Harnessing the potential: advances in cyanobacterial natural product research and biotechnology.

Covering: 2000 to 2023Cyanobacteria produce a variety of bioactive natural products that can pose a threat to humans and animals as environmental toxins, but also have potential for or inspire pharmaceutical use. As oxygenic phototrophs, cyanobacteria furthermore hold great promise for sustainable biotechnology. Yet, the necessary tools for exploiting their biotechnological potential have so far been established only for a few model strains of cyanobacteria, while large untapped biosynthetic resources are hidden in slow-growing cyanobacterial genera that are difficult to access by genetic techniques. In recent years, several approaches have been developed to circumvent the bottlenecks in cyanobacterial natural product research. Here, we summarize current progress that has been made in unlocking or characterizing cryptic metabolic pathways using integrated omics techniques, orphan gene cluster activation, use of genetic approaches in original producers, heterologous expression and chemo-enzymatic techniques. We are mainly highlighting genomic mining concepts and strategies towards high-titer production of cyanobacterial natural products from the last 10 years and discuss the need for further research developments in this field.

The Landscape of Recombination Events That Create Nonribosomal Peptide Diversity.

Nonribosomal peptides (NRP) are crucial molecular mediators in microbial ecology and provide indispensable drugs. Nevertheless, the evolution of the flexible biosynthetic machineries that correlates with the stunning structural diversity of NRPs is poorly understood. Here, we show that recombination is a key driver in the evolution of bacterial NRP synthetase (NRPS) genes across distant bacterial phyla, which has guided structural diversification in a plethora of NRP families by extensive mixing and matching of biosynthesis genes. The systematic dissection of a large number of individual recombination events did not only unveil a striking plurality in the nature and origin of the exchange units but allowed the deduction of overarching principles that enable the efficient exchange of adenylation (A) domain substrates while keeping the functionality of the dynamic multienzyme complexes. In the majority of cases, recombination events have targeted variable portions of the Acore domains, yet domain interfaces and the flexible Asub domain remained untapped. Our results strongly contradict the widespread assumption that adenylation and condensation (C) domains coevolve and significantly challenge the attributed role of C domains as stringent selectivity filter during NRP synthesis. Moreover, they teach valuable lessons on the choice of natural exchange units in the evolution of NRPS diversity, which may guide future engineering approaches.

One-Pot Chemoenzymatic Synthesis of Microviridin Analogs Containing Functional Tags.

Microviridins are a prominent family of ribosomally synthesized and posttranslationally modified peptides (RiPPs) featuring characteristic lactone and lactam rings. Their unusual cage-like architecture renders them highly potent serine protease inhibitors of which individual variants specifically inhibit different types of proteases of pharmacological interest. While posttranslational modifications are key for the stability and bioactivity of RiPPs, additional attractive properties can be introduced by functional tags. To date - although highly desirable - no method has been reported to incorporate functional tags in microviridin scaffolds or the overarching class of graspetides. In this study, a chemoenzymatic in vitro platform is used to introduce functional tags in various microviridin variants yielding biotinylated, dansylated or propargylated congeners. This straightforward approach paves the way for customized protease inhibitors with built-in functionalities that can help to unravel the still elusive ecological roles and targets of this remarkable class of compounds and to foster applications based on protease inhibition.

Deciphering Chemical Mediators Regulating Specialized Metabolism in a Symbiotic Cyanobacterium.

Genomes of cyanobacteria feature a variety of cryptic biosynthetic pathways for complex natural products, but the peculiarities limiting the discovery and exploitation of the metabolic dark matter are not well understood. Here we describe the discovery of two cell density-dependent chemical mediators, nostoclide and nostovalerolactone, in the symbiotic model strain Nostoc punctiforme, and demonstrate their pronounced impact on the regulation of specialized metabolism. Through transcriptional, bioinformatic and labeling studies we assigned two adjacent biosynthetic gene clusters to the biosynthesis of the two polyketide mediators. Our findings provide insight into the orchestration of specialized metabolite production and give lessons for the genomic mining and high-titer production of cyanobacterial bioactive compounds.

Natural 1,3-Dipolar Cycloadditions.

