life Article Natural Product Gene Clusters in the Filamentous Nostocales Cyanobacterium HT-58-2 Xiaohe Jin 1,*, Eric S. Miller 2 and Jonathan S. Lindsey 1 1 Department of Chemistry, North Carolina State University, Raleigh, NC 27695-8204, USA; [email protected] 2 Department of Plant and Microbial Biology, North Carolina State University, Raleigh, NC 27695-7615, USA; [email protected] * Correspondence: [email protected] Abstract: Cyanobacteria are known as rich repositories of natural products. One cyanobacterial- microbial consortium (isolate HT-58-2) is known to produce two fundamentally new classes of natural products: the tetrapyrrole pigments tolyporphins A–R, and the diterpenoid compounds tolypodiol, 6-deoxytolypodiol, and 11-hydroxytolypodiol. The genome (7.85 Mbp) of the Nostocales cyanobacterium HT-58-2 was annotated previously for tetrapyrrole biosynthesis genes, which led to the identification of a putative biosynthetic gene cluster (BGC) for tolyporphins. Here, bioinformatics tools have been employed to annotate the genome more broadly in an effort to identify pathways for the biosynthesis of tolypodiols as well as other natural products. A putative BGC (15 genes) for tolypodiols has been identified. Four BGCs have been identified for the biosynthesis of other natural products. Two BGCs related to nitrogen fixation may be relevant, given the association of nitrogen stress with production of tolyporphins. The results point to the rich biosynthetic capacity of the HT-58-2 cyanobacterium beyond the production of tolyporphins and tolypodiols. Citation: Jin, X.; Miller, E.S.; Lindsey, J.S. Natural Product Gene Clusters in Keywords: anatoxin-a/homoanatoxin-a; hapalosin; heterocyst glycolipids; natural products; sec- the Filamentous Nostocales ondary metabolites; shinorine; tolypodiols; tolyporphins Cyanobacterium HT-58-2. Life 2021, 11, 356. https://doi.org/10.3390/ life11040356 1. Introduction Academic Editors: Paul M. D’Agostino, Nádia Eusébio and Tolyporphins represent a class of compounds in the pigments of life family with struc- Ângela Brito tural features distinct from other prominent constituents, including heme, chlorophylls, bacteriochlorophylls, cobalamin, and coenzyme F430. Tolyporphin A, the first member of Received: 14 March 2021 the tolyporphins family, was found in the lipophilic extract of a cyanobacterial sample. The Accepted: 15 April 2021 sample, HT-58-2, was obtained from Nan Madol on the island of Pohnpei in Micronesia [1]. Published: 18 April 2021 The discovery and identification of tolyporphin A originated with a broad screen of diverse cyanobacteria for anti-cancer activity [2]. Indeed, tolyporphin A was found to exhibit efflux Publisher’s Note: MDPI stays neutral pump inhibition and photocytotoxicity toward tumor cells [3–5]. with regard to jurisdictional claims in An astonishing aspect of the discovery of tolyporphin A is the presence of a compound published maps and institutional affil- with a dioxobacteriochlorin chromophore in a cyanobacterial sample. Cyanobacteria iations. employ chlorophyll, not bacteriochlorophyll, for photosynthesis, and the presence of compounds with a bacteriochlorin chromophore in a chlorin-based photosynthetic system is unprecedented. Over the years, fractionation of lipophilic extracts from the HT-58-2 culture has led to the identification of a family of tolyporphins, now numbering 18 (A– Copyright: © 2021 by the authors. R) [6–8] (Figure1). Most of the tolyporphins are dioxobacteriochlorins, but three are Licensee MDPI, Basel, Switzerland. oxochlorins, and one is a porphyrin. The structural diversity suggests an in vivo role for This article is an open access article tolyporphins as secondary metabolites in as-yet undefined defense processes. If so, this distributed under the terms and represents a fundamentally new biological function in the pigments of life family. To date, conditions of the Creative Commons the HT-58-2 culture remains the only known producer of tolyporphins. Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Life 2021, 11, 356. https://doi.org/10.3390/life11040356 https://www.mdpi.com/journal/life Life 2021, 11, x FOR PEER REVIEW 2 of 18 Life 2021, 11, 356 2 of 17 (http://creativecommons.org/li- censes/by/4.0/). O R N HN N HN N HN NH N NH N R NH N R O O Tolyporphins A -J , L-O Tolyporphin K , Q , R Tolyporphin P R = C -glycoside, OH or OAc R = C -glycoside, OH or OAc OH OH OH OH Me Me Me Me C -glycosides O O O O OAc OH HO OH OH HO Figure 1. StructuresStructures of of dioxobacteriochlorins dioxobacteriochlorins (tolyporphins A A–J,–J, L L–O),–O), oxochlorins (tolyporphins K, Q, R), and a porphyrin (tolyporphin P). The π-chromophore-chromophore isis shownshown inin magenta,magenta, green,green, andand red,red, respectively.respectively. ToTo