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Cloning of the Bisucaberin B Biosynthetic Gene Cluster from the Marine Bacterium Tenacibaculum Mesophilum, and Heterologous Production of Bisucaberin B

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Cloning of the Bisucaberin B Biosynthetic Gene Cluster from the Marine Bacterium Tenacibaculum Mesophilum, and Heterologous Production of Bisucaberin B marine drugs Article Cloning of the Bisucaberin B Biosynthetic Gene Cluster from the Marine Bacterium Tenacibaculum mesophilum, and Heterologous Production of Bisucaberin B Masaki J. Fujita *, Yusuke Goto and Ryuichi Sakai Graduate School of Fisheries Sciences, Hokkaido University, Hakodate 041-8611, Japan; [email protected] (Y.G.); ryu.sakai@fish.hokudai.ac.jp (R.S.) * Correspondence: masakifujita@fish.hokudai.ac.jp; Tel.: +81-138-40-8806 Received: 24 August 2018; Accepted: 15 September 2018; Published: 19 September 2018 Abstract: The biosynthetic gene cluster for bisucaberin B (1, bsb gene cluster), an N-hydroxy-N-suc cinyl diamine (HSD)-based siderophore, was cloned from the marine bacterium Tenacibaculum mesophilum, originated from a marine sponge. The bsb gene cluster consists of six open reading frames (ORFs), in contrast to the four ORFs typically seen in biosynthetic gene clusters of the related molecules. Heterologous expression of the key enzyme, BsbD2, which is responsible for the final biosynthetic step of 1 resulted in production of bisucaberin B (1), but not bisucaberin (2) a macrocyclic counterpart of 1. To date, numbers of related enzymes producing macrocyclic analogues have been reported, but this work represents the first example of the HSD-based siderophore biosynthetic enzyme which exclusively produces a linear molecule rather than macrocyclic counterparts. Keywords: siderophore; biosynthesis; heterologous production; bisucaberin; desferrioxamine 1. Introduction Siderophores are microbial products that strongly chelate ferric ions, facilitating the acquisition of iron in iron-deficient environments. Iron is essential for the growth of almost all organisms and represents a key limiting factor in bioproduction; thus microorganisms utilize a variety of siderophores to compete for iron [1]. It is also reported that some bacteria utilize exogenous siderophores for their own growth, a process that has been termed “siderophore piracy” [2–4]. Recent work has revealed that avaroferrin (3), an N-hydroxy-N-succinyl diamine (HSD)-based siderophore that is produced by a marine bacterium, halts swarming of a competitive bacterium [5]. These observations suggest that siderophores contribute to complex chemical communications among environmental microorganisms. Desferrioxamines and related molecules (1–8) are representative bacterial siderophores composed primarily of N-hydroxy-N-succinyl cadaverine (HSC, 9) and N-hydroxy-N-succinyl putrescine (HSP, 10) subunits, and have been isolated from various bacterial phyla (Figure1)[ 6–10]. Desferrioxamine B(7) is used clinically as a treatment for iron poisoning [11] and several interesting bioactivities are reported for this group of molecules, including inhibition of mycobacterium biofilm formation [12] as well as enhancement of macrophage-mediated cancer cell cytolysis [13]. Mar. Drugs 2018, 16, 342; doi:10.3390/md16090342 www.mdpi.com/journal/marinedrugs Mar. Drugs 2018, 16, 342 2 of 12 Mar. Drugs 2018, 16, x 2 of 11 FigureFigure 1.1. StructuresStructures ofof thethe representativerepresentative NN-hydroxy--hydroxy-NN-succinyl-succinyl diaminediamine (HSD)-based(HSD)-based siderophoressiderophores ((11––88)) producedproduced byby variousvarious bacterialbacterial phyla,phyla, andand theirtheir monomericmonomeric precursorsprecursorsN N-hydroxy--hydroxy-NN-succinyl-succinyl cadaverinecadaverine andand NN-hydroxy--hydroxy-N-succinyl putrescin (9 and 1010,, respectively).respectively). CompoundsCompounds 2–5 and 88 areare cycliccyclic dimersdimers andand trimers,trimers, whilewhile 11 andand 66 areare linearlinear dimerdimer andand trimer,trimer, respectively.respectively. CompoundCompound 77 waswas pseudopseudo trimer,trimer, oneone ofof itsits terminalsterminals waswas cappedcapped bybyacetate acetate group. group. ToTo date,date, severalseveral genegene clustersclusters responsibleresponsible forfor thethe biosynthesisbiosynthesis ofof HSD-basedHSD-based siderophoressiderophores havehave beenbeen clonedcloned [[14–19].14–19]. These clusters generally encodeencode four proteins, with the firstfirst threethree enzymesenzymes (enzymes(enzymes A–C)A–C) catalyzingcatalyzing thethe formationformation ofof thethe commoncommon keykey intermediate,intermediate, HSDsHSDs ((9 andand 1010),), fromfrom aminoamino acidsacids (lysine (lysine and ornithine)and ornithine) by sequential by sequential decarboxylation, decarboxylation,N-hydroxylation, N-hydroxylation, and condensation and withcondensation succinyl-CoA. with succinyl-CoA. The fourth enzyme The fourth (enzyme enzyme D) catalyzes (enzyme the