Microbial Pyrrolnitrin: Natural Metabolite with Immense Practical Utility

Pyrrolnitrin (PRN) is a microbial pyrrole halometabolite of immense antimicrobial significance for agricultural, pharmaceutical and industrial implications. The compound and its derivatives have been isolated from rhizospheric fluorescent or non-fluorescent pseudomonads, Serratia and Burkholderia. They are known to confer biological control against a wide range of phytopathogenic fungi, and thus offer strong plant protection prospects against soil and seed-borne phytopathogenic diseases. Although chemical synthesis of PRN has been obtained using different steps, microbial production is still the most useful option for producing this metabolite. In many of the plant-associated isolates of Serratia and Burkholderia, production of PRN is dependent on the quorum-sensing regulation that usually involves N-acylhomoserine lactone (AHL) autoinducer signals. When applied on the organisms as antimicrobial agent, the molecule impedes synthesis of key biomolecules (DNA, RNA and protein), uncouples with oxidative phosphorylation, inhibits mitotic division and hampers several biological mechanisms. With its potential broad-spectrum activities, low phototoxicity, non-toxic nature and specificity for impacts on non-target organisms, the metabolite has emerged as a lead molecule of industrial importance, which has led to developing cost-effective methods for the biosynthesis of PRN using microbial fermentation. Quantum of work narrating focused research efforts in the emergence of this potential microbial metabolite is summarized here to present a consolidated, sequential and updated insight into the chemistry, biology and applicability of this natural molecule.


Introduction
Of 5-30 million species on the Earth, fewer than 2 million have been described and fewer than 1% have been explored for a vast repertoire of new natural products with socio-economic significance [1]. Hence, it is reasonable to expect that many more natural products not only from known species, but also from unidentified organisms are yet to come to benefit humanity and the environment [2]. Natural products offer unique structural molecules unparalleled by any other molecular family with an array of biological activities such as for drug leads. The many natural products that occupy the market today without any chemical modification are a testimony to the remarkable properties of secondary molecules produced by an array of plants, insects, animals, microbes and numerous species of marine organisms [3].

Pyrrolnitrin (PRN)
Pyrrolnitrin [3-chloro-4-(2-nitro-3-chlorophenyl) pyrrole] is a phenylpyrrole derivative containing two chlorine atoms and a nitro group. PRN, isolated from Pseudomonas pyrrocinia and various other pseudomonads, was classified as halometabolite in as early as 1964 [38]. Later, the compound was biosynthesized using tryptophan as supplement in the medium [53] and chemically synthesized by Nakano et al [54]. Biosynthesis of PRN in Pseudomonas aureofaciens ATCC 15926 has shown that L-tryptophan is a direct precursor ( Figure 1) [53]. However, Hammil et al. [55] obtained high yield of PRN in D-tryptophan amended medium. Tryptophan analogs amended in the fermentation medium can also yield a series of PRN-like derivatives [56] (Table 2) with low antimicrobial activity than the native parent compound.         Structurally, PRN possesses benzene and pyrrole rings with chlorine atoms on both of them and nitro and chlorine units to form an unusual natural skeleton. It has chlorine moiety to contribute more towards biological activity [57] in comparison to its bromine derivative [58]. Consequently, several natural congeners of PRN such as amino-pyrrolnitrin, iso-pyrrolnitrin, 2-chloropyrrolnitrin, oxy-pyrrolnitrin, 4-fluoropyrrolnitrin, and 3-fluoro-3-dechloropyrrolnitrin have been reported. Brominated derivatives of PRN can be synthesized by replacing chlorine ion with bromine in the presence of sodium bromide.
