catalysts

Review Recent Advances in Flavin-Dependent Halogenase Biocatalysis: Sourcing, Engineering, and Application

Johannes Büchler †, Athena Papadopoulou † and Rebecca Buller * Department of Life Sciences and Facility Management, Competence Center for Biocatalysis, Institute of Chemistry and Biotechnology, Zurich University of Applied Sciences, Einsiedlerstrasse 31, 8820 Waedenswil, Switzerland; [email protected] (J.B.); [email protected] (A.P.) * Correspondence: [email protected]; Tel.: +41-58-934-5438 These authors contributed equally. †


Received: 15 November 2019; Accepted: 2 December 2019; Published: 5 December 2019


Abstract: The introduction of a halogen atom into a small molecule can effectively modulate its properties, yielding bioactive substances of agrochemical and pharmaceutical interest. Consequently, the development of selective halogenation strategies is of high technological value. Besides chemical methodologies, enzymatic halogenations have received increased interest as they allow the selective installation of halogen atoms in molecular scaffolds of varying complexity under mild reaction conditions. Today, a comprehensive library of aromatic halogenases exists, and enzyme as well as reaction engineering approaches are being explored to broaden this enzyme family’s biocatalytic application range. In this review, we highlight recent developments in the sourcing, engineering, and application of flavin-dependent halogenases with a special focus on chemoenzymatic and coupled biosynthetic approaches.

Keywords: flavin-dependent halogenases; enzyme engineering; anion promiscuity; reaction engineering; co-factor recycling; biocatalysis

1. Introduction In nature, over 5000 halogenated products have been described since their first discovery over 100 years ago including potent antimicrobial agents such as vancomycin and chloramphenicol (Figure1)[ 1]. The incorporation of a halogen can significantly alter a molecule’s properties and may impact its bioactivity, metabolism, and pharmacokinetic profile [2]. As a consequence, the selective halogenation of small molecules is of great interest in the pharmaceutical and agrochemical industry. Around 20% of small molecule drugs and approximately 30% of agrochemicals are halogenated [3] Examples in the pharmaceutical industry include blockbusters such as rivaroxaban and empagliflozin with annual sales revenues in 2018 of $6.6 and $2.3 billion, respectively (Figure1)[ 4]. Furthermore, C-X motifs are useful synthetic handles for modification of the molecule in further chemical steps, making halogenated species common intermediates in synthetic manufacturing routes [5,6]. However, chemical halogenation, even though being a well-established technology, suffers from the use of hazardous or even highly toxic chemicals and sometimes poor atom efficiency [7,8]. In addition, chemical methods often lack selectivity or have specific demands on the substrate structure [8,9]. Consequently, the replacement of chemical halogenation by biological means may offer a more selective, greener, and less dangerous process route toward molecules of industrial interest. Biocatalytic halogenation is carried out by enzymes called halogenases or haloperoxidases, which can be classified according to their catalytic mechanism: heme, vanadium, and flavin-dependent halogenases follow an electrophilic mechanism, while non-heme iron halogenases halogenate through the formation of radical intermediates, and S-adenosyl-L-methionine (SAM) fluorinases react via a

Catalysts 2019, 9, 1030; doi:10.3390/catal9121030 www.mdpi.com/journal/catalysts Catalysts 2019, 9, 1030 2 of 20

nucleophilic pathway [10]. This review will focus on work carried out on flavin-dependent halogenases (electrophilic halogenases), which in contrast to heme- and vanadium-dependent halogenases, Catalysts 2019, 9, 1030 2 of 21 selectively derivatize small molecules, making them especially interesting for applications.

Figure 1. SelectionFigure of 1. valuableSelection of halogenated valuable halogenated agrochem agrochemicals,icals, pharmaceuticals pharmaceuticals and natural and products. natural products. Flavin-dependent halogenases (Fl-Hals) use reduced flavin, bound in the FAD binding site of the Biocatalyticenzyme, halogenation to create a C4a-hydroperoxyflavin is carried out speciesby en uponzymes reaction called with oxygenhalogenases [11]. The generated or haloperoxidases, peroxy species cannot react directly with the substrate as the flavin binding site and the substrate which can bebinding classified site are separatedaccordin byg an approximatelyto their catalytic 10 Å long tunnelmechanism: [12–15]. Instead, heme, crystal vanadium, structures of and flavin- dependent halogenasesflavin-dependent follow halogenases an showelectrophilic the presence ofmechanism, a chloride binding while site near non-heme the flavin binding iron halogenases halogenate throughsite [15,16 the], and formation mechanistic studies of suggestradical that in a nucleophilictermediates, attack ofand the chlorideS-adenosyl-L-methionine on the peroxyflavin (SAM) may create hypohalous acid (HOX) [11]. The hypohalous acid then travels to the substrate binding fluorinases reactsite via and a is nucleophilic believed to interact pathway with an active [10]. site This lysine review either by will reaction focus or via on hydrogen work carried bonding out on flavin- dependent halogenases(Figure2)[ 16, 17(electrophilic]. It is assumed that halogenases), this electrophilic chlorine which species in iscontrast responsible to for heme- electrophilic and vanadium- aromatic substitution of the proximally bound substrates. Notably, an active site glutamate residue dependent halogenases,has also been foundselectively important derivatize for catalysis; however,small molecules, its precise role inmaking the reaction them mechanism especially still interesting for applications.remains debated [2,18]. Flavin-dependent halogenases (Fl-Hals) use reduced flavin, bound in the FAD binding site of the enzyme, to create a C4a-hydroperoxyflavin species upon reaction with oxygen [11]. The generated peroxy species cannot react directly with the substrate as the flavin binding site and the substrate binding site are separated by an approximately 10 Å long tunnel [12–15]. Instead, crystal structures of flavin-dependent halogenases show the presence of a chloride binding site near the flavin binding site [15,16], and mechanistic studies suggest that a nucleophilic attack of the chloride on the peroxyflavin may create hypohalous acid (HOX) [11]. The hypohalous acid then travels to the substrate binding site and is believed to interact with an active site lysine either by reaction or via hydrogen bonding (Figure 2) [16,17]. It is assumed that this electrophilic chlorine species is responsible for electrophilic aromatic substitution of the proximally bound substrates. Notably, an active site glutamate residue has also been found important for catalysis; however, its precise role in the reaction mechanism still remains debated [2,18]. Catalysts 2019, 9, 1030 3 of 20 Catalysts 2019, 9, 1030 3 of 21

FigureFigure 2. Reaction 2. Reaction mechanism mechanism of flavin-dependent of flavin-dependent halo halogenases.genases. Reduced Reduced flavin flavin reacts reacts with with oxygen oxygen to formto form a peroxyflavin a peroxyflavin species, species, which which in turn in turn reac reactsts with with a halide a halide ion, ion, generating generating free free hypohalous hypohalous acid.acid. The Thehypohalous hypohalous acid acid travels travels through through an a anpproximately approximately 10 Å 10 tunnel Å tunnel before before reacting reacting with with an an activeactive site site lysine. lysine. Through Through an electrophilicelectrophilic aromatic aromatic substitution substitution mechanism, mechanism, the halogenatedthe halogenated aromatic aromaticcompound compound is formed. is formed.