[3+2] in the wild: Biomimetic natural product syntheses and theoretical considerations have indicated that 1,3-dipolar cycloadditions take place in nature. Now, the structure, biosynthesis, and function of a heavily modified prenylated flavin cofactor have been elucidated. In the azomethine ylide form, it undergoes [3+2] cycloadditions with aromatic acids and promotes their decarboxylation.

Terpenoid biosynthesis off the beaten track: unconventional cyclases and their impact on biomimetic synthesis.

Terpene and terpenoid cyclizations are counted among the most complex chemical reactions occurring in nature and contribute crucially to the tremendous structural diversity of this largest family of natural products. Many studies were conducted at the chemical, genetic, and biochemical levels to gain mechanistic insights into these intriguing reactions that are catalyzed by terpene and terpenoid cyclases. A myriad of these enzymes have been characterized. Classical textbook knowledge divides terpene/terpenoid cyclases into two major classes according to their structure and reaction mechanism. However, recent discoveries of novel types of terpenoid cyclases illustrate that nature's enzymatic repertoire is far more diverse than initially thought. This Review outlines novel terpenoid cyclases that are out of the ordinary.

Bacterial Synthesis of Unusual Sulfonamide and Sulfone Antibiotics by Flavoenzyme-Mediated Sulfur Dioxide Capture.

Sulfa drugs, such as sulfonilamide and dapsone, are classical antibiotics that have been in clinical use worldwide. Despite the relatively simple architectures, practically no natural products are known to feature such aromatic sulfonamide or diarylsulfone substructures. We report the unexpected discovery of three fully unprecedented, sulfonyl-bridged alkaloid dimers (sulfadixiamycins A-C) from recombinant Streptomyces species harboring the entire xiamycin biosynthesis gene cluster. Sulfadixiamycins exhibit moderate antimycobacterial activities and potent antibiotic activities even against multidrug-resistant bacteria. Gene inactivation, complementation, and biotransformation experiments revealed that a flavin-dependent enzyme (XiaH) plays a key role in sulfadixiamycin biosynthesis. XiaH mediates a radical-based, three-component reaction involving two equivalents of xiamycin and sulfur dioxide, which is reminiscent of radical styrene/SO2 copolymerization.

Biosynthetic code for divergolide assembly in a bacterial mangrove endophyte.

Divergolides are structurally diverse ansamycins produced by a bacterial endophyte (Streptomyces sp.) of the mangrove tree Bruguiera gymnorrhiza. By genomic analyses a gene locus coding for the divergolide pathway was detected. The div gene cluster encodes genes for the biosynthesis of 3-amino-5-hydroxybenzoate and the rare extender units ethylmalonyl-CoA and isobutylmalonyl-CoA, polyketide assembly by a modular type I polyketide synthase (PKS), and enzymes involved in tailoring reactions, such as a Baeyer-Villiger oxygenase. A detailed PKS domain analysis confirmed the stereochemical integrity of the divergolides and provided valuable new insights into the formation of the diverse aromatic chromophores. The bioinformatic analyses and the isolation and full structural elucidation of four new divergolide congeners led to a revised biosynthetic model that illustrates the formation of four different types of ansamycin chromophores from a single polyketide precursor.

Bioinspired total synthesis of sespenine.

The first total synthesis of sespenine, a rare indole sesquiterpenoid from a mangrove endophyte, has been accomplished. A bioinspired aza-Prins/Friedel-Crafts/retro Friedel-Crafts cascade reaction assembles the bridged tetrahydroquinoline core. Further investigations on the aza-Prins cyclization imply that the C3 configuration of the hydroxyindolenine intermediate is crucial to the biosynthesis of sespenine and its congener xiamycin A.

Heterologous expression of the cryptic mdk gene cluster and structural revision of maduralactomycin A.

After conducting an in silico analysis of the cryptic mdk cluster region and performing transcriptomic studies, an integrative Streptomyces BAC Vector containing the mdk gene sequence was constructed. The heterologous expression of the mdk cluster in Streptomyces albus J1074 resulted in the production of the angucyclic product, seongomycin, which allowed for the assesment of its antibacterial, antiproliferative, and antiviral activities. Heterologous production was further confirmed by targeted knock-out experiments involving key regulators of the biosynthetic pathways. We were further able to revise the core structure of maduralactomycin A, using a computational approach.