Biomolecules 2019, 9, x FOR PEER REVIEW 6 of 26 possible for PRN and green synthesis routes could also support industrial processes for PRN production.  Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used "conceptualization, X.X. and Y.Y.; methodology, X.X.; software, X.X.; validation, X.X., Y.Y. and Z.Z.; formal analysis, X.X.; investigation, X.X.; resources, X.X.; data curation, X.X.; writing-original draft preparation, X.X.; writing-review and editing, X.X.; Figure 1. Biosynthetic steps in the synthesis of pyrrolnitrin. 7-chlorotryptophan is formed from tryptophan due to flavin-dependent halogenation catalyzed by the enzyme tryptophan 7-halogenase (PrnA). Further, the enzyme PrnB (monodechloroaminopyrrolnitrin synthase catalyzes formation of monodechloroaminopyrrolnitrin from 7-chlorotryptophan while the enzyme PrnC leads to catalytic reaction for the conversion of monodechloroaminopyrrolnitrin into aminopyrrolnitrin. In the last step, aminopyrrolnitrin is converted to pyrrolnitrin with the help of the enzyme PrnD (aminopyrrolnitrin oxygenase).

Pyrrolnitrin: Chemical Synthesis
PRN is positive towards Ehrlich's reagent where pyrrole ring gets condensed with p-dimethylaminobenzaldehyde to form the violet color complex. Pauly's coupling reaction yields red color [59] and gives a negative reaction to the ferric chloride nitro group detection test. PRN can be oxidized by chromic acid to form corresponding compound which on oxidation with permanganate, yields carboxylic acid [38].

Microbial Pyrrolnitrin Production and Recovery
Microbial synthesis of PRN is easy, reliable and eco-friendly and requires low-cost medium constituents, ambient conditions for growth and production, the least additional energy requirements and minimum expensive equipment. This is the major reason microbial synthesis of PRN has become the preferred alternative to chemical processes [49]. After initial isolation of PRN from Pseudomonas pyrrocinia [38] and thereafter reports from different fluorescent and non-fluorescent Pseudomonas species [53,64], several strains of Burkholderia cepacia, Corallococcus exiguus, Cystobacter ferrugineus, Enterobacter agglomerans, Myxococcus fulvus, Serratia spp. and Actinosporangium vitaminophilum have been classified to produce PRN in varying quantities [65][66][67][68]. Serratia plymuthica [69] and S. ruhidaea [70] are identified for enhanced production of PRN. Recently, a strain belonging to Burkholderia cepacia complex, JKB9, showing broad-spectrum antifungal activity, was held responsible for suppressing growth of Phytophthora capsici, Fusarium oxysporum and Rhizoctonia solani [71]. This strain, which has shown stronger antifungal activity than Burkholderia strains KCTC2973 and ATCC25416 against Phytophthora blight, was confirmed for PRN production using thin layer chromatography (TPC), high performance liquid chromatography (HPLC) and Nuclear Magnetic Resonance (NMR) spectrometric studies. Complete genome sequencing of Burkholderia pyrrocinia 2327 T revealed insights into the cells possessing antibiotic capabilities for the biosynthesis of PRN [72]. Cloning of gene clusters responsible for encoding enzymes involved in the production of pyrrolnitrin in organisms has greatly helped in marking of the biosynthetic routes. Using an antibiotic producing strain of P. fluorescens [73] cloned four gene clusters to elucidate biochemistry of these molecules and to link it with the enzymes that may offer the routes for the synthesis of new chemical structures. Earlier, prnABCD operon from P. protegens Pf-5 was co-expressed in tomato plants with universal vector IL-60 and successfully demonstrated resistance to damping-off disease caused by R. solani [74].