2. Enzyme2. Enzyme Diversity Diversity and and Substrate Substrate Scope Scope To Todate, date, a plethora a plethora of flavin-dependent of flavin-dependent halogenase halogenasess (Fl-Hals) (Fl-Hals) has has been been identified. identified. Fl-Hals Fl-Hals accept accept a a rangerange of didifferentfferent substratessubstrates but but preferentially preferentially have have been been found found to halogenate to halogenate L-tryptophan L-tryptophan (Figure 3: (Figurered square) 3: red [19square)], pyrrole-like [19], pyrrole-like compounds (Figurecompou3:nds green (Figure triangle) 3: [ green20], phenolic triangle) compounds [20], phenolic (Figure 3: compoundsblue circle) (Figure [21], and 3: blue indole circle) (Figure [21],3: and orange indole square) (Figure [ 22 ].3: Inorange this section, square) we [22]. will In give this ansection, overview we of willthe give natural an overview diversity of flavin-dependentthe natural diversit halogenasesy of flavin-dependent emphasizing halogenases the recently discoveredemphasizing members the recentlyof this discovered enzyme family. members of this enzyme family. TheThe first first flavin-dependent flavin-dependent halogenaseshalogenases were were identified identified in thein mid-1990sthe mid-1990s [19,23]. [19,23]. PrnA, discovered PrnA, discoveredin Pseudomonas in Pseudomonas fluorescens influorescens 1998, is for in example 1998, involvedis for example in the biosynthesis involved ofin pyrrolnitrin,the biosynthesis a secondary of pyrrolnitrin,metabolite a secondary derived from metabolite L-tryptophan derived with from a L-tryptophan strong antifungal with a activity strong antifungal [19]. PrnA activity catalyzes [19]. the PrnAchlorination catalyzes atthe the chlorination 7-position of at the the indole 7-position moiety ofof L-tryptophanthe indole moiety (Product of TypeL-tryptophan I, Figure3 ).(Product Following Typethis I, discovery,Figure 3). Following research intensified this discovery, and manyresearch additional intensified L-tryptophan and many additional Fl-Hals (Trp-Fl-Hals) L-tryptophan were Fl-Halsdescribed (Trp-Fl-Hals) [19,24–27 were]. The described widely applied[19,24–27]. 7-tryptophan The widely halogenase applied 7-tryptophan RebH, for example,halogenase stems RebH, from for theexample, bacterium stemsLechevalieria from the bacterium aerocolonigenes Lechevalieriaand its naturalaerocolonigenes function and isthe its chlorinationnatural function of precursors is the chlorinationof the antitumor of precursors agent rebeccamycin of the antitumor [24]. Anotheragent rebeccamycin Fl-Hal able to[24]. halogenate Another atFl-Hal the 7-position able to is halogenateKtzQ found at the in Kutzneria7-positionsp. is KtzQ 744 [26 found]. This in halogenaseKutzneria sp. is involved744 [26]. This in the halogenase biosynthesis is involved of Kutzneride, in thean biosynthesis antifungal of nonribosomal Kutzneride, an hexadepsipeptide. antifungal nonribosomal After chlorination hexadepsipeptide. at the 7-position, After chlorination tryptophan at is further chlorinated at the 6-position by KtzR, which has a 120-fold preference for 7-Cl-L-Trp over the 7-position, tryptophan is further chlorinated at the 6-position∼ by KtzR, which has a ∼120-fold preferenceL-Trp [26 for]. Other7-Cl-L-Trp halogenases over L-Trp have [26]. a natural Other preference halogenases to chlorinate have a natural at the pr 6-positioneference ofto L-Tryptophan, chlorinate at thesuch 6-position as Thal fromof L-Tryptophan,Streptomyces such albogriseolus as Thal from[25] and Streptomyces a thermophilic albogriseolus tryptophan [25] and halogenase a thermophilic (Th-Hal) tryptophanfound in Streptomyceshalogenase (Th-Hal) violaceusniger found[28 in]. Furthermore,Streptomyces SttHviolaceusniger found in Streptomyces[28]. Furthermore, toxytricini SttH[29 found] and the in recentlyStreptomyces identified toxytricini halogenases [29] and in Saccharomonosporathe recently identifiedsp. CNQ-490 halogenases (Tar14) in [30 Saccharomonospora], as well as a halogenase sp. CNQ-490found through(Tar14) [30], metagenomic as well as a screening halogenase of foun soild samples through from metagenomic the Anza-Borrego screening desert of soil (BorH)samples [31 ] fromare the able Anza-Borrego to chlorinate desert and brominate (BorH) [31] at are the able 6-position to chlorinate of this and aromatic brominate amino at acid. the 6-position The 5-position of thisof aromatic the indole amino moiety acid. ofThe tryptophan 5-position of can the be in modifieddole moiety by of PyrH tryptophan from Streptomyces can be modified rugosporus by PyrH[27 ], fromSpmH Streptomyces from Streptomyces rugosporussp. [27], SCSIO SpmH 03032 from [32] andStreptomyces XszenFHal sp. identified SCSIO 03032 in Xenorhabdus [32] and szentirmaiiXszenFHal[33 ]. identifiedRecently, in aXenorhabdus novel set of szentirmaii flavin-dependent [33]. Recently, halogenases a novel called set of BrvH flavin-dependent [22], xcc-b100_1333, halogenases xcc-b100_4156, called BrvHand [22], xcc-b100_4345 xcc-b100_1333, [34] were xcc-b100_4156, described. Theseand xcc-b100_4345 enzymes were found[34] were to have described. a high sequenceThese enzymes identity to wereknown found Trp-Fl-Hals, to have a buthigh could sequence not convert identity L-tryptophan. to known Trp-Fl-Hals, This property but was could ascribed not toconvert the fact L- that tryptophan.certain amino This acidsproperty required wasto ascribed stabilize to tryptophan the fact that within certain the activeamino site acids pocket required were missingto stabilize [22,34 ]. tryptophanInstead, thesewithin halogenases the active site can pocket accept were smaller missing substrates [22,34]. such Instead, as indole these (Product halogenases Type can III, Figureaccept 3), smaller substrates such as indole (Product Type III, Figure 3), which triggered us to name these enzymes “Indole Fl-Hals” in the frame of this review, and prefer bromination over chlorination. BrvH Catalysts 2019, 9, 1030 4 of 20 which triggered us to name these enzymes “Indole Fl-Hals” in the frame of this review, and prefer brominationCatalysts 2019, over9, 1030 chlorination. BrvH was found in Brevundimonas BAL3, whereas the three xcc-b1004 of 21 enzymes stem from Xanthomonas campestris (Xcc). The plant pathogen Xcc belongs to a group of was found in Brevundimonas BAL3, whereas the three xcc-b100 enzymes stem from Xanthomonas Proteobacteria that is known to infect over 350 plant species by secreting a characteristic yellow pigment campestris (Xcc). The plant pathogen Xcc belongs to a group of Proteobacteria that is known to infect called xanthomonadin, a mixture of brominated aryl polyene esters. Finally, in 2019, a viral halogenase, over 350 plant species by secreting a characteristic yellow pigment called xanthomonadin, a mixture named VirX1, was discovered in a cyanophage and was shown to act on 6-azaindole (Product Type II, of brominated aryl polyene esters. Finally, in 2019, a viral halogenase, named VirX1, was discovered Figurein a cyanophage3)[35]. and was shown to act on 6-azaindole (Product Type II, Figure 3) [35].

FigureFigure 3. 3.Phylogenetic Phylogenetictree treedepicting depicting aa selectionselection ofof flavin-dependentflavin-dependent halogenases and their native products.products. TheThe tree was was constructed constructed using using the the maxi maximummum likelihood likelihood method, method, a bootstrap a bootstrap of 1000, of 1000,with withthe thealignment alignment based based on amino on amino acid acidss by byMEGAX MEGAX [36] [36 and] and MUSCLE MUSCLE [37]. [37 Branches]. Branches corresponding corresponding to topartitions partitions reproduced reproduced in in less less than than 50% 50% of of bootstrap bootstrap replicates replicates are are collapsed. collapsed.

