounds may prove useful as lead structures for the further development of pharmaceutical agents capable of modulating biological activity of FGF-2.     

Microwave-assisted extraction,HPLC analysis,and inhibitory effects on carbonic anhydrase I,II, VA,and VII isoforms of 14 blueberry Italian cultivars

Abstract

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.     

Production of Skatole and para-Cresol by a Rumen Lactobacillus sp

The objective of this study was to examine the substrate specificity of several ruminal strains of a Lactobacillus sp. which previously was shown to produce skatole (3-methylindole) by the decarboxylation of indoleacetic acid. A total of 13 compounds were tested for decarboxylase activity. The Lactobacillus strains produced p-cresol (4-methylphenol) by the decarboxylation of p-hydroxyphenylacetic acid, but did not produce either o-cresol or m-cresol from the corresponding hydroxyphenylacetic acid isomers. These strains also decarboxylated 5-hydroxyindoleacetic acid to 5-hydroxyskatole and 3,4-dihydroxyphenylacetic acid to methylcatechol. Skatole and p-cresol were produced in a 0.5:1 ratio, when indoleacetic acid and p-hydroxyphenylacetic acid were combined in equimolar concentrations. Competition studies with indoleacetic acid and p-hydroxyphenylacetic acid suggested that two different decarboxylating enzymes are involved in the production of skatole and p-cresol by these strains. This is the first demonstration of both skatole production and p-cresol production by a single bacterium.     

Novel targets of pentacyclic triterpenoids in Staphylococcus aureus: A systematic review

BackgroundStaphylococcus aureus is an important pathogen both in community-acquired and healthcare-associated infections, and has successfully evolved numerous strategies for resisting the action to practically all antibiotics. Resistance to methicillin is now widely described in the community setting (CMRSA), thus the development of new drugs or alternative therapies is urgently necessary. Plants and their secondary metabolites have been a major alternative source in providing structurally diverse bioactive compounds as potential therapeutic agents for the treatment of bacterial infections. One of the classes of natural secondary metabolites from plants with the most bioactive compounds are the triterpenoids, which comprises structurally diverse organic compounds. In nature, triterpenoids are often found as tetra- or penta-cyclic structures.AimThis review highlights the anti-staphylococcal activities of pentacyclic triterpenoids, particularly α-amyrin (AM), betulinic acid (BA) and betulinaldehyde (BE). These compounds are based on a 30-carbon skeleton comprising five six-membered rings (ursanes and lanostanes) or four six-membered rings and one five-membered ring (lupanes and hopanes).MethodsElectronic databases such as ScienceDirect, PubMed and Scopus were used to search scientific contributions until March 2018, using relevant keywords. Literature focusing on the antimicrobial and antibiofilms of effects of pentacyclic triterpenoids on S. aureus were identified and summarized.ResultsPentacyclic triterpenoids can be divided into three representative classes, namely ursane, lupane and oleananes. This class of compounds have been shown to exhibit analgesic, immunomodulatory, anti-inflammatory, anticancer, antioxidant, antifungal and antibacterial activities. In studies of the antimicrobial activities and targets of AM, BA and BE in sensitive and multidrug-resistant S. aureus, these compounds acted synergistically and have different targets from the conventional antibiotics.ConclusionThe inhibitory mechanisms of S. aureus in novel targets and pathways should stimulate further researches to develop AM, BA and BE as therapeutic agents for infections caused by S. aureus. Continued efforts to identify and exploit synergistic combinations by the three compounds and peptidoglycan inhibitors, are also necessary as alternative treatment options for S. aureus infections.     

Carnivorous Plants as a Source of Potent Bioactive Compound: Naphthoquinones

Carnivorous plants are among the curiosities of nature being different from the normal plants in their mode of nutrition. These plants have fascinated several researchers for centuries. They are also characterized by synthesis of bioactive compounds which are used as a mechanism for self defense. These compounds possess a broad spectrum of biological activities such as antiparasitic, antibacterial, insecticidal, fungicidal, anti-inflammatory, antipyretic, antiproliferative activities. Although, several antimicrobial drugs have been introduced during the recent decades, the problems of microbial infections resistant to synthetic pesticides still exist which necessitate the introduction of novel antimicrobial agents with additional modes of actions than the currently available therapeutic agents. Naphthoquinones are one of the most studied bioactive compounds which have been reported to inhibit the growth of proliferative cells and microbes. Efforts have been made to induce the biosynthesis of naphthoquinone in different species of carnivorous plants. It has been demonstrated that the accumulation of naphthoquinones in carnivorous plants was increased by injecting chitin into the plant tissues. Also, their biosynthesis could be enhanced by the incorporation of elicitors in in vitro cultures of plants. In the present review, we discuss the applications of naphthoquinones and its biosynthesis in carnivorous plants.     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Initiation of an Aquaculture of Sponges for the Sustainable Production of Bioactive Metabolites in Open Systems: Example, Geodia cydonium

Among Metazoa, sponges (phylum Porifera) are the richest source for different bioactive compounds. The availability of the raw material is, however, restricted. To obtain enough of the bioactive compounds for application in human therapy, sponges have to be cultured in in vitro systems. One technique for the establishment of a long-term cell culture from sponges has recently been elaborated. Here, we present a procedure to cultivate tissue samples from sponges in an open system. The species Geodia cydonium, which produces bioactive compounds, has been selected. Tissue samples of approximately 10 g were attached to the bottoms of cultivation trays. After 2 to 3 days, the tissue samples formed a robust contact with the metal support. Subsequently, sets of trays, called tray batteries, either remained in huge aquaria at the Center for Marine Research or were transferred to the vicinity of a fish and mussel farm. The growth rates of the samples remained unchanged within the first month; however, after 3 and 6 months, they increased to 147% and 189%, respectively. In parallel, extracts were prepared from the tissue samples and tested for cytotoxicity in a mouse lymphoma cell assay system. Extracts from cultured tissue initially had a low inhibitory potency; however, after cultivation for 3 or 6 months, values comparable to those of extracts from sponges taken from the biotope were found. In addition, a molecular marker was applied to document the response (health state) of the tissue and the identity of the material in culture. The CD63 molecule was chosen because the expression of this molecule in mammalian systems changes with the age of the animals. The corresponding complementary DNA was isolated from Geodia cydonium. With this probe, the level of expression in cultured tissue samples decreased immediately after starting cultivation; after a cultivation period of 6 months, however, values were similar to those found in controls. These data show that sponge species that produce bioactive compounds can be cultivated in open systems, in which they retain their potency to produce bioactive compounds as well as their health state. Received September 16, 1998; accepted June 18, 1999