Microbial wild type strains secrete PRN in low quantity (Table 3) and production varies with the medium constituents. P. aureofaciens ATCC 15926 strain when grown in minimal medium, secreted PRN in low concentration (<0.3 µg mL −1 ). Even optimized variation of constituents in growth medium could not increase PRN production. However, the production enhanced by 30-fold when P. aureofaciens ATCC 15926 was mutated with N-methyl-N'-nitro-N-nitrosoguanidine [75]. Addition of DL-tryptophan (1 mg mL −1 ) in CMM medium also doubled PRN production after 120 h but additional amount of tryptophan resulted in less yield [76]. Besides intracellular production of PRN from Pseudomonas spp., the excretion of the compound was also detected in the supernatant of fermented medium of Serratia marcescens strain ETR17 [85]. B. cepacia yielded 0.54 mg L −1 of PRN in monosodium glutamate medium at 27 • C as quantified by preparative HPLC [66]. Initially, Elander et al. [64] reported that only 27.58% Pseudomonas spp. secreted PRN in shake flask fermentation propagated in CMM, C, or E media. The authors concluded that P. multivorans C653 (ATCC 17760) showed maximum PRN production in medium C, followed by E and then CMM. P. aureofaciens was shown to secrete moderate PRN in CMM medium (40-80 µg mL −1 ). The PRN concentration increased in D-tryptophan amended medium where it was incorporated in the biosynthesis of PRN.
While growing P. aureofaciens in isotopically labeled tryptophan (at different positions) containing medium, Martin et al. [86] demonstrated that amino nitrogen of D-tryptophan became the nitro group of PRN. The two chlorine atoms in PRN, C3 of side chain became pyrrole and C2 of the indole nucleus got retained during biosynthesis (Figure 1). Furthermore, Chang et al. [77] confirmed that H-2 and H-α of the indole and side chain give rise to H-5 and H-2 of PRN, respectively, and, thus, proposed that L-tryptophan is the immediate precursor in PRN biosynthetic pathway. PRN formation using labeled tryptophan showed that L-rather than D-tryptophan was the immediate precursor of PRN [87]. 7-chloroindole-3-acetic acid, 3-chloroanthranilate detected in fermented medium revealed that 7-chlorotryptophan served as a common precursor for PRN [88].
Variety of production media and their pH remained a key parameter to influence PRN secretion. Shake flask fermentation of P. cepacia LT4-12-W revealed that the final yield (at 168 h) of PRN almost doubled at pH 5.8. Amendment of MS medium with glutamate salt of sodium yielded 60.50 mg mL −1 of PRM secretion [89]. The effect of different physicochemical conditions on plasmid-mediated PRN secretion has also been reported from Acinetobacter haemolyticus A19 isolate from wheat rhizosphere [83].
Recovery strategy of PRN involves cell growth in appropriate medium, extraction in acetone followed by removal of oily matter from concentrated acetone solution using petroleum benzene [38]. From fermented broth at pH 10 or 11 (6 mL) with NaOH, cell pellet centrifugation following sonication with acetone (600 µL) for 1 min, separation of acetone supernatant, re-extraction of pellets again in acetone (300 µL) and drying of acetone extract also yield PRN extract [59]. Further, fermented cultures were extracted after 48 h with equal volume of ethyl acetate [90] and centrifuged. Pellets sonicated twice with ethyl acetate (5 mL) for 3 min then recovery of organic phase [91] resulted in PRN rich dried extract [92]. Majumdar et al. [83] reported lysis of 18 h culture of Acinetobacter haemolyticus A19 using 1% SDS followed by sonication for 5-15 min and supernatant collection for PRN. In the case of bioactivity and characterization study, chromatographic separation techniques such as column chromatography and flash column with different mobile phases were explored (Table 4).

Analytical Characteristics of Pyrrolnitrin
PRN is chemically substituted with 3-phenyl pyrrole derivative containing two chlorine atoms and a nitro group [57].
