Specialized Metabolites from Methylotrophic Aaron W. Puri*

Department of Chemistry and the Henry Eyring Center for Cell and Genome Science, University of Utah, Salt Lake City, UT, USA. *Correspondence: [email protected] htps://doi.org/10.21775/cimb.033.211

Abstract these compounds and strategies for determining Biosynthesized small molecules known as special- their biological functions. ized metabolites ofen have valuable applications Te explosion in bacterial genome sequences in felds such as medicine and agriculture. Con- available in public databases as well as the availabil- sequently, there is always a demand for novel ity of bioinformatics tools for analysing them has specialized metabolites and an understanding of revealed that many bacterial are potentially their bioactivity. Methylotrophs are an underex- untapped sources for new molecules (Cimerman- plored metabolic group of that have several cic et al., 2014). Tis includes organisms beyond growth features that make them enticing in terms those traditionally relied upon for natural product of specialized metabolite discovery, characteriza- discovery, and recent studies have shown that tion, and production from cheap feedstocks such examining the biosynthetic potential of new spe- as and gas. Tis chapter will cies indeed reveals new classes of compounds examine the predicted biosynthetic potential of (Pidot et al., 2014; Pye et al., 2017). Tis strategy these organisms and review some of the specialized is complementary to synthetic biology approaches metabolites they produce that have been character- focused on activating BGCs that are not normally ized so far. expressed under laboratory conditions in strains traditionally used for natural product discovery, such as Streptomyces (Rutledge and Challis, 2015). Introduction Tis chapter will focus on the methylotrophic Specialized metabolites, also known as secondary Proteobacteria, which use reduced carbon com- metabolites or natural products, form the basis of pounds with no carbon–carbon bonds as their many bioactive compounds essential to modern sole sources of carbon and energy. Specifcally, the medicine and agriculture (Demain and Sanchez, Proteobacteria are some of the most well-studied 2009; Cantrell et al., 2012; Newman and Cragg, organisms within this metabolic group (Chis- 2016). Tis makes intuitive sense because produc- toserdova et al., 2009). While several molecules tion of these compounds is genetically encoded in have been isolated from methylotrophic Proteo- biosynthetic gene clusters (BGCs) and therefore bacteria that have had an impact on the feld of the resulting structures have had millennia to natural products (Kenney and Rosenzweig, 2018a; evolve their roles. Te great value of these mol- Khmelenina et al., 2015), our understanding of the ecules in manipulating biological systems means specialized of these organisms is still there is a constant demand for new sources of in its infancy.