Today,Today, the the biocatalytic biocatalytic toolbox toolbox contains contains flavin-dependent flavin-dependent halogenases halogenases that can that convert can molecules convert beyondmolecules L-tryptophan beyond L-tryptophan derivatives. derivatives. PrnC and PrnC PltA foundand PltA in Pseudomonasfound in Pseudomonas fluorescens fluorescens, for example,, for convertexample, pyrrole-like convert pyrrole-like substrates substrates [19,20]. While [19,20]. PltA Whil requirese PltA requires its substrate its substrate to be tethered to be tethered to a carrier to a proteincarrier [ 20protein], PrnC [20], acts onPrnC a free-standing acts on a free-standing intermediate intermediate in the biosynthesis in the ofbiosynthesis pyrrolnitrin. of After pyrrolnitrin. the PrnA catalyzedAfter the chlorination PrnA catalyzed of L-tryptophan chlorination toof form L-tryptophan 7-chloro-L-tryptophan to form 7-chloro-L-tryptophan (vide infra), which ( isvide followed infra), bywhich a ring is rearrangement followed by a andring a rearrangement decarboxylation and step, a decarboxylation PrnC chlorinates step, the 3-positionPrnC chlorinates of the pyrrolethe 3- ringposition to form of aminopyrrolnitrinthe pyrrole ring to (Productform aminopyrroln V, Figure3)[itrin19 ].(Product Phenols V, are Figure an additional 3) [19]. Phenols molecule are class an thatadditional is known molecule to be converted class that by is halogenasesknown to be such converted as Rdc2, by RadH, halogenases Bmp5, such and ChlAas Rdc2, [38 –RadH,41]. Phenolic Bmp5, halogenasesand ChlA [38–41]. typically Phenolic originate halogenases from fungi typically except originate for PltM, from which fungi was except isolated for from PltM,Pseudomonas which was fluorescensisolated fromPf-5 Pseudomonas [42]. Initially, fluorescens PltM was Pf-5 identified [42]. Initially, as one PltM of three was identified putative halogenases as one of three encoded putative in thehalogenases biosynthetic encoded gene cluster in the ofbiosynthetic pyoluteorin gene (one cluster of the of others pyoluteorin being PltA), (one of an the antifungal others being compound PltA), an antifungal compound containing a dichloropyrrole moiety [42]. Almost twenty years later, it was shown that PltM catalyzes mono- and di-chlorination of phloroglucinol (Product Type VI, Figure 3), yielding a compound that serves as a transcriptional factor of the pyoluteorin biosynthesis rather Catalysts 2019, 9, 1030 5 of 20 Catalysts 2019, 9, 1030 5 of 21 Catalysts 2019, 9, 1030 5 of 21 Catalysts 2019, 9, 1030 5 of 21 than a biosynthetic intermediate [43]. MalA’, a rather unusual halogenase from Malbranchea Catalyststhancontaining a 2019biosynthetic, 9,a 1030 dichloropyrrole intermediate moiety [43]. [42 ].MalA’, Almost a twenty rather years unusual later, halogenase it was shown from that PltMMalbranchea catalyzes5 of 21 thangraminicola a biosynthetic, can act onintermediate the bulky molecule [43]. MalA’, premalbranchea a rather mideunusual (Product halogenase Type VII, from Figure Malbranchea 3) [44]. graminicolamono- and, can di-chlorination act on the bulky of molecule phloroglucinol premalbranchea (Productmide Type (Product VI, Figure Type3), yieldingVII, Figure a compound3) [44]. graminicolaExcept, can for act the on very the electronegative bulky molecule fluoride premalbranchea ion, flavin-dependentmide (Product haloge Typenases VII, Figure have been 3) [44]. shown thanthat Excepta servesbiosynthetic for as the a very transcriptional intermediate electronegative factor[43]. fluoride MalA’, of the ion, pyoluteorina ratherflavin-dependent unusual biosynthesis halogenase haloge rathernases from than have aMalbrancheabeen biosynthetic shown to introduceExcept for all the common very electronegative halides into small fluoride molecules. ion, flavin-dependent However, certain haloge anionnases preferences have been exist: shown The graminicolatointermediate introduce, can all [commonact43]. on MalA’, the halides bulky a rather moleculeinto unusual small premalbranchea molecules. halogenase However, frommideMalbranchea (Product certain anion Type graminicola preferencesVII, Figure, can 3)exist: act [44]. on The the towell introduce describedCatalysts all common Trp-Fl-Hals 2019, 9, 1030 halides RebH, into Thal small and molecules. PyrH prefer However, chlorination certain anionover brominationpreferences exist:[24,25,27],5 of The 21 wellbulky Exceptdescribed molecule for theTrp-Fl-Hals premalbrancheamide very electronegative RebH, Thal fluoride (Productand PyrH ion, Type prefer flavin-dependent VII, chlorination Figure3)[44 haloge]. over nasesbromination have been [24,25,27], shown wellwhile described BrvH and Trp-Fl-Hals VirX1 prefer RebH, bromination Thal and over PyrH chlo preferrination chlorination [22,35]. For over the brominationthree xcc-b100 [24,25,27], enzymes, towhile introduce BrvHExcept CatalystsCatalysts andCatalystsall for common VirX1the 2019 20192019 very,, , 9 99 ,, prefer, 1030 10301030 halides electronegative bromination into small fluoride overmolecules. chlo ion,rination However, flavin-dependent [22,35]. certain For halogenasesanionthe three preferences xcc-b100 have been exist:enzymes,555 ofofof 21 shown21 21The whileno chlorination BrvHthan and aVirX1was biosynthetic observed, prefer brominationintermediate and iodination over[43]. was chloMalA’, onlyrination a found rather [22,35]. for unusual Bmp5, For thehalogenasePltM, three and xcc-b100 VirX1from Malbranchea[34,35,40,45] enzymes, wellnoto chlorination introducedescribed allTrp-Fl-Halswas common observed, RebH, halides and iodinationThal into and small PyrHwas molecules. only prefer found chlorination However, for Bmp5, certain overPltM, bromination and anion VirX1 preferences [34,35,40,45] [24,25,27], exist: (Table 1). graminicolathanthanthan aaa biosyntheticbiosyntheticbiosynthetic, can act on theintermediateintermediateintermediate bulky molecule [43].[43].[43]. MalA’,MalA’,premalbrancheaMalA’, aaa ratherratherrather mide unusualunusualunusual (Product halogenasehalogenasehalogenase Type VII, fromfromfrom Figure MalbrancheaMalbrancheaMalbranchea 3) [44]. no Thechlorination well described was observed, Trp-Fl-Hals and RebH,iodination Thal was and only PyrH found prefer for chlorination Bmp5, PltM, over and bromination VirX1 [34,35,40,45] [24,25,27 ], while(Table BrvH 1). graminicolaandgraminicolaExcept VirX1 ,for ,can canprefer the act act very on on bromination the theelectronegative bulky bulky molecule molecule over fluoride chlopremalbranchea premalbranchearination ion, flavin-dependent [22,35].midemide (Product (Product For thehaloge Type Type threenases VII, VII, xcc-b100 Figure Figurehave been 3) 3) enzymes,[44]. [44]. shown (TableLooking 1). graminicola forward,, can the act ondiscovery the bulky ofmolecule flavin-depen premalbrancheadent halogenasesmide (Product with Type VII,alternative Figure 3) [44].substrate nowhile chlorinationLooking BrvHto introduce andforward, wasExceptExceptExcept VirX1 observed, all for forfor thecommon preferthe thethe very verydiscoveryvery and bromination electronegative electronegativehalideselectronegative iodination ofinto flavin-depen small over was fluoride fluoridefluoride molecules. chlorinationonly ion, ion,ion, founddent flavin-dependent flavin-dependentflavin-dependent However, halogenases for [22 Bmp5,,35 certain]. For PltM, haloge halogehaloge withanion the andnasesnasesnases three alternativepreferences VirX1 have havehave xcc-b100 been beenbeen [34,35,40,45] exist: substrateshown shownshown enzymes, The preferencesLooking willforward, be fueled the bydiscovery the rapidly of flavin-depen expanding amountdent halogenases of genetic withinformation alternative available substrate from (Tablepreferencesno chlorination 1). well tototowill introduce introduce introducedescribed be was fueledobserved, all allall Trp-Fl-Hals common commoncommon by the and halides halides halidesrapidly RebH, iodination into intointo Thalexpand small smallsmall and was molecules. molecules.molecules. ingPyrH only amount prefer found However, However,However, chlorinationof for genetic Bmp5, certain certaincertain information anion overPltM,anionanion bromination preferences preferencespreferences and VirX1 available exist: exist:exist:[24,25,27], [34, 35 The TheThe from,40 , 45 ] preferencespublic databases. will be As fueled gene bysynthesi the rapidlys is becoming expand ingmore amount cost effective of genetic and information high throughput available assays from are public(TableLooking databases.1).whilewellwellwell forward, BrvH describeddescribeddescribed As andgene the VirX1Trp-Fl-HalsTrp-Fl-HalsTrp-Fl-Hals synthesi discovery prefers RebH, RebH, RebH,isbromination becomingof Thalflavin-depenThalThal andandand over more PyrHPyrHPyrH chlo dentcost preferpreferpreferrination effectivehalogenases chlorinationchlorinationchlorination [22,35]. and For overhighoverover withthe brominationthree brominationbrominationthroughput alternative xcc-b100 [24,25,27],[24,25,27],[24,25,27], assaysenzymes,substrate are publicbeing databases.established,nowhilewhile chlorination BrvH AsBrvH putative gene and and was synthesiVirX1 VirX1 halogenasesobserved, prefer prefers is brominationand brominationbecoming caniodination be verifi over moreover was chlo chloed costonly quicklyrinationrination effectivefound [22,35]. [22,35]. infor an Bmp5,and experimentalFor For high the PltM,the three threethroughput and xcc-b100 xcc-b100 VirX1 setup. [34,35,40,45] enzymes, assaysenzymes, In a recent are preferencesbeing established,Looking whilewill forward,be BrvH putative fueled and theVirX1 by halogenases discoverythe prefer rapidly bromination can of expand flavin-dependentbe verifi overing edchlo amount quicklyrination of halogenases [22,35].in genetic an experimental For information the with three alternativexcc-b100 setup. available enzymes,In a substrate recent from beingstudy established, from(Table nonoFisherno chlorination chlorinationchlorination 1). putative et al. 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[46], gene halogenasesthe synthesis authors is usedcan becoming be a genomeverifi moreed miningquickly cost eff approachinective an experimental and to high identify throughput setup. 128 putative In assaysa recent Fl- are Hals. 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[with46 putative AsAs],As enzyme the genegenegenethe authors synthesisynthesichaperoneshalogenasessynthesis were usedsss isis isscreened becomingbecomingbecoming acan genomeGroES be verifi moretowardsmoremoreand mininged costcost costquicklyGroEL, effectiveeffective approachelectroneffective in 87 an andandand halogenaseexperimentalrich to highhighhigh identify substrates throughputthroughputthroughput 128 candidatessetup. putativeknown assaysassaysassays In a recent areareare Fl-Hals.towere be (11commonly s/analysis). convertedbeing The established, soluble by Fl-Hals, enzymeputative as halogenasess wellwere as screened more can becomplex verifitowardsed substrates.quickly electron in an With experimentalrich substratesthis approach, setup. known In 39a recent new to be Fl- obtainedcommonlyBy co-expression instudy converted beingbeingsufficient from established,established, with Fisher bysoluble theFl-Hals, et putativeputative chaperonesal. amounts [46], as halogenases thehalogenaseswell authors GroESforas morethe usedcancan andlate complex bebe aGroEL,r verifigenomeverifi analysiseded substrates. quickly87quicklymining halogenasevia inhighinapproach anan With experimentalexperimentalthroughput candidates thisto identify approach, setup.setup. LC-MS 128 were InputativeIn 39 a obtaineda recentanalysis recentnew Fl- Fl- in commonlyHals were converted studystudystudyidentified, from fromfrom by Fisher FisherFisher Fl-Hals,some et etet al. al.al.of [46], [46],[46],as which well the thethe authors authors authorsas showed more used usedused complex a ahaa genome genomegenomelogenation substrates. mining miningmining of approach approachapproach Withcomplex thisto toto identify identifyidentify approach,compounds, 128 128128 putative putativeputative 39 newoffering Fl- Fl-Fl- Fl- (11Halssu s/analysis).ffi werecientHals. solubleidentified, TheBy co-expression amountssoluble some enzyme forof with thewhichs later thewere chaperones analysisshowed screened viaha GroES logenationtowards high and throughput electronGroEL, of complex 87 LC-MSrich halogenase substrates compounds, analysis candidates known (11 soffering/analysis). wereto be advantageousHals.Hals. starting ByBy co-expressionco-expression points for with withfurther thethe evolutionchaperoneschaperones [46]. GroESGroES andand GroEL,GroEL, 8787 halogenasehalogenase candidatescandidates werewere HalsThe were soluble obtainedidentified,Hals. enzymes By in co-expression sufficient weresome screened ofsoluble withwhich towardsamountsthe showedchaperones electronfor theha GroESlogenationlate richr analysis substratesand GroEL, of via complex knownhigh87 halogenase throughput to compounds, be commonly candidates LC-MS offeringanalysis convertedwere commonlyadvantageous convertedobtainedobtained starting inin by sufficientsufficientpoints Fl-Hals, for solublesoluble furtheras well amountsamounts asevolution more forfor complexthethe [46]. latelate rr analysis analysissubstrates. viavia highhigh With throughputthroughput this approach, LC-MSLC-MS 39 analysisanalysis new Fl- advantageous(11obtained s/analysis).starting in pointssufficient The soluble for soluble further enzyme amounts evolutions were for screenedthe [46]. late r analysistowards viaelectron high throughputrich substrates LC-MS known analysis to be Halsby Fl-Hals,were (11identified, as s/analysis). well as moresome The soluble complexof which enzyme substrates. showeds were screened Withhalogenation this towards approach, electronof complex 39 rich new substrates compounds, Fl-Hals known were identified,offeringto be commonly(11(11 s/analysis).s/analysis). converted TheThe soluble solubleby Fl-Hals,Table enzymeenzyme 1.as Halide wellss werewere as versatility more screenedscreened complex towardstowardsof Fl-Hals. substrates. electronelectron With richrich thissubstratessubstrates approach, knownknown 39 new toto bebe Fl- advantageoussome of whichcommonly starting showed converted points halogenation for by Fl-Hals,furtherTable of 1. as complexevolutionHalide well as versatility more compounds, [46]. complex of Fl-Hals. substrates. offering advantageousWith this approach, starting 39 new points Fl- for Halscommonlycommonly were identified, convertedconverted bybysome Fl-Hals,Fl-Hals, of whichasas wellwell asshowedas moremore complex complexhalogenation substrates.substrates. of complex WithWith thisthis approach,compounds,approach, 3939 newnewoffering Fl-Fl- HalsHals werewere identified,identified, someTablesome of of1. Halidewhichwhich showedversatilityshowed haha oflogenationlogenation Fl-Hals. ofof complexcomplex compounds,compounds, offeringoffering further evolutionadvantageousEnzymeHals were [46 identified,]. starting pointsHalogen some for of further (whichX) evolutionshowedLiterature ha[46].logenation of complex compounds, offering Enzymeadvantageous starting Halogenpoints for further(X) evolutionLiterature [46]. EnzymeRebHadvantageousadvantageous startingstartingHalogen Cl pointspointsTable > Br forfor 1.( X furtherfurther Halide) Literature evolutionevolution [24]versatility [46].[46]. of Fl-Hals. RebH Cl > TableBr 1. Halide [24] versatility of Fl-Hals. RebHThal Cl Cl > >Br Br Table 1. [24]Halide [25] versatility of Fl-Hals. EnzymeThal Halogen Cl > Br (TableTableTableX) 1. 1.1. Halide LiteratureHalide [25]Halide versatility versatilityversatility of ofof Fl-Hals. Fl-Hals.Fl-Hals. ThalPyrH Enzyme Enzyme Cl Cl > Halogen > Br Br Halogen (X) (X [25] [27]) Literature Literature RebHPyrH EnzymeEnzymeEnzyme Cl > Br HalogenHalogenHalogen ( ((XXX [24][27]))) LiteratureLiteratureLiterature PyrHKtzQ RebH ClCl > Br Cl > Br [27][26] [24] ThalKtzQRebH RebHRebHRebH Cl Cl> Br> Br Cl Cl Cl > >> Br BrBr [25][26] [24 [24] [24] [24]] KtzQPrnA Thal ClCl Cl > Br [26][19] [25] PyrHPrnAThal ThalThalThal Cl Cl> Br> Br Cl Cl Cl > >> Br BrBr [27][19] [25 [25] [25] [25]] Th-Hal PyrH Cl, Br Cl > Br [47] [27] PrnAPyrH PyrHPyrHPyrH Cl Cl > Br Cl Cl Cl > >> Br BrBr [19] [ 27 [27] [27] [27]] KtzQTh-Hal KtzQ ClCl, Br Cl [26][47] [26] Th-HalKtzRKtzQ KtzQKtzQKtzQ Cl,Cl Cl Br ClClCl [47][26] [ 26 [26][26][26]] Tryptophan-Fl-Hal PrnAKtzRPrnA PrnA Cl ClCl [19][26] [19 [19]] Tryptophan-Fl-Hal KtzRSttH PrnAPrnAPrnA ClCl, Br ClClCl [26][29] [19][19][19] Tryptophan-Fl-Hal Th-Hal Th-Hal Cl, Br Cl, Br [47] [47] SttHTh-Hal Th-HalTh-HalTh-Hal Cl, Cl, Br Br Cl,Cl,Cl, Br BrBr [29] [47 [47][47][47]] SttHTar14 Cl,Cl, Br Br [29][30] KtzRTar14KtzR KtzRKtzRKtzRKtzR ClCl, Cl Br ClClClCl [26][30] [26 [26][26][26]] Tryptophan-Fl-Hal Tryptophan-Fl-HalTryptophan-Fl-HalTryptophan-Fl-Hal Tar14SpmH Cl,Cl, Br Br [30][32] SttHSpmHSttH SttHSttHSttHSttH Cl, Cl, Br Br Cl,Cl,Cl,Cl, Br Br BrBr [29][32] [29 [29][29][29]] SpmHXszenFHalTar14 Tar14 Cl,Cl, Cl,Br Br Br Cl, Br [32][33] [ 30 [30]] Tar14XszenFHal Tar14Tar14Tar14 Cl, Br Cl,Cl,Cl, Br BrBr [30][33] [30][30][30] XszenFHalBorHSpmH SpmH SpmHSpmH Cl,Cl, Cl,Br Br Br Cl,Cl,Cl, BrBr Br [33][31] [ 32 [32][32]] SpmHBorH SpmH Cl, Br Cl, Br [32][31] [32] BorHBrvHXszenFHal XszenFHalXszenFHalXszenFHalXszenFHal Cl, Cl Cl, Br< Br BrCl,Cl, Cl,Cl, Br Br BrBr [31] [22] [ 33 [33][33][33]] XszenFHalBrvHBorH Cl, Cl Cl,