Separation of PRN from bacterial media extract using TLC utilized various stationary phases such as silica gel G, GF 254 , 60 F 254 , KCI8 F, C18 Glass and several mobile phases. PRN can be detected on TLC under UV transilluminator [83,100] and visualized by spraying diazotized sulfanilic acid (DSA) or Pauly's, Ehrlich's and van Urk's reagent to develop maroon and violet color, respectively [101,102] or H 2 SO 4 on Silica Gel G plate [64]. The R f value for various TLC system served to identify PRN from different bacterial species. The compound has been analyzed by retention time in gradient HPLC system [65] but isocratic solvent system of 45% water, 30% acetonitrile, and 25% methanol also separated pyrrolnitrin at 252 nm in preparative HPLC [102]. Modifications in the polarity of solvents, mobile-stationary phase and elution methods are key strategies to quantify PRN using HPLC (Table 5). Yellow colored PRN molecule isolated from Pseudomonas pyrrocinia absorbs at 252 nm with molar extinction coefficient of ε = 7500 in ethanol [26]. Myxobacterial PRN also showed λ max at 252 nm in methanol [94]. Functional group stretching in FTIR vary with different PRN derivatives due to its structural features. Typical bond stretching at 1530 and 1375 cm −1 characterized for nitro group [38] while 3489 cm −1 represent pyrrole ring. Similarly, PRN isolated from supernatant of fermented medium inoculated by Myxococcus fulvus strain Mx f147 indicated infrared spectrum to confirm pyrrole ring (3460), nitro group (1530 and 1375), CH 3 (stretch) (1460) and C=C aromatic weak intensity (1600) [94]. Mass spectroscopy (MS) of PRN is ascertain using different ionization techniques. MS of PRN isolated from Pseudomonas cepacia B37w showed molecular ion at m/z 256 with the formula C 10 H 6 C 12 N 2 O 2 [59]. Electrospray mass spectroscopy (negative ion spectrum) of PRN further confirmed (mass-to-charge ratio; m/z) at 256 [66]. High-resolution mass spectrometry of the two molecular ions gave m/z 255.9826 and 257.9777, respectively, indicating the molecular formula C 10   NMR spectroscopy is widely used for analytical measurement of microbial metabolites. The PRN is confirmed by NMR spectrum [59] [89]. NMR spectrum of purified PRN secreted by plasmid-mediated A. haemolyticus A19 revealed the values δ: 6.2-6.6 (m, 2H, H-2, H-5), 6.77 (q 1H, H6), 7.03 (m, 1H, H-4), 7.38 (m2H, Ha, Hc) compared with standard 1H NMR spectrum of [65]. PRN synthesized from Myxococcus fulvus strain Mx f147 showed 13C NMR spectrum (in acetone-d6; Bruker 400 MHz) [94]. Structural investigation of PRN with X-ray analysis revealed the presence of two molecules with observed density of 1.74 g/cm 3 that lie opposite to each other about the center of symmetry. It further confirmed the location of two Cl atoms in the asymmetric unit with 3D Patterson function, dihedral angle of the pyrrole, the benzene rings and chlorine substitution on pyrrole ring located apart from the nitro group [109].

Biochemistry of Pyrrolnitrin
Microbial synthesis of PRN requires D-tryptophan, but cost of precursor amino acid and intracellular secretion limits its large-scale production. The NO 2 group is derived from anthranilic acid, phenylalanine and tryptophan that could serve as a precursor for PRN secretion [57]. However, anthranilic acid and L-phenylalanine usually decrease PRN secretion in P. aureofaciens and B. cepacian [66], while tryptophan stimulates PRN production [57,101]. In the medium, L-tryptophan gets quick intake within the cells than the D-isomer but addition of L-isomer could not yield more PRN secretion [110]. In actinomycetes, D-tryptophan enhances secretion of PRN when added separately in the culture medium [101] and maximum accumulation was observed at stationary phase after 120 h [66,101]. It indicated that the L-isomer of tryptophan enter cells quickly and participate in the protein synthesis, while D-tryptophan enter slowly and available at the time of antibiotic secretion [55]. Besides, L-glutamic acid amended medium showed maximum antifungal activity, which substantially declined with the addition of L-tryptophan, L-valine, L-serine, L-phenylalanine and L-cysteine [66]. In brief, D-tryptophan and L-glutamic acid are more direct precursors of PRN than any other amino acids.