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Methylotrophic bacteria as an biosynthetic potential in a past analysis of bacte- underexplored resource for rial genomes (Cimermancic et al., 2014). Many specialized metabolite discovery including methylotrophic Methylotrophic bacteria are an atractive source for species rely on the ethylmalonyl-CoA pathway to the discovery of new specialized metabolites for assimilate acetyl-CoA into central metabolism, and several reasons. this pathway involves high fux through CoA-linked intermediates that are also ofen used in the synthe- 1 Obligate methylotrophs, including many spe- sis of specialized metabolites such as polyketides cies of methane-oxidizing bacteria, may have (Alber, 2011). Tis makes the exploration of the been overlooked during traditional ‘grind and specialized metabolism of these organisms particu- fnd’ searches for new compounds that focused larly intriguing. on the isolation of specifc genera like Strepto- Bacteriocin and terpene BGCs were most myces using rich media. commonly predicted within the categories clas- 2 Te lack of complex carbon substrate in sifed by the antibiotic and secondary metabolite methylotroph growth medium can aid in the analysis shell (antiSMASH) (Blin et al., 2017). rapid analysis of new compounds produced However, the majority of predicted clusters were by a culture without laborious prefractiona- detected using ClusterFinder, which relies on more tion procedures, which may also help in high probabilistic (and less stringent) methods for BGC throughput screening approaches. identifcation (Cimermancic et al., 2014). Tis 3 Te chemically defned nature of the growth could suggest that methylotrophs produce diferent medium and simplicity of the carbon source compounds than those made by traditionally stud- also allows substrates with heavy labels such as ied organisms, which would be promising for the 15 13 13 NO3 and CH4 or CH3OH to be used for discovery of new classes of compounds. However, minimal cost, which can aid in structural elu- sequence homology-based comparisons to char- cidation using mass spectrometry and NMR. acterized BGCs and experimental verifcation will 4 If a molecule with atractive properties is be essential to exploring the hypothesis that these identifed, its methylotroph producer can gene clusters are producing novel products. be grown at scale using a feedstock such as methane or methanol, both of which have recently re-emerged as substrates of interest in Intercellular chemical industrial microbiology (Schrader et al., 2009; communication Henard and Guarnieri, 2018). Quorum sensing It is therefore worthwhile to discover more Quorum sensing (QS) allows bacteria to con- molecules produced by this ecologically important trol gene expression in a cell density-dependent group of bacteria. manner. In Proteobacteria, a canonical QS signal Genome sequencing eforts supported by enti- is the acyl-homoserine lactone (acyl-HSL), which ties including the Joint Genome Institute (JGI) can vary in its acyl chain length, saturation, and oxi- and Organization for Genome dation. Tese signals are produced by LuxI-family Analysis (OMeGA) have enabled a detailed exami- acyl-HSL synthases, and detected by LuxR-family nation of the predicted biosynthetic potential of receptor/transcription factors. For reviews please many methylotrophic species (Fig. 12.1). Amongst see Papenfort and Bassler (2016) as well as White- the strains analysed here, more BGCs are generally ley et al. (2017). predicted in methanotroph genomes compared Te frst QS system to be characterized in a meth- with the genomes of non-methanotrophic ylotroph was in the model strain methylotrophs. Overall the Alphaproteobacteria, extorquens AM1 (Penalver et al., 2006). Research- including members of the Methylobacterium ers found that AM1 possesses two LuxI-family among the non-, appear to possess synthases, termed MlaI and MsaI. While MsaI pro-

a large number of predicted BGCs. Te Methylobac- duces the short chain acyl-HSLs C6- and C8-HSL, terium genus was also highlighted for its substantial MlaI produces the more unusual unsaturated long

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Figure 12.1 Predicted biosynthetic gene clusters in genomes of methylotrophic Proteobacteria. Predictions were made using antiSMASH 4.1.0 (Blin et al., 2017) with ClusterFinder (CF) (Cimermancic et al., 2014) enabled with default settings. HSL, homoserine lactone; NRPS, non-ribosomal peptide synthetase; PKS, polyketide synthase.

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chain molecules 7Z-C14-HSL and 2E,7Z-C14-HSL tundrenone (see below) (Puri et al., 2018). Te fact only when AM1 is grown on methanol (Table that tundrenone is produced in a QS-dependent 12.1). Te short chain acyl-HSLs produced by M. manner fts the paradigm that QS systems ofen extorquens AM1 increase the production of exopol- activate the production of extracellular factors such ysaccharide (Penalver et al., 2006), and therefore as antibiotics (McGowan et al., 1995; Duerkop could have a role in bioflm formation. In support et al., 2009) and proteases (Pearson et al., 1997), of this, recently a highly homologous QS system in thereby allowing bacteria to afect their surround- a Methylobacterium populi strain was found to regu- ing environment at high cell density. late the structure and adherence of bioflms made It will be exciting to characterize more methy- by this organism (Morohoshi et al., 2018). lotroph QS systems in the future and determine Many Methylobacterium species have been their biological roles. It should be noted that found to produce acyl-HSLs as detected by bio- methylotrophs occupy diverse ecological niches, assays (Poonguzhali et al., 2007). In one study including both acidic (Dedysh et al., 2000) and researchers found that the orange tree symbiont alkaline (Khmelenina et al., 1997) environments. Methylobacterium mesophilicum SR 1.6/6 produces Te HSL ring is subject to base-catalysed hydroly- several signals, including novel variants (Table sis (Schaefer et al., 2000; Dong et al., 2001), which 12.1) (Pomini et al., 2009). Tere is some evidence may provide one explanation for why methylo- that increased concentrations of these molecules trophs isolated from alkaline environments such as may regulate the transcription of metabolic genes some Methylomicrobium species do not possess in this bacterium (Dourado et al., 2013), however acyl-HSL-based quorum sensing systems in their the precise biological role of these QS signals will genomes (Fig. 12.1). require further investigation. A QS system was recently discovered and Interspecies chemical characterized in the methane-oxidizing Gam- communication maproteobacterium Methylobacter tundripaludum Specialized metabolites biosynthesized by methy- 21/22, which was isolated from lake sediment lotrophs have also been reported to be involved (Table 12.1) (Puri et al., 2017). Tis bacterium pro- in interspecies interactions. Te copper-binding