Cyanobacterial Genome Sequencing, Annotation, and Bioinformatics.

Cyanobacteria are collectively a globally important monophyletic phylum of bacteria. They have attracted a lot of attention, not only because they are rich sources of natural bioactive products, including toxic substances, but also because they play an important role in global nitrogen and carbon cycles, and are capable of maintaining versatile environmental niche adaptations. A vast number of cyanobacterial genomes have become available due to fast development of sequencing technologies, but effort is still needed to comprehensively understand the molecular basis of their diversity. Here, we introduce a basic pipeline for the cyanobacterial genome sequencing project that can be employed to complete the whole cyanobacterial genome. The pipeline includes DNA extraction from the cyanobacterial culture of interest, hybrid genome sequencing, and genome assembly and annotation. At the end of the chapter, we briefly introduce genome mining tools and one successful genome mining example from our laboratory. This chapter provides general guidance regarding the sequencing project and thus includes several references for alternative methods and tools so that the reader can easily modify the pipeline according to the needs of the laboratory.

Gene Cluster Activation in a Bacterial Symbiont Leads to Halogenated Angucyclic Maduralactomycins and Spirocyclic Actinospirols.

Growth from spores activated a biosynthetic gene cluster in Actinomadura sp. RB29, resulting in the identification of two novel groups of halogenated polyketide natural products, named maduralactomycins and actinospirols. The unique tetracyclic and spirocyclic structures were assigned based on a combination of NMR analysis, chemoinformatic calculations, X-ray crystallography, and 13C labeling studies. On the basis of HRMS2 data, genome mining, and gene expression studies, we propose an underlying noncanonical angucycline biosynthesis and extensive post-polyketide synthase (PKS) oxidative modifications.

Unlocking the Spatial Control of Secondary Metabolism Uncovers Hidden Natural Product Diversity in Nostoc punctiforme.

Filamentous cyanobacteria belong to the most prolific producers of structurally unique and biologically active natural products, yet the majority of biosynthetic gene clusters predicted for these multicellular collectives are currently orphan. Here, we present a systems analysis of secondary metabolite gene expression in the model strain Nostoc punctiforme PCC73102 using RNA-seq and fluorescence reporter analysis. Our data demonstrate that the majority of the cryptic gene clusters are not silent but are expressed with regular or sporadic pattern. Cultivation of N. punctiforme using high-density fermentation overrules the spatial control and leads to a pronounced upregulation of more than 50% of biosynthetic gene clusters. Our data suggest that a combination of autocrine factors, a high CO2 level, and high light account for the upregulation of individual pathways. Our overarching study not only sheds light on the strategies of filamentous cyanobacteria to share the enormous metabolic burden connected with the production of specialized molecules but provides an avenue for the genome-based discovery of natural products in multicellular cyanobacteria as exemplified by the discovery of highly unusual variants of the tricyclic peptide microviridin.

Cryptic indole hydroxylation by a non-canonical terpenoid cyclase parallels bacterial xenobiotic detoxification.

Terpenoid natural products comprise a wide range of molecular architectures that typically result from C-C bond formations catalysed by classical type I/II terpene cyclases. However, the molecular diversity of biologically active terpenoids is substantially increased by fully unrelated, non-canonical terpenoid cyclases. Their evolutionary origin has remained enigmatic. Here we report the in vitro reconstitution of an unusual flavin-dependent bacterial indoloterpenoid cyclase, XiaF, together with a designated flavoenzyme-reductase (XiaP) that mediates a key step in xiamycin biosynthesis. The crystal structure of XiaF with bound FADH2 (at 2.4 Å resolution) and phylogenetic analyses reveal that XiaF is, surprisingly, most closely related to xenobiotic-degrading enzymes. Biotransformation assays show that XiaF is a designated indole hydroxylase that can be used for the production of indigo and indirubin. We unveil a cryptic hydroxylation step that sets the basis for terpenoid cyclization and suggest that the cyclase has evolved from xenobiotics detoxification enzymes.