Catalysts 2019, 9, 1030 6 of 20

Catalysts 2019, 9, 1030 6 of 21 3. Cofactor and Reaction Engineering 3. Cofactor and Reaction Engineering As can be deduced from the reaction mechanism (Figure2), flavin-dependent halogenases require As can be deduced from the reaction mechanism (Figure 2), flavin-dependent halogenases a constant supply of reduced FADH2 to function. In nature, the oxidized flavin species is reduced require a constant supply of reduced FADH2 to function. In nature, the oxidized flavin species is by a flavin reductase (Fre). In the case of halogenase RebH, for example, the corresponding flavin reduced by a flavin reductase (Fre). In the case of halogenase RebH, for example, the corresponding reductase called RebF was identified in the native organism Lechevalieria aerocolonigenes [24]. Likewise, flavin reductase called RebF was identified in the native organism Lechevalieria aerocolonigenes [24]. a flavin halogenase–flavin reductase pair, PrnA and PrnF, was identified in Pseudomonas fluorescens. Likewise, a flavin halogenase–flavin reductase pair, PrnA and PrnF, was identified in Pseudomonas For in vitro studies of halogenases, however, alternative flavin reductases may be used. Examples fluorescens. For in vitro studies of halogenases, however, alternative flavin reductases may be used. comprise a thermophilic Fre from Bacillus subtilis WU-S2B [47], a Fre from Pseudomonas fluorescens [22], Examples comprise a thermophilic Fre from Bacillus subtilis WU-S2B [47], a Fre from Pseudomonas or a Fre from the Escherichia coli strain K12 [33]. Flavin reductases employ NADH to reduce flavin; fluorescens [22], or a Fre from the Escherichia coli strain K12 [33]. Flavin reductases employ NADH to therefore, biocatalytic applications may include an additional enzyme for the reduction of NADH such reduce flavin; therefore, biocatalytic applications may include an additional enzyme for the reduction as a glucose dehydrogenase (GDH) (Figure4) or an alcohol dehydrogenase (ADH). By adding the of NADH such as a glucose dehydrogenase (GDH) (Figure 4) or an alcohol dehydrogenase (ADH). necessary auxiliary enzymes (Fre and GDH or ADH), halogenation biocatalysis reactions can run on By adding the necessary auxiliary enzymes (Fre and GDH or ADH), halogenation biocatalysis the inexpensive substrate glucose or isopropanol, while the cost intensive cofactors FAD and NAD+ reactions can run on the inexpensive substrate glucose or isopropanol, while the cost intensive are recycled. cofactors FAD and NAD+ are recycled.

Figure 4. For the regeneration of FADH2, which is needed for the production of the reactive species Figure 4. For the regeneration of FADH2, which is needed for the production of the reactive species hypohaloushypohalous acid, acid, two two auxiliary auxiliary enzymes enzymes can can be be used: used: a a flavin flavin reductase reductase (Fre) (Fre) for for the the reduction reduction of of FAD FAD andand a a glucose glucose dehydrogenase dehydrogenase for for the the reduction reduction of NAD +. .Overall, Overall, the the co-substrate co-substrate glucose glucose is is used used in in excess,excess, whereas whereas the the more more expensive expensive cofactors cofactors FAD FAD and NAD + cancan be be recycled. recycled.

DespiteDespite the the described described advantages, advantages, the the introduction introduction of of a a cofactor-recycling cofactor-recycling system system renders renders the the biocatalyticbiocatalytic reaction reaction more more complicated, complicated, less less robust, robust, and and harder harder to scale-up. Consequently, Consequently, several several studiesstudies addressing addressing the the drawbacks drawbacks of of cofactor cofactor regene regenerationration have have been been carried carried out. out. Andorfer Andorfer et et al. al. constructedconstructed a a fusion fusion protein protein of of a a flavin flavin halogenase halogenase and and a a reductase reductase by by linking linking the the enzymes enzymes RebH RebH and and RebF.RebF. Interestingly, thethe fusion fusion construct construct showed showed higher higher product product titers titersin vivo in vivofor nativefor native and non-nativeand non- nativesubstrates substrates than the than co-expression the co-expression of the individual of the individual enzymes enzymes [48]. Furthermore, [48]. Furthermore, the authors the suggested authors suggestedthat the creation that the of thecreation fusion of protein the fusion could protein simplify could evolution simplify assays evolution because assays the reductase because must the reductasenot be regularly must not prepared be regularly and purified. prepared Following and purified. an alternative Following strategy,an alternative Frese strategy, and Sewald Frese linked and Sewalda halogenase linked and a thehalogenase auxiliary and enzymes the auxiliary after translation enzymes by after employing translation the bifunctional by employing chemical the bifunctionalglutaraldehyde. chemical In their glutaraldehyde. study, the authors In their overexpressed study, the authors RebH overexpressed (Fl-Hal), PrnF (Fre),RebH and(Fl-Hal), an alcohol PrnF (Fre),dehydrogenase and an alcohol (use ofdehydrogenase isopropanol instead(use of ofisopropanol glucose for instead the regeneration of glucose for of NADthe regeneration+) individually of NADand subsequently+) individually crosslinked and subsequently the three crosslinked enzymes to the so-called three enzymes combi cross-linked to so-called enzymescombi cross-linked aggregates enzymes(combiCLEAs) aggregates (Figure (combiCLEAs)5)[ 49]. By creating (Figure combiCLEAs, 5) [49]. By creating the system combiCLEAs, could be reused the system at least could ten times be reusedand stored at least for severalten times months, and stored whereas for theseveral purified months, free RebHwhereas showed the purified a considerable free RebH loss showed in activity a considerableafter being stored loss in for activity 12 weeks after at being 4 ◦C[ stored49]. for 12 weeks at 4 °C [49]. Going beyond traditional co-factor recycling schemes, Schroeder et al. showed that the need for auxiliary enzymes can be entirely overcome by using EDTA and light for the regeneration of FADH2 (Figure6). In their study, the authors reduced enzyme bound FAD with EDTA as the sacrificial electron donor and light at 450 nm. In this way, 58% of a 0.5 mM L-Trp solution was chlorinated within 25 h by Catalysts 2019, 9, 1030 7 of 21 Catalysts 2019, 9, 1030 7 of 21