duces and responds to the signal 3-OH-C10-HSL, small molecule methanobactin (see below) is pro- which activates expression of a BGC co-located duced by many methanotrophs, but its structure with the QS genes in M. tundripaludum. Te same varies between species (Kenney and Rosenzweig, group of researchers subsequently identifed the 2018a). It was reported that methanobactin pro- specialized metabolite product of this BGC, named duced by Methylocystis sp. str. SB2 may regulate the

Table 12.1 Acyl-homoserine lactone quorum sensing signals produced by methylotrophic Proteobacteria Producing strain Acyl-homoserine lactone signal Reference

Methylobacterium extorquens AM1 C6-HSL Penalver et al. (2006) C8-HSL 7Z-C14-HSL 2E,7Z-C14-HSL

Methylobacterium mesophilicum SR 1.6/6 C12-HSL Pomini et al. (2009) C13-HSL C14-HSL 2E-C12-HSL 7Z-C14-HSL 2E,7Z-C14-HSL

Methylobacter tundripaludum 21/22 3-OH-C10-HSL Puri et al. (2017)

Methylobacterium populi P-1M 3-OH-C14-HSL Morohoshi et al. (2018) a C14:1-HSL

Note: While the full stereochemistry has not been determined for all molecules, all biologically produced acyl- homoserine lactone (acyl-HSL) signals where stereochemistry has been determined possess an (S)-N- confguration. aStereochemistry of unsaturated bond not determined.

Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Methylotroph Specialized Metabolites | 215 expression of genes related to methane oxidation in and Rosenzweig, 2018a). Te formation of the Methylosinus trichosporium OB3b via mechanisms oxazole ring and thioamide in methanobactin was beyond simply altering copper availability (Farhan recently characterized, and involves the protein Ul-Haque et al., 2015). Te ability of methano- complex MbnBC, which contains domains of previ- trophs to interact with methanobactin produced by ously unrecognized function (Kenney et al., 2018). other species points to piracy between these organ- Understanding the molecular mechanisms of isms and suggests a ferce competition for copper in methanobactin biosynthesis may ultimately enable the environment (El Ghazouani et al., 2012; Das- the production of methanobactin analogues with sama et al., 2016; DiSpirito et al., 2016). varying properties. Te presence of volatile specialized metabolites Applications of methanobactin include its use in interactions between methanotrophs and non- as a chelation therapy for copper toxicity such as methylotrophic heterotrophs has also been surveyed in Wilson disease (Lichtmannegger et al., 2016). (Veraart et al., 2018). Te methanotroph Methylo- Methanobactin from M. trichosporium OB3b also bacter luteus was reported to produce the volatile binds other metals including gold with high afnity bicyclic terpenoids cadinene and alpha-murolene (Choi et al., 2006), making it useful for biomining in response to the presence of the heterotroph Pseu- as well as nanoparticle preparations (DiSpirito et domonas mandelli. Te role of these compounds in al., 2016). Tis versatile compound also has growth interactions between metabolically linked metha- inhibitory activity against a range of bacteria (DiS- notrophs and non-methanotrophic species will pirito et al., 2007), which is a less discussed property require further investigation. of methanobactin but which may play a role in shaping the composition of methane-oxidizing bacterial communities in nature. Methanobactin Examples of molecules production is activated by low copper availability, produced by methylotrophs making it similar to other specialized metabolites that have tightly regulated production (Kim et al., Methanobactin 2004; Kenney et al., 2016; Gu and Semrau, 2017). Te particulate (pMMO) Genome mining has also identifed methanobactin is found in almost all methane-oxidizing bacteria, BGCs in other non-methyltrophic species (Kenney and it requires copper to catalyse the oxidation and Rosenzweig, 2013), and it will be interesting to of methane to methanol (Balasubramanian et al., see what role methanobactins play in these bacteria. 2010). Consequently, methanotrophs have a great need for copper and use specialized mechanisms Toblerols for its acquisition (Semrau et al., 2010; Kenney and Modular, non-iterative polyketide synthases Rosenzweig, 2018b). Several methanotrophs that (PKSs) follow an assembly line logic that ofen are members of the Alphaproteobacteria secrete makes it possible to predict the structure of the a ribosomally produced and post-translationally metabolite product (Till and Race, 2016). Te modifed peptide (RiPP) that tightly binds copper products of trans-acyltransferase (trans-AT) PKSs termed methanobactin (Table 12.2) (Kim et al., can be more difcult to predict, however detailed 2004). Because of its relation to iron binding sidero- studies of the genes in these BGCs has resulted in phores, methanobactin is known as a chalkophore. a set of product prediction rules for these modular Methanobactin is perhaps the most well known of synthases as well (Nguyen et al., 2008). Recently, all characterized specialized metabolites produced researchers investigated an unusual trans-AT PKS by methylotrophs. For more comprehensive gene cluster in M. extorquens AM1 that could not be reviews of methanobactin please see DiSpirito et al. classifed using previously developed approaches (2016) as well as Kenney and Rosenzweig (2018a). (Ueoka et al., 2017). Te products of the cluster Although the exact structure of metha- were found to be a family of novel molecules nobactin varies between species, all isolated containing cyclopropanol moieties, and these com- versions coordinate copper using oxazolone rings pounds were subsequently named the toblerols or heterocycles with similar functionality and (Table 12.2). Te authors discovered that toblerol neighbouring enethiol/thioamide groups (Kenney C suppresses the production of an additional factor

Curr. Issues Mol. Biol. (2019) Vol. 33 caister.com/cimb Table 12.2 Selected specialized metabolites produced by methylotrophic Proteobacteria Compound name Structure Examples of producing strain(s) Reference

Methylobacterium rhodium, 4,4 -Diapolycopene-4,4 -dioic acid Kleinig et al., (1979), Tao et al. (2005) ′ ′ Methylomonas sp. str. 16a caister.com/cimb

Ectoine Methylomicrobium alcaliphilum 20Z Reshetnikov et al. (2006)

T Gibberellic acid (GA3) Methylobacillus arboreus Iva Agafonova et al. (2018)

Methylobacterium mesophilicum DSM Indole-3-acetic acid (IAA) Ivanova et al. (2001) 1708 Curr. Issues Mol. Biol. (2019) Vol. 33 Table 12.2 Continued

Compound name Structure Examples of producing strain(s) Reference caister.com/cimb

Methylosinus trichosporium OB3b Methanobactin (from OB3b) (structural variants produced by other Kim et al. (2004), Behling et al. (2008) species)

Toblerol C Methylobacterium extorquens AM1 Uekoa et al. (2017)

Tundrenone Methylobacter tundripaludum 21/22 Puri et al. (2018)