PRN biosynthesis was unraveled in P. aureofaciens [77,101]. Later, genes (prnABCD operon) and corresponding enzymes involved were delineated in P. fluorescens BL915 (Figure 2) [90,111]. The biosynthesis of PRN occurs in four sequential steps: chlorination by prnA, rearrangement and decarboxylation by prnB, chlorination by prnC and oxidation by prnD enzyme (Figure 1). This involves regioselective halogenation of tryptophan through the addition of chlorine into D-tryptophan by tryptophan 7-halogenase (prnA) following nucleophilic and electrophilic reactions [112] and activation of intermediate lysine-chloramine species as the first step [113,114]. Further, the reaction catalyzed by prnB shows structural similarity with two-domain indoleamine 2,3-dioxygenase enzyme (IDO) and involves several intermediary steps. The second step forms a binary complex that combines with L-tryptophan or 7-Cl-L-tryptophan to create a ternary complex. The third step in PRN biosynthetic pathway of P. fluorescens leads to catalytic conversion of mono-chloro-deamino-pyrrolnitrin into amino-pyrrolnitrin by regioselectivity using halogenating and chlorinating enzyme [115]. In the last step, prnD catalyzes the oxidation of amino group of aminopyrrolnitrin to nitro group and thus forms PRN [90,111,116].
(PrnA). Further, the enzyme PrnB (monodechloroaminopyrrolnitrin synthase catalyzes formation of monodechloroaminopyrrolnitrin from 7-chlorotryptophan while the enzyme PrnC leads to catalytic reaction for the conversion of monodechloroaminopyrrolnitrin into aminopyrrolnitrin. In the last step, aminopyrrolnitrin is converted to pyrrolnitrin with the help of the enzyme PrnD (aminopyrrolnitrin oxygenase). Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used "conceptualization, X.X. and Y.Y.; methodology, X.X.; software, X.X.; validation, X.X., Y.Y. and Z.Z.; formal analysis, X.X.; investigation, X.X.; resources, X.X.; data curation, X.X.; writing-original draft preparation, X.X.; writing-review and editing, X.X.; Aminopyrrolnitrin oxidase or arylamine oxygenase (rieske N-oxygenase) catalyzes oxidation of an arylamine into the arylnitro group.
Except prnB, trptophan-7-halogenase (prnA), monodechloroaminopyrrolnitrin (prnC) and aminopyrrolnitrin oxidase (prnD) enzymes require flavin reductase (prnF) gene located close to the prnABCD operon which is considered as a part of the cluster [117]. Bioinformatics clubbed with the biochemical tools identified the role of prnF gene in prnD-catalyzed unusual arylamine oxidation in Pseudomonas fluorescens Pf-5 [118]. The prnF and prnD genes form a two-component oxygenase system, in which the gene product enzyme prnF supplies the reduced flavin to prnD. The prnF requires NADH as an electron donor to reduce FAD so that reduced FAD supplies electrons from NADPH to the prnD oxygenase component through protein-protein interactions in order to protect the flavin from oxidation.

Biological Activity
Structure-activity mechanism reveals that the primary target of PRN lies in the cell membrane to impede protein, RNA, DNA synthesis and uncouple the normal electron flow in the respiratory electron transport chain [128]. The metabolite has demonstrated biological activity at low concentration and act as an uncoupler of oxidative phosphorylation in Neurospora crassa. High concentration of PRN causes impairment of electron transport in flavin region and cytochrome c oxidase; accumulation of glycerol; synthesis of triacyl glycerol leading to leakage of cell membrane and inhibition of cell growth; in vitro activity against bacteria and fungi in the range of 1-100 µg mL −1 ; in vitro activity against leukemia and melanoma cell lines; and moderate antimycobacterial activity at 8 µg mL −1 [129]. The halometabolite was used as a drug lead for fenipoclonil and fludioxonil synthesis [130]. The amino derivative of PRN was identified as an androgen receptor antagonist [131]. PRN has the unique property to persist actively in the soil over a month, and can be readily diffused and slowly released after lysis of host bacterial cell [132]. However, the compound is sensitive to decomposition due to light [98].