Catalysts 2019, 9, 1030 7 of 20 using the flavin halogenase PyrH. Although the light driven cofactor recycling approach had a clearly 1 1 lower turnover frequency (0.72 h− ) compared to the enzymatic regeneration (3.0 h− ), this study described the production of chlorinated compounds by applying only light, halide salt, air, and FADH2 inCatalysts aqueous 2019, solution9, 1030 [50]. 7 of 21

Figure 5. Bromination of L-Trp using RebH cross-linked with the auxiliary enzymes PrnF and ADH Figure 5. Bromination of L-Trp using RebH cross-linked with the auxiliary enzymes PrnF and ADH (combi cross-linked enzymes aggregates (combiCLEAs)). (combi cross-linked enzymes aggregates (combiCLEAs)). Going beyond traditional co-factor recycling schemes, Schroeder et al. showed that the need for Going beyond traditional co-factor recycling schemes, Schroeder et al. showed that the need for auxiliary enzymes can be entirely overcome by using EDTA and light for the regeneration of FADH2 auxiliary enzymes can be entirely overcome by using EDTA and light for the regeneration of FADH2 (Figure 6). In their study, the authors reduced enzyme bound FAD with EDTA as the sacrificial (Figure 6). In their study, the authors reduced enzyme bound FAD with EDTA as the sacrificial electron donor and light at 450 nm. In this way, 58% of a 0.5 mM L-Trp solution was chlorinated electron donor and light at 450 nm. In this way, 58% of a 0.5 mM L-Trp solution was chlorinated within 25 h by using the flavin halogenase PyrH. Although the light driven cofactor recycling within 25 h by using the flavin halogenase PyrH. Although the light driven cofactor recycling approach had a clearly lower turnover frequency (0.72 h−1) compared to the enzymatic regeneration approach had a clearly lower turnover frequency (0.72 h−1) compared to the enzymatic regeneration (3.0 h−1), this study described the production of chlorinated compounds by applying only light, halide (3.0 h−1), this study described the production of chlorinated compounds by applying only light, halide salt, Figureair, and 5.5. FADH Bromination2 in aqueous of L-Trp solution usingusing RebHRebH [50]. cross-linkedcross-linke d withwith thethe auxiliaryauxiliary enzymesenzymes PrnFPrnF andand ADHADH salt, (combi(combiair, and cross-linkedcross-linked FADH2 in enzymes enzymesaqueous aggregatesaggregates solution (combiCLEAs)).[50].(combiCLEAs)).

Going beyond traditional co-factor recycling schemes, Schroeder et al. showed that the need for auxiliary enzymes can be entirely overcome by using EDTA and light for the regeneration of FADH2 (Figure 6). In their study, the authors reduced enzyme bound FAD with EDTA as the sacrificial electron donor and light at 450 nm. In this way, 58% of a 0.5 mM L-Trp solution was chlorinated within 25 h by using the flavin halogenase PyrH. Although the light driven cofactor recycling approach had a clearly lower turnover frequency (0.72 h−1) compared to the enzymatic regeneration (3.0 h−1), this study described the production of chlorinated compounds by applying only light, halide salt, air,Figure and 6. FADH Chlorination2 in aqueous of L-Trp solution by applying [50]. the flavin-dependent halogenase PyrH, EDTA, and Figure 6. Chlorination of L-Trp by applying the flavin-dependent halogenase PyrH, EDTA, and Figurelight. 6. Chlorination of L-Trp by applying the flavin-dependent halogenase PyrH, EDTA, and light. light. Another approach to to circumvent circumvent the the use use of of auxiliary auxiliary enzymes enzymes was was developed developed by byIsmail Ismail et al. et Another approach to circumvent the use of auxiliary enzymes was developed by Ismail et al. al.[51]. [51 Employing]. Employing a set a of set three of three tryptophan tryptophan halogena halogenasesses (PyrH, (PyrH, Thal, Thal, and RebH) and RebH) in combination in combination with [51]. Employing a set of three tryptophan+ halogenases (PyrH, Thal, and RebH) in combination with withan NADH an NADH mimic mimic for FAD for+ FADreduction,reduction, L-tryptophan L-tryptophan could be could selectively be selectively halogenated halogenated at the 5-, at 6-, the or an NADH mimic for FAD+ reduction, L-tryptophan could be selectively halogenated at the 5-, 6-, or 5-,7-position 6-, or 7-position of the indole of the moiety, indole respectively moiety, respectively (Figure 7). (Figure Notably,7). Notably,the biocatalysis the biocatalysis reactions reactionsusing the 7-position of the indole moiety, respectively (Figure 7). Notably, the biocatalysis reactions using the usingNADH the mimic NADH showed mimic showeda 3.5–4.7 a 3.5–4.7times timesfaster faster initial initial rate rate compared compared to tothe the enzymatic enzymatic cofactor NADH mimic showed a 3.5–4.7 times faster initial rate compared to the enzymatic cofactor regenerationregeneration systemsystem [[51].51]. regeneration system [51]. Figure 6. Chlorination of L-Trp by applying the flavin-dependent halogenase PyrH, EDTA, and light.

Another approach to circumvent the use of auxiliary enzymes was developed by Ismail et al. [51]. Employing a set of three tryptophan halogenases (PyrH, Thal, and RebH) in combination with an NADH mimic for FAD+ reduction, L-tryptophan could be selectively halogenated at the 5-, 6-, or 7-position of the indole moiety, respectively (Figure 7). Notably, the biocatalysis reactions using the NADH mimic showed a 3.5–4.7 times faster initial rate compared to the enzymatic cofactor regeneration system [51].

Figure 7. Chlorination of L-Trp using different Fl-Hals (PyrH, Thal, and RebH) applying an NADH mimic for the regeneration of FAD.

Catalysts 2019, 9, 1030 8 of 20

4. Strategies to Scale-up Biocatalytic Aromatic Halogenations Flavin-dependent halogenases selectively install halides into small molecules under mild reaction conditions, thus providing a promising green alternative to the often toxic catalysts used in traditional organic halogenations. However, restrictions associated with the biocatalytic approach such as low expression yields and limited stability of the enzymes, as well as a narrow substrate scope currently still hinder industrial application. In order to broaden the application potential of flavin-dependent halogenases, several studies addressing the above enumerated drawbacks have been carried out in the last decade.

4.1. Protein Overexpression and Stabilization The first attempt to upscale enzymatic halogenation was carried out by the group of Lewis [52]. In this study, the authors increased the expression level of the soluble halogenase RebH on a scale sufficient for preparative biocatalysis. Co-expression of chaperones (GroEL and GroES) in tandem with cell culture condition optimization led to a 10-fold increase of enzyme concentration in E. coli 1 (111 mg L− ) compared to previously reported values [24]. In the same study, a regeneration system utilizing glucose dehydrogenase (GDH) together with a fusion protein of reductase RebF and maltose binding protein (MBP) for preparative scale reactions (10 mg) was developed. High halogenation yields were observed for the transformation of several non-natural bioactive substrates including indoles and naphthalenes highlighting the synthetic utility of the enzyme (Figure8). In order to explore the accessible synthesis scale, 100 mg of L-tryptophan were used as a substrate for chlorination by crude cell lysate of RebH. Although the substrate was not fully converted, the enzyme remained active under these conditions, and a 69% final yield could be obtained [52]. Catalysts 2019, 9, 1030 9 of 21

Figure 8. ProductProduct structure structure and and isolated isolated yields yields of of pr preparativeeparative scale scale RebH RebH catalyzed halogenation. halogenation. HPLC conversion yields are shown in brackets.