D) formationcatalyzes the of multipleformation amide-bond of multiple linkageamide-bond between linkage the HSD betw monomerseen the HSD (9, 10 monomers) as well as ( the9, 10 subsequent) as well head-to-tailas the subsequent cyclization head-to-tail reaction, yieldingcyclization the reaction, final macrocyclic yielding products the final (Figure macrocyc2)[ 20lic]. Theseproducts amide (Figure bond-formation 2) [20]. These enzymes amide (enzyme bond- Ds)formation comprise enzymes a novel (enzyme group of Ds) non-ribosomal comprise a novel peptide gr synthetasesoup of non-ribosomal [20]. It is also peptide reported synthetases that enzyme [20]. DsIt is accept also reported a wide rangethat enzyme of HSDs Ds as accept their substratesa wide range to produceof HSDs aas variety their substrates of macrocyclic to produce final productsa variety withof macrocyclic diverse bioactivities final products [21–24 ],with but fordiverse which bioactivities the molecular [21–24], mechanisms but for of which the regulation the molecular of the oligomerizationmechanisms of reactionsthe regulation and macrocyclization of the oligomerizat reactionion reactions are largely and unknown. macrocyclization reaction are largelyDuring unknown. the survey of novel siderophores from marine bacteria, we found a marine bacterium TenacibaculumDuring the mesophilum survey of, whichnovel belongssiderophores to the from phylum marine Bacteroidetes bacteria, we isolated found from a marine an unidentified bacterium PalauanTenacibaculum marine mesophilum sponge,, that which exclusively belongs to produces the phylum a linear Bacteroidete HSD-baseds isolated siderophore, from an unidentified bisucaberin B(Palauan1), but marine does sponge, not produce that exclusively the macrocyclic produces counterpart a linear HSD-based2 [10]. Other sidero bacteriaphore, bisucaberin reported so B ( far1), producebut does mainly not produce the macrocyclic the macrocyclic forms; linearcounterpart molecules 2 [10]. are thoughtOther bacteria to be biosynthetic reported so intermediates far produce ormainly shunt the by-products macrocyclic (Figure forms;2 linear). These molecules observations are thought suggested to be biosynthetic that T. mesophilum intermediatesmay or encode shunt theby-products first example (Figure of a2). HSD-based These observations siderophore suggested biosynthetic that machineryT. mesophilum that may inherently encodelacks the first the finalexample macrocyclizing of a HSD-based activity. siderophore Therefore, webiosynthetic expected thatmachinery a detailed that analysis inherently of the lacks enzymes the fromfinal T.macrocyclizing mesophilum would activity. provide Therefore, information we expected regarding that the moleculara detailedmechanism analysis of of the the enzymes macrocyclization from T. reaction.mesophilum Here, would we report provide cloning information of the biosynthetic regarding the gene molecular cluster of mechanism bisucaberin Bof ( 1the) from macrocyclizationT. mesophilum, reaction. Here, we report cloning of the biosynthetic gene cluster of bisucaberin B (1) from T. mesophilum, confirmation of the enzymatic function by heterologous expression, and a brief sequential analysis of the cloned enzymes. Mar. Drugs 2018, 16, 342 3 of 12 confirmation of the enzymatic function by heterologous expression, and a brief sequential analysis of the cloned enzymes. Mar. Drugs 2018, 16, x 3 of 11 FigureFigure 2.2.Proposed Proposed biosynthetic biosynthetic pathway pathway of N-hydroxy- of N-hydroxy-N-succinylN-succinyl diamine (HSD)-based diamine (HSD)-based siderophores, siderophores,and schematic and organization schematic oforganization the typical biosyntheticof the typica genel biosynthetic cluster; this gene organization cluster; this is organization conserved in is a conservedwide range in of a bacterialwide range phyla. of bacterial phyla. 2.2. Results Results and and Discussion Discussion 2.1.2.1. Cloning Cloning of of the the Bisucaberi Bisucaberinn B B Biosynthetic Biosynthetic Gene Gene Cluster Cluster ThoughThough HSD-based HSD-based siderophore siderophore biosynthetic biosynthetic ge genesnes have have not not been been reported reported from from phylum phylum Bacteroidetes,Bacteroidetes, taking taking advantage advantage of of the the highly highly co conservednserved amino amino acid acid sequ sequenceence among among amide-bond amide-bond formationformation enzymes enzymes (enzyme (enzyme Ds) Ds) from from diverse diverse bacter bacterialial taxa, taxa, we we cloned cloned the the conserved conserved part part by by PCR PCR amplificationamplification using using a a degenerate degenerate primer primer set. set. An An amplified amplified DNA DNA fragment fragment from from the the genomic genomic DNA DNA of of T.T. mesophilum showedshowed high high similarity similarity to to the the known known biosynth biosyntheticetic genes, genes, suggesting suggesting the the presence presence of
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