Methybacterium mesophilicum DSM 1708, Methylovorus mays VKM Ivanova et al. (2000), Koenig et al. trans-Zeatin-riboside B-2221, Methylobacterium extorquens (2002) DSM 1337 Curr. Issues Mol. Biol. (2019) Vol. 33 218 | Puri

that inhibits the growth of other Methylobacterium and characterized that functionalizes the termini isolates. Te actual factor that causes growth inhibi- of the precursor molecule 4,4′-diapolycopene with tion is currently under investigation, but the results aldehyde moieties (Tao et al., 2005). Tis enzyme so far point to an intriguing hierarchy of bioac- may be useful for future modifcation strategies for tive small molecule production and regulation in these pigments to be employed in semisynthetic these bacteria. Tis study highlights an example processes.

where a methylotroph contains a non-canonical Production of the more traditional C40 carot- BGC that produces novel specialized metabolites, enoids has also been studied in methylotrophs.

demonstrating the utility of methylotrophs as an Methylobacterium species are known to produce C40 underexplored source of these molecules. carotenoids such as oscillaxanthin (Van Dien et al., 2003; Konovalova et al., 2007). Additionally, Meth- Tundrenone ylomonas sp. str. 16a has been used as a chassis for

Tundrenone is a novel specialized metabolite the production of the C40 carotenoid astaxanthin produced by the methanotroph M. tundripaludum (Ye et al., 2007). An astaxanthin-producing metha- in a quorum sensing-dependent manner (Puri et notroph could be used in aquaculture as a single cell al., 2017, 2018). Tundrenone contains a tetrahy- protein source that simultaneously enhances the droindenone core predicted to be derived from pigmentation of the farmed animals. the primary metabolite chorismate, as well as a lipid tail with unusual modifcations (Table 12.2). Ectoine Tese notable structural features suggest that an Ectoine is a cyclic imino acid that functions as understanding of tundrenone’s biosynthesis may an osmoprotectant in many species of halotol- uncover new enzymatic reactions that are useful for erant and halophilic bacteria, including several understanding and engineering the biosynthesis methylotrophic species found in ecosystems of other small molecules. Te biological function with high salinity (Table 12.2) (Galinski et al., of tundrenone is currently unknown, however the 1985; Reshetnikov et al., 2011). Te biosynthesis BGC responsible for tundrenone production is of ectoine has been studied in the halotolerant conserved in M. tundripaludum isolates from across methane-oxidizing bacterium Methylomicrobium the globe (Puri et al., 2017), suggesting that this alcaliphilum 20Z (Reshetnikov et al., 2005, 2006), molecule may have an ecologically signifcant role which was isolated from a soda lake in Russia for this organism. (Khmelenina et al., 1997; Kalyuzhnaya et al., 2008). Ectoine is produced from aspartic acid Carotenoids by the products of the ectABC biosynthetic gene Many methylotrophs are pigmented, as the respon- cluster, and its production markedly increases sible carotenoids can provide antioxidant efects under osmotic stress (Reshetnikov et al., 2011). including protecting their hosts from UV damage As with many specialized metabolites, the biosyn- (Birben et al., 2012; Vorholt, 2012). Tis fact also thesis of ectoine is transcriptionally regulated. Te has led to the commonly used descriptive group- ectABC BGC also contains a co-located MarR- ing of pink pigmented facultative methylotrophs type transcriptional repressor, which is partially (PPFMs), which includes many species of the responsible for regulation of ectABC expression Methylobacterium genus that are found in asso- at diferent salt concentrations (Mustakhimov ciation with plants (see below). Methylobacterium et al., 2010). Production of ectoine is of com- rhodium (formerly Pseudomonas rhodos) produces mercial interest because this compatible solute is

C30-carotenoids including 4,4′-diapolycopene-4,4′- valuable for the preservation of biologics includ- dioic acid diesters that contain conjugated sugars ing those used in cosmetics and medicine, and (Table 12.2) (Kleinig et al., 1979). Tese unusual currently ectoine is produced biosynthetically molecules are not known to be produced by many for commercial means (Graf et al., 2008; Kunte bacterial species, but they were also found to be et al., 2014). Consequently there is interest in produced by methane-oxidizing bacteria of the production of this specialized metabolite from genus Methylomonas (Tao et al., 2005). An oxidase methane gas, a cheap and abundant feedstock for in strain 16a termed CrtNb has been identifed industrial biotechnology.

