Cytotoxic Compounds Derived from Marine Algicolous and Spongicolous Endophytic Fungi: A Review

Endophytes have gained particular interest in the search of potential pharmaceutical candidates for a long time due to their diversity, species richness and bioprospecting nature. They generally produce the essential metabolites for their expansion inside the plant which is involved in various biotransformation processes of utilizing host nutrients and cell components to continue microbial growth, sustenance, and reproduction. In above processes, they produce a huge amount of both structurally and functionally diverse secondary metabolites for maintaining an effective symbiosis with hosts. These compounds are proven to have significant bioactive properties like antibacterial, antifungal, antiviral, anti-inflammatory, antioxidants, antitumor activities. Despite the proven significance, a little is exploited so far about endophytes. Particularly marine fungal endophytes which are the centre of attention in this review have gained much less importance. Due to unique environmental feature, fungal endophytes derived from marine environment offer vast diversity in different bioactive secondary metabolites. This review has focused on algicolous endophytes and bioactive secondary metabolites discovered during the last two decades. Particular importance has been given to cytotoxic and antimicrobial metabolites. Due to intensive studies during last several years, an extensive number of publications are now available on cytotoxic compounds derived from endophytic fungi of marine algicolous and spongicolous origin that have been summarized in this review. Dhaka Univ. J. Pharm. Sci. 20(2): 247-265, 2021 (December)

Antifouling Activity of Halogenated Compounds Derived from the Red Alga Sphaerococcus coronopifolius: Potential for the Development of Environmentally Friendly Solutions

Nowadays, biofouling is responsible for enormous economic losses in the maritime sector, and its treatment with conventional antifouling paints is causing significant problems to the environment. Biomimetism and green chemistry approaches are very promising research strategies for the discovery of new antifouling compounds. This study focused on the red alga Sphaerococcus coronopifolius, which is known as a producer of bioactive secondary metabolites. Fifteen compounds, including bromosphaerol (1), were tested against key marine biofoulers (five marine bacteria and three microalgae) and two enzymes associated with the adhesion process in macroalgae and invertebrates. Each metabolite presented antifouling activity against at least one organism/enzyme. This investigation also revealed that two compounds, sphaerococcinol A (4) and 14R-hydroxy-13,14-dihydro-sphaerococcinol A (5), were the most potent compounds without toxicity towards oyster larvae used as non-target organisms. These compounds are of high potential as they are active towards key biofoulers and could be produced by a cultivable alga, a fact that is important from the green chemistry point of view.

Novel Alkaloids from Marine Actinobacteria: Discovery and Characterization

The marine environment is an excellent resource for natural products with therapeutic potential. Its microbial inhabitants, often associated with other marine organisms, are specialized in the synthesis of bioactive secondary metabolites. Similar to their terrestrial counterparts, marine Actinobacteria are a prevalent source of these natural products. Here, we discuss 77 newly discovered alkaloids produced by such marine Actinobacteria between 2017 and mid-2021, as well as the strategies employed in their elucidation. While 12 different classes of alkaloids were unraveled, indoles, diketopiperazines, glutarimides, indolizidines, and pyrroles were most dominant. Discoveries were mainly based on experimental approaches where microbial extracts were analyzed in relation to novel compounds. Although such experimental procedures have proven useful in the past, the methodologies need adaptations to limit the chance of compound rediscovery. On the other hand, genome mining provides a different angle for natural product discovery. While the technology is still relatively young compared to experimental screening, significant improvement has been made in recent years. Together with synthetic biology tools, both genome mining and extract screening provide excellent opportunities for continued drug discovery from marine Actinobacteria.

Hemimycalins C–E; Cytotoxic and Antimicrobial Alkaloids with Hydantoin and 2-Iminoimidazolidin-4-one Backbones from the Red Sea Marine Sponge Hemimycale sp.

In the course of our continuing efforts to identify bioactive secondary metabolites from Red Sea marine sponges, we have investigated the sponge Hemimycale sp. The cytotoxic fraction of the organic extract of the sponge afforded three new compounds, hemimycalins C–E (1–3). Their structural assignments were obtained via analyses of their one- and two-dimensional NMR spectra and HRESI mass spectrometry. Hemimycalin C was found to differ from the reported hydantoin compounds in the configuration of the olefinic moiety at C-5–C-6, while hemimycalins D and E were found to contain an 2-iminoimidazolidin-4-one moiety instead of the hydantoin moiety in previously reported compounds from the sponge. Hemimycalins C–E showed significant antimicrobial activity against Escherichia coli and Candida albicans and cytotoxic effects against colorectal carcinoma (HCT 116) and the triple-negative breast cancer (MDA-MB-231) cells.