pursue questions of biosynthesis and in vivo functionfunction of tolyporphins, in late 2015 we obtainedobtained thethe tolyporphin-producing tolyporphin-producing culture culture HT-58-2. HT-58 Subsequent-2. Subsequent studies stud haveies have revealed re- vealedthe following: the following: (1) the HT-58-2(1) the HT culture-58-2 isculture dominated is dominated by a single by filamentousa single filamentous cyanobacterium cyano- bacteriumin a non-axenic in a non cyanobacterial–microbial-axenic cyanobacterial consortium–microbial (Figureconsortium2A,B) (Figure [ 9,10]; (2) 2A several-weeks,B) [9,10]; (2) severalgrowth-weeks under growth stress imparted under stress by theimparted deprivation by the ofdeprivation aqueous-soluble of aque nitrateous-soluble profoundly nitrate profoundlyincreases the increases production the of production tolyporphins, of tolyporphins, reaching a level reaching rivalling a thatlevel of rivalling chlorophyll that [11 of]; chlorophyll(3) tolyporphins [11]; are(3) presenttolyporphins in the sheathare present and cellin the septa sheath of the and filamentous cell septa cyanobacterium, of the filamen- tousas revealed cyanobacterium, by hyperspectral as revealed confocal by hyperspectral fluorescence confocal imaging fluorescence [12]; (4) the imaging cyanobacterium [12]; (4) thecontains cyanobacterium a circular genome contains (7.85 a circular Mbp) withgenome genes (7.85 for Mbp) biosynthesis with genes of heme, for biosynthesis chlorophyll ofa, heme,phycocyanobilin, chlorophyll anda, phycocyanobilin, cobalamin (in part), and ascobalamin well as a (in putative part), as biosynthetic well as a putative gene cluster bio- synthetic(here termed gene BGC-T) cluster for (here the termed biosynthesis BGC- ofT) tolyporphinsfor the biosynthesis (Figure 2ofE) tolyporphins [9]. Notably, (Figure BGC-T 2E)contains [9]. Notably, all genes BGC in the-T corecontains pathway all genes (from in L-glutamic the core pathway acid to protoporphyrinogen (from L-glutamic acid IX) to of protoporphyrinogentetrapyrrole biosynthesis, IX) of except tetrapyrrolehemD, whichbiosynthesis, is located except elsewhere hemD, inwhich the genome. is located else- whereIn in an the initial genome. report in 1992 [1], the Nostocales cyanobacterium HT-58-2 was categorized as Tolypothrix nodosa on the basis of visual inspection of the characteristic filamentous morphology (Figure2C,D). The term “tolypothrix” refers to “a hairy ball of yarn,” whereas “nodosa” denotes the presence of nodules, which are believed to be responsible for nitrogen fixation. The availability of the sequence information enabled phylogenomic analysis, which showed the HT-58-2 cyanobacterium to be more closely aligned with the genus Brasilonema according to 16S rRNA [9]. Scrutiny of the lipophilic extracts of the HT-58-2 culture has revealed other new natural products. The natural products include tolypodiol (Figure3), the first diterpenoid compound obtained from cyanobacteria [13]. A monoacetate derivative of tolypodiol was subsequently synthesized by chemical means [13]. Both tolypodiol and the synthetic O-acetate derivative showed potent anti-inflammatory activity in a mouse ear edema assay. Recently, two tolypodiol analogues, 6-deoxytolypodiol and 11-hydroxytolypodiol, were also found in extracts from the HT-58-2 culture. The structures and absolute configuration of the two tolypodiol analogues were determined, and the compounds were assayed for anti-inflammatory activity [14]. Prior work [9] annotating the genome of the Nostocales HT-58-2 suggested numerous genes for diverse functions, but did not delve deeply into the BGCs of natural products, including those for tolypodiols. Life 2021, 11, 356 3 of 17 Life 2021, 11, x FOR PEER REVIEW 3 of 18 Life 2021, 11, x FOR PEER REVIEW 4 of 18 FigureFigure 2. ((AA)) Optical Optical image image of of the the HT-58-2 HT-58-2 culture culture on on a a BG-11 BG-11 agar agar plate plate shows shows community community bacteria bacteria (red (red arrows) arrows) in in the the presence of filamentous cyanobacteria.Recently, two (B) Scanningtolypodiol electron analogues, microscopy 6-deoxytolypodiol image shows community and 11-hydroxytolypodiol, bacteria (red arrows) were presence of filamentous cyanobacteria. (B) Scanning electron microscopy image shows community bacteria (red arrows) attached to the sheath of thealso filamentous found in bacteria. extracts (C from,D) Optical the HT micrographs-58-2 culture. show The the structures clumping of and HT-58-2 absolute cyanobacteria configuration attached to the sheath of the filamentous bacteria. (C,D) Optical micrographs show the clumping of HT-58-2 cyanobacteria (Neofluar 40X 0.75, Zeiss) grownof the in two BG-11 tolypodiol for 35 days. analogues (E) Genes