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Phytohormones Proteobacteria have led to the discovery of novel Plant stomata release methanol during growth as structures, molecular functions, and enzymatic a by-product of pectin demethylation (Nemecek- transformations. Tese eforts also underscore the Marshall et al., 1995; Galbally and Kirstine, 2002), continued utility of isolating bacterial strains and and this methanol is in turn catabolized by resi- characterizing the compounds they produce natu- dent methylotrophs that occupy the phyllosphere rally. While culturing these organisms may involve (Vorholt, 2012). In addition to this metabolic link, specialized knowledge or equipment, doing so can the relationship between plants and methylotrophs aid in the discovery of a metabolite’s biological is thought to be mutualistic, with methylotrophs activity due to biological context. Isolating naturally providing phytohormones to their plant hosts produced compounds can also add some certainty (Holland et al., 2002; Kutschera, 2007; Fedorov et that a molecule of interest is the actual product of al., 2011). the native pathway, which can be an issue for dis- In support of the close link between plants and covery eforts based on heterologous expression. methylotrophs, Methylobacterium species have been Te predicted biosynthetic potential of methylo- found to promote the growth of plants, and the trophic Proteobacteria, along with several practical molecular details of these interactions have been advantages based on the growth conditions of these investigated. For example, strains of M. mesophili- bacteria, make this an exciting time to investigate cum and Methylovorus mays were found to produce the specialized metabolism of these organisms. substances that promote the growth of Amarantus caudatus seedlings, including the cytokinin zeatin (Ivanova et al., 2000). trans-Zeatin biosynthesis in Future directions Methylobacterium spp. was later found to involve It has become increasingly clear that many bacterial tRNA precursors (Koenig et al., 2002); however, BGCs are not expressed in axenic cultures grown removal of trans-zeatin alone from bacterial cul- under laboratory conditions (Milshteyn et al., 2014; tures had no efect on soybean seed germination Rutledge and Challis, 2015). Consequently, biolog- stimulation. ical context is necessary for both the production of Methylotrophs have also been reported to many specialized metabolites and an understand- synthesize additional phytohormones, including ing of their bioactivity. Methylotrophic bacteria auxins and gibberellins. Researchers found that M. occupy a diversity of niches, and one particularly mesophilicum and strains intriguing future direction for the study of special- produce the canonical auxin indole-3-acetic acid ized metabolites produced by these organisms is in (IAA) (Ivanova et al., 2001), which exerts major the context of microbe-microbe and microbe–host infuences on plant growth and development (Duca interactions. Examples include plant–microbe et al., 2014). Furthermore, a Methylobacterium interactions, where recently a Methylophilus strain oryzae strain (Siddikee et al., 2010) as well as the was found to produce factor(s) that inhibit the obligate methylotroph Methylobacillus arboreus growth of several other phyllosphere constituents (Agafonova et al., 2018) have been reported to pro- (Helfrich et al., 2018). Methylotrophs are also duce bioactive gibberellic acid GA3, and GA3 from symbionts of marine animals (Cavanaugh et al., the later strain stimulated the sprouting of letuce 1987; Petersen and Dubilier, 2009), and a sponge seeds. symbiont with a large biosynthetic repertoire was Tese molecules point to a complex chemical recently predicted to have the ability to catabolize conversation between methylotrophs and their methanol (Lackner et al., 2017). Finally, the meta- plant hosts, as plants are canonical producers of bolic relationships between methane-oxidizing natural products. bacteria and the non-methanotrophic heterotrophs they support in the environment may provide con- text for the discovery of new molecules (Oshkin et Conclusions al., 2014; Krause et al., 2017; Veraart et al., 2018). It Te specialized metabolites that have been isolated will be exciting to see the developments in each of and characterized to date from methylotrophic these areas in the years to come.

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