The preparative efficiency efficiency of RebH has also b beeneen explored by Frese and Sewald who developed a multifunctional biocatalytic system exploiting the technique of cross-linked enzyme aggregates (combiCLEAs, vide infra). Using Using this enzymatic preparation, complete bromination of L-tryptophan was achieved after eight days, and 1.813 g of of L-7-bromotryptophan L-7-bromotryptophan TFA TFA were isolated in high purity (isolated yieldyield 92%). 92%). CombiCLEA CombiCLEA formation formation was was found found to increase to increase significantly significantly the long-term the long-term stability stabilityof RebH, of thus RebH, facilitating thus facilitating the enzymatic the enzymatic regioselective regioselective halogenation halogenation for gram scale for gram synthesis scale [49 synthesis]. In the [49].same In manner, the same the Micklefield manner, the group Micklefield halogenated grou non-naturalp halogenated substrates non-natura such asl anthranilamidesubstrates such and as anthranilamidetryptophol using and CLEAs tryptophol of four diusingfferent CLEAs Fl-Hals asof thefour first different step of aFl-Hals chemoenzymatic as the first reaction step [of5]. a chemoenzymaticThe instability reaction of flavin-dependent [5]. halogenases in certain reaction conditions has also been addressedThe instability by directed of evolution.flavin-dependent The Lewis halogenases group was in amongcertain thereaction first to conditions generate ahas thermostable also been addressedhalogenase by variant directed by employingevolution. threeThe Lewis rounds group of error was prone among PCR the and first recombinations. to generate a thermostable The obtained halogenaseRebH variant, variant which by containedemploying eight three mutations,rounds of error was prone characterized PCR and by recombinations. an 18 ◦C increased The obtained melting RebH variant, which contained eight mutations, was characterized by an 18 °C increased melting temperature compared to the wildtype enzyme. However, the catalytic efficiency of the mutant was significantly decreased at 21 °C owing to a lower conformational flexibility of the enzyme [53]. Exploiting a high throughput assay for rapid library screening, Schnepel et al. also evolved thermostable flavin-dependent halogenases. In their study, a doubly mutated variant was found with a 10 °C increase of its Tm compared to the wildtype enzyme. Unlike the wildtype enzyme, which significantly lost its activity after 2–3 h of incubation at 40 °C, the new variant, named Thal-GR, remained active even after 6 h of incubation at this temperature. In addition to the increased catalyst stability, the specific activity of the mutant Thal-GR was also 2.5 times higher than that of the wildtype [54]. Based on these results, the same group published a comprehensive study to further improve the thermostability of the generated variant Thal-GR three years later. Combining different mutagenesis strategies, the authors identified seven variants with higher thermostability compared to the native enzyme. A variant called Thal-GLV exhibited a good stability-activity relationship as it showed a significant increase in thermostability (ΔΤ50 = 16 °C, ΔΤm = 23.5 °C) compared to the wildtype, while exhibiting a 2.4-fold increase in activity at 25 °C. The stabilizing effect of the mutations has been linked to an increased association of the halogenase monomers to form homodimers in solution. Moreover, a crucial site with a strong effect on substrate selectivity was also identified. The single mutant Thal-S359G showed a distinct preference towards L-tryptophan, and only 17% bromination of D-tryptophan was observed compared to >99% in the case of the wildtype enzyme [55]. In a complementary approach to identify thermostable halogenases, the Micklefield group identified a thermophilic tryptophan halogenase (Th-Hal) from a thermophilic and halotolerant strain of Streptomyces via computational genome mining. The newly discovered thermostable flavin halogenase was characterized by an approximately 10 °C higher melting temperature (Tm = 47.8 °C) than other known Fl-Hals such as RebH, SttH, PrnA, KtzR, and PyrH. Combining this thermostable enzyme with a thermophilic flavin reductase (Th-Fre) from Bacillus subtilis WU-S2, the authors established a more efficient and robust biocatalytic system that led to 74% Catalysts 2019, 9, 1030 9 of 20 temperature compared to the wildtype enzyme. However, the catalytic efficiency of the mutant was significantly decreased at 21 ◦C owing to a lower conformational flexibility of the enzyme [53]. Exploiting a high throughput assay for rapid library screening, Schnepel et al. also evolved thermostable flavin-dependent halogenases. In their study, a doubly mutated variant was found with a 10 ◦C increase of its Tm compared to the wildtype enzyme. Unlike the wildtype enzyme, which significantly lost its activity after 2–3 h of incubation at 40 ◦C, the new variant, named Thal-GR, remained active even after 6 h of incubation at this temperature. In addition to the increased catalyst stability, the specific activity of the mutant Thal-GR was also 2.5 times higher than that of the wildtype [54]. Based on these results, the same group published a comprehensive study to further improve the thermostability of the generated variant Thal-GR three years later. Combining different mutagenesis strategies, the authors identified seven variants with higher thermostability compared to the native enzyme. A variant called Thal-GLV exhibited a good stability-activity relationship as it showed a significant increase in thermostability (∆T50 = 16 ◦C, ∆Tm = 23.5 ◦C) compared to the wildtype, while exhibiting a 2.4-fold increase in activity at 25 ◦C. The stabilizing effect of the mutations has been linked to an increased association of the halogenase monomers to form homodimers in solution. Moreover, a crucial site with a strong effect on substrate selectivity was also identified. The single mutant Thal-S359G showed a distinct preference towards L-tryptophan, and only 17% bromination of D-tryptophan was observed compared to >99% in the case of the wildtype enzyme [55]. In a complementary approach to identify thermostable halogenases, the Micklefield group identified a thermophilic tryptophan halogenase (Th-Hal) from a thermophilic and halotolerant strain of Streptomyces via computational genome mining. The newly discovered thermostable flavin halogenase was characterized by an approximately 10 ◦C higher melting temperature (Tm = 47.8 ◦C) than other known Fl-Hals such as RebH, SttH, PrnA, KtzR, and PyrH. Combining this thermostable enzyme with a thermophilic flavin reductase (Th-Fre) fromCatalystsBacillus 2019, 9, subtilis1030 WU-S2, the authors established a more efficient and robust biocatalytic system10 of 21 that led to 74% conversion of a 0.5 mM solution of tryptophan at 45 ◦C in contrast to other known halogenases,conversion of whicha 0.5 mM gave solution significantly of tryptophan lower at conversions 45 °C in contrast (around to other 40% known for PyrH) halogenases, using the which same reactiongave significantly conditions lower [47]. conversions (around 40% for PyrH) using the same reaction conditions [47].

4.2. Substrate Scope and Regioselectivity To assessassess thethe substrate substrate scope scope of of flavin-dependent flavin-dependent halogenases, halogenases, Frese Frese etal. et showedal. showed that that RebH RebH can halogenatecan halogenate substrates substrates beyond beyond the native the native L-tryptophan L-tryptophan such as such C-5- as and C-5- C-6-substituted and C-6-substituted fluoro-, fluoro-, amino-, chloro-,amino-, hydroxy-,chloro-, hydroxy-, and methyl-tryptophans and methyl-tryptophans [56]. Subsequently, [56]. Subsequently, Shepherd etShepherd al. successfully et al. successfully submitted thesubmitted non-indolic the non-indolic substrates substrates kynurenine, kynurenine, anthranilamide, anthranilamide, anthranilic anthranilic acid, and acid, other and anilines other anilines to the regiocomplementaryto the regiocomplementary tryptophan tryptophan halogenases halogenase PyrHs and PyrH PrnA and highlighting PrnA highlighting that also that these also enzymes these possessenzymes substrate possess promiscuity,substrate promiscuity, which can which be exploited can be inexploited applications in applications (Figure9)[ (Figure57]. 9) [57].

Figure 9.9. Non-indolic substrates accepted for chlorination. X: X: PyrH PyrH halogenation halogenation site, Y: PrnA halogenation site.site.

The Tsodikov group investigated the accepted substrate spectrum of the halide versatile phenolic halogenase PltM. The authors found that PltM can halogenate several small phenolic and aniline derivatives. Encouraged by these findings, the study was successfully continued towards more bulky compounds including FDA approved drugs and natural products such as terbutaline, fenoterol, resveratrol, and catechin (Figure 10) [45]. Notably, no halogenation activity was observed in the case of a nitrobenzene derivative due to the strongly electron withdrawing nitro group that prevented the halogenation of the substrate. Tailoring the substrate scope of enzymes towards desired molecules has also been approached through protein engineering. Interestingly, even enzymes exhibiting very little initial substrate promiscuity could be altered in this way. Following a substrate walking approach, Payne et al., for example, extended the substrate scope of the natural tryptophan halogenase RebH towards the significantly larger biologically active compounds yohimbine and carvedilol (Figure 11). Starting with a thermostable mutant of RebH (1-PVM), the authors performed three rounds of random mutagenesis and screening and could generate variants that selectively halogenate a number of unnatural substrates with a range of sizes, substitutions, and biological activities. Although the majority of the beneficial mutations were found close to the active site, a crucial mutation (A442V) was located distal to the active center. This finding highlights the worth of random mutagenesis in directed evolution strategies as this technique may identify amino acid positions that are difficult to predict via rational design strategies [58]. Catalysts 2019, 9, 1030 10 of 20

The Tsodikov group investigated the accepted substrate spectrum of the halide versatile phenolic halogenase PltM. The authors found that PltM can halogenate several small phenolic and aniline derivatives. Encouraged by these findings, the study was successfully continued towards more bulky compounds including FDA approved drugs and natural products such as terbutaline, fenoterol, resveratrol, and catechin (Figure 10)[45]. Notably, no halogenation activity was observed in the case of a nitrobenzene derivative due to the strongly electron withdrawing nitro group that prevented the halogenation of the substrate. Catalysts 2019, 9, 1030 11 of 21

Figure 10. FDA approved drugs (terbutaline, fenoterol) fenoterol) and and natural natural products products (resveratrol, (resveratrol, catechin) catechin) accepted by the phenolic halogenase PltM [45]. [45].