Inhibitory effect of PRN is seen on the mitochondrial electron transport system of Neurospora crassa 74A [66]. Studies using N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD) confirmed that PRN block transfer of electron between the dehydrogenases and cytochrome c-oxidase components of the respiratory chain. At low concentrations, PRN uncouples oxidative phosphorylation in Neurospora mitochondria and impedes electron transport in both the Flavin region and cytochrome C oxidase at high concentration [133]. PRN also function as a signal molecule, beyond its role as a bioactive molecule to suppress fungal and affected cell motility [134]. Antifungal activity of the compound increased at pH 6.0, became maximum at pH 10 or 11 and declined after pH 11. Temperature influence on antifungal activity was maximum at 28 • C. Similarly, 2% NaCl content in the medium showed maximum activity. Such studies indicated more scope for medium modifications for obtaining maximum PRN production followed by maximizing biological activity of the compound.

23.
Caenorhabditis elegans 0.1 [84] Bacterial growth inhibition by PRN forms complex with phospholipids of cell membranes that eventually cease cellular respiration [138]. Furthermore, PRN causes leakage of A260 mµ absorbing material inside the cells and impairs synthesis of protein, DNA and RNA [138]. However, in vitro protein synthesis in PRN treated Rhizoctonia solani and Escherichia coli remained unaffected [139]. It bursts protoplast of Bacillus megaterium KM at growth inhibitory concentration [138]. The multitudes and range of activity of PRN makes it a preferred bioactive compound for agricultural chemical sector.
PRN found most prolific applications in controlling damping-off disease of cotton and cucumber, tan spot of wheat, storage molds of pome fruits, seedling disease of cotton, dry rot of potato and sclerotinia wilt of sunflower [73]. More usage of the compound lies in its significant antibiotic activity and low toxicity to mammalian species [146]. Wounds on apple and pear were challenged with a conidial suspension of antagonist grey mold B. cinerea and blue mold Penicillium expansum to investigate the efficacy of pyrrolnitrin (6-200 µg mL −1 ) to control diseases at 2 and 24 • C after harvest. High concentrations of PRN proved effective at 24 • C on both diseases of apple and pear, while low concentrations appeared effective at cold temperature [147]. Hence, PRN is an attractive strategy to control postharvest diseases on fruits, vegetables and other agricultural products being produced at low temperature conditions. In a preliminary field experiment on strawberries, postharvest treatment with PRN (250 mg L −1 ) at low storage temperature delayed development of post-harvest rot by 2-4 days, but did not reduce rate of development [79] and spoilage to acceptable levels.
In greenhouse studies, PRN showed prominent activity against Pyricularia oryzae and Botrytis cinerea [148]. The PRN producer P. chlororaphis O6 has shown antifungal activities both in vitro and in planta [82] on tomato against late blight disease and demonstrated major antagonism. In addition, biocontrol of fungal disease Fusarium Head Blight (FHB) caused by F. graminearum on wheat heads in growth chamber conditions was studied using strain Pseudomonas chlororaphis G05 co-treated with: (i) wild-type strain G05; (ii) phz-deleted mutant G05∆phz; and (iii) mutant G05∆prn. The experiment showed wheat heads were infected with F. graminearum at rates of 5-8% and 80-90%, respectively, when co-sprayed with wild-type strain G05 and mutant G05∆prn [144], and PRN of wild type strain was found to be vigorously active against FHB disease.
The glasshouse experiments with talc-based formulation of S. marcescens ETR17 were similar to in vitro studies. Incidence of root rot in bacteria treated tea plants were considerably lower in comparison to untreated control as well as the fungicide treated sets. Additionally, ETR17 formulation also increased the root and shoot length of the tea seedlings under both sterile and unsterile soil conditions in comparison to the untreated controls [85].