Chemoenzymatic One-Pot Process for the Synthesis of Tetrahydroisoquinolines

1,2,3,4-Tetrahydyroisoquinolines form a valuable scaffold for a variety of bioactive secondary metabolites and commercial pharmaceuticals. Due to the harsh or complex conditions of the conventional chemical synthesis of this molecular motif, alternative mild reaction pathways are in demand. Here we present an easy-to-operate chemoenzymatic one-pot process for the synthesis of tetrahydroisoquinolines starting from benzylic alcohols and an amino alcohol. We initially demonstrate the oxidation of 12 benzylic alcohols by a laccase/TEMPO system to the corresponding aldehydes, which are subsequently integrated in a phosphate salt mediated Pictet–Spengler reaction with m-tyramine. The reaction conditions of both individual reactions were analyzed separately, adapted to each other, and a straightforward one-pot process was developed. This enables the production of 12 1,2,3,4-tetrahydyroisoquinolines with yields of up to 87% with constant reaction conditions in phosphate buffer and common laboratory glass bottles without the supplementation of any additives.

Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>

Discovery, Characterisation and Engineering of Non-Ribosomal Peptide Synthetases and Phosphopantetheinyl Transferase Enzymes

<p>Non-ribosomal peptide synthetases (NRPSs) are multi-modular biosynthetic enzymes that are responsible for the production of many bioactive secondary metabolites produced by microorganisms. They are activated by phosphopantetheinyl transferase (PPTase) enzymes, which attach an essential prosthetic group to a specific site within a “carrier protein” (CP) domain that is an integral part of each NRPS module. Of particular importance in this work is the NRPS BpsA, which produces a blue pigment called indigoidine; but only when BpsA has first been activated by a PPTase. BpsA can be used as a reporter for PPTase activity, to identify PPTases and/or measure their activity. Several CP-substituted BpsA variants were used, in order to study and identify PPTases which may recognise different CP domains. The first part of the research described in this thesis examined the features of foreign CP interactions within BpsA that made these functional substitutions possible. Two key residues, the +4 and +24 positions relative to an invariant serine, were found to be highly important; with appropriate substitutions at these positions yielding active CP-substituted variants. Wild type BpsA and the CP-substituted variants were then used as the basis of a screen to discover new PPTase genes, and associated natural product biosynthetic genes, from metagenomic libraries. The vast majority of bacteria that produce bioactive secondary metabolites are unable to be cultured under laboratory conditions; screening metagenomic libraries is a way to access this untapped biodiversity in order to discover new natural products. Two environmental DNA libraries were screened, and PPTase genes were identified via their ability to activate BpsA, giving rise to blue colonies in high throughput agar plate screens. This screen proved to be a powerful enrichment strategy with almost half of the novel 21 PPTase genes recovered also linked to biosynthetic gene clusters. Using the evolved CP-substituted BpsA variants (and thereby altering the PPTase recognition site) enabled a wider variety of hits to be found. This led to the hypothesis that some of the PPTases discovered via this screening method would have non-overlapping substrate specificities, a beneficial property for certain PPTase applications. The 21 PPTase genes discovered via metagenomic screening were characterised further, using a series of assays involving BpsA to measure their activity. As is common for PPTase enzymes, there were difficulties in obtaining enough soluble protein via purification to perform a detailed analysis of each. Those that were able to be purified had much lower activity than other previously characterised PPTases, and were also not as specific for their CP substrates as they had first appeared to be. Due to these low activity levels, several other previously characterised PPTases were also studied further using the BpsA methods. All PPTases showed a relatively broad activity across a range of CP substrates. The desire to obtain PPTases with more specific substrate specificities led to the development of a directed evolution screen to alter PPTase CP specificity. In a proof-of-principle study the E. coli PPTase EntD was evolved to lose activity with the BpsA CP while retaining activity with its native CP. This screen, the first of its kind to evolve PPTases for greater CP substrate specificity, was successful in recovering several improved variants. These variants had either completely abolished or vastly decreased activity for the WT BpsA CP while retaining the ability to activate the native (EntF) CP domain. The general strategy developed here can be applied to the evolution of other PPTases and CP substrates.</p>