EnzymeTailoring engineering the substrate has scope also been of enzymes employed towards to alter desired the regioselectivity molecules has of also halogenases been approached in order tothrough control protein the site of engineering. halogenation Interestingly, in small molecule evens. enzymes In a first example exhibiting of this very approach, little initial the substratestructure ofpromiscuity PrnA, a tryptophan could be 7-halogenase, altered in this was way. analyzed, Following and three a substrate aromatic walking residues approach, around the Payne enzyme’s et al., activefor example, site were extended mutated the to substratealanine. One scope identified of the natural variant, tryptophan F103A, was halogenase found to halogenate RebH towards both the at thesignificantly C7 and C5 larger position biologically of the activetryptophan compounds indole yohimbine ring exhibiting and carvedilol a 2:1 ratio (Figure of the 11 ).products. Starting withThe exchangea thermostable of the mutant large amino of RebH acid (1-PVM), phenylalanine the authors with performed the smaller three alanine rounds allowed of random the mutagenesissubstrate to adoptand screening its “native” and orientation could generate but variantsalso bind that in a selectively similar mode halogenate as in the a tryptophan number of unnatural 5-halogenase substrates PyrH [59].with In a rangea similar of sizes, manner, substitutions, structure guided and biological mutagene activities.sis was employed Although theby Shepherd majority ofet theal. in beneficial 2016 to altermutations the regioselectivity were found close of totryptophan the active site,6-halogenase a crucial mutationSttH. The (A442V) generated was locatedtriple mutant distal toSttH the L460F/P461E/P462Tactive center. This finding transformed highlights 75% the of worth the supplied of random tryptophan mutagenesis into in the directed C5-halogenated evolution strategiesproduct, whereasas this technique the wildtype may identifyenzyme aminoexhibited acid almost positions the that same are activity difficult towards to predict the via C6-halogenated rational design productstrategies [60]. [58 ].In the same year, Lewis and co-workers published a thorough study in which RebH wasCatalysts engineered 2019, 9, 1030 for altered regioselectivity. High throughput MALDI-MS analysis was used to12 screen of 21 the transformation of deuterium substituted probe substrates. Utilizing a previously evolved variant, which was able to chlorinate tryptamine with 99% selectivity towards the C7 position, the authors identified two variants, named 8F and 10S, which could selectively chlorinate tryptamine at the C6 and C5 site, respectively. Additional reverse mutations (mutations that lead to the initial starting sequence) proved beneficial for the enzymes activity while maintaining the achieved selectivity [61]. In a structure-based protein engineering approach, Moritzer et al. substituted five residues in the substrate binding site of Thal by the corresponding amino acids of the closely related tryptophan halogenase RebH to switch regioselectivity from the 6- to 7-position. The resulting variant, named Thal-RebH5, exhibited a 95% change in regioselectivity towards C7-halogenation for both chlorination and bromination of L-tryptophan, further confirming the importance of the binding pose of the substrate for the regioselectivity outcome [62]. Building on previous learnings, Payne et al. recently reported the first enantioselective desymmetrization of methylenedianinelines catalyzed by engineered RebH variants. Docking simulations and structure guided mutagenesis afforded mutants with FigureimprovedFigure 11. 11.RebH RebHactivity catalyzedcatalyzed and altered halogenationhalogenation selectivity of variousvarious [63]. substratessubstrates indicating the the isolated isolated product product yields yields andand conversion conversion yieldsyields (in(in brackets).brackets). aa Multiple closely eluting eluting products products were were observed observed and and were were not not individuallyindividually isolated. isolated.

4.3. Biosynthetic Pathways Fueled by the discovery of novel halogenases with interesting properties, the incorporation of halogenases into biosynthetic pathways has become an attractive tool to increase the diversity of accessible products and to synthesize bioactive molecules or valuable intermediates. In 2010, Roy et al. carried out the first introduction of a halogenase gene into a synthetically unrelated pathway. In their study, the authors complemented the biosynthesis of the uridyl peptide antibiotic (UPA) pacidamycin from Streptomyces coeruleorubidus with the prnA gene expressing the tryptophan 7-halogenase PrnA. The resulting strain could produce the “unnatural” chloropacidamycin in a yield of 1 mg per liter culture with a ratio to its non-chlorinated analogue as high as 4:1. The installed chlorine then served as a handle, enabling the selective chemical functionalization of the antibiotic to yield a variety of derivatives [64]. Today, halogenase genes have also been integrated into medicinal plant metabolisms in order to produce chlorinated indole alkaloids with altered and/or improved pharmacological properties. Two prokaryotic halogenase genes encoding 7-tryptophan halogenase RebH or 5-tryptophan halogenase PyrH together with RebF as the partner reductase were introduced into Catharanthus roseus. It was reported that both halogenases function productively in the plant cell environment leading to the formation of the corresponding halotryptophans (7- or 5-halotryptophan), which subsequently enter the metabolic pathway of the plant cells to form chlorinated indole alkaloids. However, the efficiency of tryptophan decarboxylase, an enzyme that converts tryptophan to tryptamine, was significantly lower towards the halogenated substrates. This led to an accumulation of substantial levels of a non-natural intermediate, which had an adverse effect on the morphology and the growth rate of tissues [65]. To overcome this negative outcome, Glenn et al. employed site directed mutagenesis to generate a RebH mutant, which preferentially halogenates tryptamine rather than the native substrate tryptophan. Using this approach, the accumulation of 7-chlorotryptophan and its negative effect on the cell metabolism could be bypassed [66]. The potential application of plants as platforms for the manufacture of rare and valuable halogenated products has been also studied by Fräbel et al. Tryptophan 6-halogenase from Streptomyces toxytricini SttH has been successfully integrated into tobacco plants allowing the formation of chlorotryptophan in the cytosol when co-expressed with the flavin reductase RebF. The introduction of an additional flavin-dependent halogenase (RebH) into the metabolic setup of the plant led to the formation of 6,7-dichlorotryptophan, while a novel precursor of monoterpenoid indole alkaloids, 6-chlorotryptamine, was observed when SttH was co-expressed with tryptophan decarboxylase (TDC). Notably, SttH and RebH remained active even in the absence of the partner reductase when localized in chloroplasts, indicating the presence of sufficient reduction equivalents in these organelles for halogenation reactions [67]. Catalysts 2019, 9, 1030 11 of 20

Enzyme engineering has also been employed to alter the regioselectivity of halogenases in order to control the site of halogenation in small molecules. In a first example of this approach, the structure of PrnA, a tryptophan 7-halogenase, was analyzed, and three aromatic residues around the enzyme’s active site were mutated to alanine. One identified variant, F103A, was found to halogenate both at the C7 and C5 position of the tryptophan indole ring exhibiting a 2:1 ratio of the products. The exchange of the large amino acid phenylalanine with the smaller alanine allowed the substrate to adopt its “native” orientation but also bind in a similar mode as in the tryptophan 5-halogenase PyrH [59]. In a similar manner, structure guided mutagenesis was employed by Shepherd et al. in 2016 to alter the regioselectivity of tryptophan 6-halogenase SttH. The generated triple mutant SttH L460F/P461E/P462T transformed 75% of the supplied tryptophan into the C5-halogenated product, whereas the wildtype enzyme exhibited almost the same activity towards the C6-halogenated product [60]. In the same year, Lewis and co-workers published a thorough study in which RebH was engineered for altered regioselectivity. High throughput MALDI-MS analysis was used to screen the transformation of deuterium substituted probe substrates. Utilizing a previously evolved variant, which was able to chlorinate tryptamine with 99% selectivity towards the C7 position, the authors identified two variants, named 8F and 10S, which could selectively chlorinate tryptamine at the C6 and C5 site, respectively. Additional reverse mutations (mutations that lead to the initial starting sequence) proved beneficial for the enzymes activity while maintaining the achieved selectivity [61]. In a structure-based protein engineering approach, Moritzer et al. substituted five residues in the substrate binding site of Thal by the corresponding amino acids of the closely related tryptophan halogenase RebH to switch regioselectivity from the 6- to 7-position. The resulting variant, named Thal-RebH5, exhibited a 95% change in regioselectivity towards C7-halogenation for both chlorination and bromination of L-tryptophan, further confirming the importance of the binding pose of the substrate for the regioselectivity outcome [62]. Building on previous learnings, Payne et al. recently reported the first enantioselective desymmetrization of methylenedianinelines catalyzed by engineered RebH variants. Docking simulations and structure guided mutagenesis afforded mutants with improved activity and altered selectivity [63].

4.3. Biosynthetic Pathways Fueled by the discovery of novel halogenases with interesting properties, the incorporation of halogenases into biosynthetic pathways has become an attractive tool to increase the diversity of accessible products and to synthesize bioactive molecules or valuable intermediates. In 2010, Roy et al. carried out the first introduction of a halogenase gene into a synthetically unrelated pathway. In their study, the authors complemented the biosynthesis of the uridyl peptide antibiotic (UPA) pacidamycin from Streptomyces coeruleorubidus with the prnA gene expressing the tryptophan 7-halogenase PrnA. The resulting strain could produce the “unnatural” chloropacidamycin in a yield of 1 mg per liter culture with a ratio to its non-chlorinated analogue as high as 4:1. The installed chlorine then served as a handle, enabling the selective chemical functionalization of the antibiotic to yield a variety of derivatives [64]. Today, halogenase genes have also been integrated into medicinal plant metabolisms in order to produce chlorinated indole alkaloids with altered and/or improved pharmacological properties. Two prokaryotic halogenase genes encoding 7-tryptophan halogenase RebH or 5-tryptophan halogenase PyrH together with RebF as the partner reductase were introduced into Catharanthus roseus. It was reported that both halogenases function productively in the plant cell environment leading to the formation of the corresponding halotryptophans (7- or 5-halotryptophan), which subsequently enter the metabolic pathway of the plant cells to form chlorinated indole alkaloids. However, the efficiency of tryptophan decarboxylase, an enzyme that converts tryptophan to tryptamine, was significantly lower towards the halogenated substrates. This led to an accumulation of substantial levels of a non-natural intermediate, which had an adverse effect on the morphology and the growth rate of tissues [65]. To overcome this negative outcome, Glenn et al. employed site directed mutagenesis to generate a RebH mutant, which preferentially halogenates tryptamine rather than the native substrate Catalysts 2019, 9, 1030 12 of 20 tryptophan. Using this approach, the accumulation of 7-chlorotryptophan and its negative effect on the cell metabolism could be bypassed [66]. The potential application of plants as platforms for the manufacture of rare and valuable halogenated products has been also studied by Fräbel et al. Tryptophan 6-halogenase from Streptomyces toxytricini SttH has been successfully integrated into tobacco plants allowing the formation of chlorotryptophan in the cytosol when co-expressed with the flavin reductase RebF. The introduction of an additional flavin-dependent halogenase (RebH) into the metabolic setup of the plant led to the formation of 6,7-dichlorotryptophan, while a novel precursor of monoterpenoid indole alkaloids, 6-chlorotryptamine, was observed when SttH was co-expressed with tryptophan decarboxylase (TDC). Notably, SttH and RebH remained active even in the absence of the partner reductase when localized in chloroplasts, indicating the presence of sufficient reduction equivalents in these organelles for halogenation reactions [67]. Following a combinatorial biosynthesis approach, Wang et al. combined enzymes from different sources in Escherichia coli to generate valuable compounds. Using a multiple vector system, the fungal Fl-Hal Rdc2 was co-expressed with tyrosine ammonia-lyase (TAL), 4-coumarate:CoA ligase (4CL), and stilbene synthase (STS), resulting in a novel chlorinated derivative in addition to resveratrol. This novel compound was identified as 2-chloro-resveratrol highlighting the broad substrate scope of Rdc2 [68].