Pharmaceutical Applications
Pyrrolnitrin demonstrated strong protecting activity against various pathogenic fungi, especially against dermatophytosis [149]. It has been recommended for the treatment of superficial fungal infection of dermatophytic Trychophyton in Japan [150,151]. A patent has been granted on antifungal composition containing pyrrolnitrin and antimycotic imidazole compound in 1987 [152]. The product was commercialized under trade name Pyro-Ace W powder Spray by Fujisawa Pharmaceutical Company Ltd., Osaka. This was marketed by Pharmacia in Italy as "Micutrin" and, in combination with betamethasone valerate, it was formulated as "Beta Micutrin" for athlete's foot and ring worm diseases. The derivative, 3-cyanopyrroles, is more biologically active as pyrrolnitrin and very stable under light [153]. Jespers and co-workers (1993) reported a Fenpiclonil (CGA 142705) with more cytotoxicity for the representatives of Ascomycetes, Basidiomycetes, and Deuteromycetes. PRN formulated with carboxymethyl cellulose (5%) was injected intraperitoneally into mice [154] and LD 50 was observed at a dosage of 500 mg Kg −1 [38].
In pharmacology, in vitro radioactive studies of pyrrolnitrin reflected that pyrrole ring is readily oxidized by enzymes undetected in urine and bile after administration [96]. Along with this, surface antigens of Candida albicans were released after treatment with PRN [38]. It also showed cytotoxicity at 10 µg mL −1 after 24 h and highest after 72 h on rat clonal pancreatic β-lines, INS-1. Thus, the compound becomes diabetogenic but appears nontoxic and insulinotropic at lower concentration [146]. PRN affected physiology of Caenorhabditis elegans, acted as repellent for adult nematodes to lower egg hatching by almost <50% at higher concentrations of PRN (1, 5, and 10 µg mL −1 ) after 24 h of exposure [84].

Conclusions
Natural bioactive PRN from different subgroups of rhizobacterial species display an array of biological properties, most prominently being the antifungal activity. Besides the leads on the formulation development and commercialization of the products for human and plant disease management, there exists tremendous scope with this small molecule for future research on making prominent functional derivatives with unmatched biological properties. The knowledge about metabolic route for biosynthesis, network of genes and enzymes linked with the intermediates, optimization of process parameters, assessment of efficient producer strains and optimized nutrient requirements of microbial species for improved PRN production need further improvement. We systematically rationalized chemistry and biological applications of PRN. However, the search for hypersecretory bacterial strains from the rhizosphere and soil habitat for economic production is being realized for maximum optimization of productivity of the molecule. Microbial systems tolerant to a wide range of organic solvents of industrial use might be a new route to economic PRN biosynthesis. Application of halogenase from high yielding bacteria could help to overcome issues of regioselectivity, dependency on chemical synthetic route and low yield of PRN. Besides, organic solvent tolerant halogenases for tailor-made synthesis and simplified downstream operations possible for PRN and green synthesis routes could also support industrial processes for PRN production.
Author Contributions: For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used "conceptualization, X.X. and Y.Y.; methodology, X.X.; software, X.X.; validation, X.X., Y.Y. and Z.Z.; formal analysis, X.X.; investigation, X.X.; resources, X.X.; data curation, X.X.; writing-original draft preparation, X.X.; writing-review and editing, X.X.; visualization, X.X.; supervision, X.X.; project administration, X.X.; funding acquisition, Y.Y.", please turn to the CRediT taxonomy for the term explanation. Authorship must be limited to those who have contributed substantially to the work reported. SP and AC prepared the MS. DPS has edited and reviewed the MS. RS has discussed biosynthetic pathways for PRN production. RP has contributed in shaping the MS in journal's style and referencing pattern.

Acknowledgments:
In this section you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments). DPS and RS are thankful to Indian Council of Agricultural Research, India for institutional support. RP is thankful to Department of Science and Technology, Government of India for WOS-B project funding.
Conflicts of Interest: Declare conflicts of interest or state "The authors declare no conflict of interest." Authors must identify and declare any personal circumstances or interest that may be perceived as inappropriately influencing the representation or interpretation of reported research results. Any role of the funders in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript, or in the decision to publish the results must be declared in this section. If there is no role, please state "The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results". The authors declare no Conflict of interest.