4.4. Chemoenzymatic Synthesis Chemoenzymatic synthesis has proven to be a powerful tool for the manufacture of complex compounds offering numerous advantages such as higher yields, decreased costs, environmental benefits, and high selectivity compared to purely chemical strategies [69]. In this context, the site-specific enzymatic halogenation of natural products can provide halogenated compounds that are of interest for the further functionalization by cross-coupling chemistry. The combination of chemo- and enzyme catalysis can thus lead to the formation of C-C, C-N, C-F, and C-O bonds from C-H positions, which were beyond the scope of chemocatalytic activation chemistry. Chemical modification through Pd catalyzed cross-coupling of a biosynthetically halogenated natural product has been reported by the group of Goss. Employing mild reaction conditions to account for the thermal instability of the substrate chloropacidamycin, the authors obtained various 7-aryl-pacidamycin derivatives in high yields. This functionalization approach could not only be applied on purified natural compounds, but also worked when using crude extracts of the halogenated substrates [64]. In a similar manner, Pd catalyzed Suzuki–Miyaura cross-coupling (SMC) reactions have been adopted by the O’Connor group to synthesize aryl and heteroaryl analogues of monoterpene indole alkaloids. Exploiting the technique of mutasynthesis in C. roseus hairy root cultures, the authors introduced a chemical halogen handle into the indole moiety of the monoterpene indole alkaloids followed by the subsequent chemical derivatization using the crude cell extracts. Based on their promising results, the authors further scaled up the cross-coupling reactions using 16 g of the crude hairy root extract to obtain milligram quantities of three alkaloid analogs [70]. Durak et al. further expanded the application scope of the chemoenzymatic strategy by derivatizing non-native bioactive small aromatic molecules such as carvedilol, pindolol, thenalidine, and tryptoline and its derivatives. Two RebH variants, 3-SS and 4-V, were used for the selective halogenation of the aforementioned compounds followed by subsequent Pd catalyzed cross-coupling reactions with boronic acids, amines, and trifluoroethanol on the crude biocatalysis extracts to generate new C-C, C-N, and C-O bonds [71]. Although the advantages of chemoenzymatic reactions for the synthesis of demanding and complex compounds has been successfully demonstrated, the detrimental effects of protein contaminants on the metal catalyst require an intermediate extraction step of the halogenated precursor that limits process scale-up. To address this challenge, the group of Sewald utilized combiCLEAs of several Fl-Hals in a single pot approach [72]. In this study, a bromine substituent was introduced biocatalytically at the C5, Catalysts 2019, 9, 1030 13 of 20

C6, or C7 position of L-tryptophan on a preparative scale. After filtration of the biocatalyst, a SMC reaction led to an efficient modification of the halogenated compounds, and the resulting cross-coupling product could subsequently be Boc protected and purified for further applications. This three-step one pot approach is an important proof-of-concept for the development of more sustainable and consecutive biohalogenation-chemocatalytic cross-coupling processes. Latham et al. further simplified the one pot halogenase-SMC cascade by separating the protein from the Pd catalyst using polydimethylsiloxane membranes (PDMS). Compartmentalization was achieved thanks to the hydrophobic nature of the PDMS membranes, which allowed for the diffusion of non-polar compounds such as substrates and intermediates while the diffusion of polar compounds (enzymes, Pd catalysts, and cofactors) was prevented (Figure 12). Combining this method with halogenase CLEAs led to higher concentrations of the aryl halide and reduced Pd loading (2 mol%) Catalysts 2019, compared9, 1030 to free pure protein reactions (Pd loading: 10 mol%) [5]. 14 of 21

Figure 12. PolydimethylsiloxaneFigure 12. Polydimethylsiloxane (PDMS) (PDMS) compartmentaliz compartmentalization.ation. PDMS PDMS membrane membrane permits diff usionpermits of diffusion of non-polarnon-polar compounds compounds (substrates, (substrates, intermediates),intermediates), while polarwhile compounds polar compounds such as enzymes, such Pd catalysts, as enzymes, Pd catalysts, andand cofactors cofactors are are blocked. blocked. This compartmentalization This compartmentalization overcomes the incompatibilityovercomes the of the incompatibility enzyme and of the transition metal that reduces the efficiency of the chemoenzymatic process. Figure adapted from [5]. enzyme and transition metal that reduces the efficiency of the chemoenzymatic process. Figure adapted fromFurther [5]. pioneering the one pot chemoenzymatic reactions, the Goss group combined synthetic biology with synthetic chemistry. After an optimization of the cultivation media of the engineered strains to minimize the impact of media components on the cross-coupling efficiency, the authors Furtherdeveloped pioneering a biological the one system pot for chemoenzymatic the generation of brominated reactions, metabolites the Goss that can group synchronously combined be synthetic biology withmodified synthetic through chemistry. cross-coupling After in living an optimization cultures (Figure 13 of). Thisthe approachcultivation provides media the advantage of the engineered strains to minimizeof a simplified the product impact isolation of media and a continuouslycomponen producingts on the biosynthetic cross-coupling system [6 ].efficiency, the authors developed a biological system for the generation of brominated metabolites that can synchronously be modified through cross-coupling in living cultures (Figure 13). This approach provides the advantage of a simplified product isolation and a continuously producing biosynthetic system [6].

Catalysts 2019, 9, 1030 14 of 20

Catalysts 2019, 9, 1030 15 of 21

.

Figure 13. InFigure culture 13. In biosynthesis culture biosynthesis and and cross-coupling cross-coupling of of Br-pacidamycin-D. Br-pacidamycin-D. The engineered The engineered strain of strain of S. coelicolor RG-1104S. coelicolor grownRG-1104 grown in the in presence the presence of of sodium sodium br bromideomide is able is ableto produce to produce Br-pacidamycin-D. Br-pacidamycin-D. The brominated compound is subsequently modified through Pd catalyzed Suzuki−Miyaura cross- The brominated compound is subsequently modified through Pd catalyzed Suzuki Miyaura coupling reactions under mild conditions. − cross-coupling reactions under mild conditions. 5. Conclusion 5. Conclusions The selective halogenation of small molecules is of high interest for many industries and is The selectiveespecially halogenation sought after in of the small pharmaceutical molecules industry is of high and interestagrochemistry for many [2,3]. Today, industries biocatalytic and is especially halogenations are promising tools for the preparation of small quantities of selectively derivatized sought afterproducts in the pharmaceutical (Table 2), which can industry be used in andmedicinal agrochemistry chemistry, or [serve2,3]. as Today, intermediates biocatalytic for the total halogenations are promisingsynthesis tools forof complex the preparation molecules (Chapter of small 4.4). quantities However, of to selectivelyour knowledge, derivatized more than productsa gram (Table2), which can bequantity used inof halogenated medicinal target chemistry, compound or has serve not yet as been intermediates prepared (Table for 2), the illustrating total synthesis two of the of complex current challenges in aromatic halogenase catalysis: the enzymes suffer from low productivity and molecules (Chapter 4.4). However, to our knowledge, more than a gram quantity of halogenated target limited scalability. Thus, in order to expand the application scope of flavin-dependent halogenases, compound hasfurther not studies yet been aimed prepared to broaden (Tablethe substrate2), illustrating scope and increase two of the the robustness current of challengesthese enzymes in aromatic halogenase catalysis:are required. the These enzymes can entail su bioinformaticffer from low studies productivity to identify additional and limited members scalability. of the flavin-Thus, in order to expand thedependent application halogenase scope superfamily of flavin-dependent with an extended halogenases,substrate scope [46] further or higher studies thermostability aimed to broaden [47]. Additionally, approaches such as the “catalophore” strategy might be successful in the the substrateidentification scope and of novel increase flavin-dependent the robustness halogenases of these[73]. In this enzymes bioinformatic are required.method, Steinkellner These can entail bioinformaticet al. studies carried toout identify a structure additional guided search members for the discovery of the of flavin-dependent ene-reductases by predicting halogenase enzyme superfamily with an extendedactivity substratebased on three-dimensional scope [46] or constellations higher thermostability of functional groups. [47 ]. Additionally, approaches such Following the discovery of promising wildtype enzymes, engineering strategies that should as the “catalophore”equally consider strategy enzyme mightstability and be successfulactivity will, atin least the to some identification extent, inform of about novel the structure- flavin-dependent halogenasesfunction [73]. Inrelationship this bioinformatic in flavin-dependent method, halogena Steinkellnerses. In this respect, et al. in carried silico design out abased structure on guided search for theenergy discovery functions of might ene-reductases be a very valuable by predictingtool, as methods enzyme such as activityRosetta_ddg based and FireProt on three-dimensional have been proven to optimize protein structures successfully [74–78]. The obtained knowledge may then constellations of functional groups. lead to the development of tailored enzyme variants, which through their increased stability, activity and, robustness may facilitate industrial usage. Looking forward, engineering flavin-dependent halogenasesTable 2. Examples towards the of preparativeacceptance of scale moreapplications electron poor ofsubstrates flavin-dependent [79] through halogenases. increasing the electrophilicity of the halogenating species will be a way to further enhance their applicability. As Scale Halogen Conversion Yield %/ Enzymeproposed by Latham et al., reaction engineeringSubstrate aspects, such as optimizing oxygen concentration in Ref. (mg) Insertion Isolated Product Yield % PyrH 136 Anthranilamide (combiCLEAs)

PyrH and SttH 15 3-Indolepropionic acid Br n.d. [a] [5] (combiCLEAs)

RebH (combiCLEAs) 24 Tryptophol 16 L-tryptophan -/62.5 18 1-methyl-L-tryptophan -/12 17 5-hydroxy-L-tryptophan -/16 Th-Hal (purified) Cl [47] 16 L-kynurenine, -/21 11 Anthranilic acid -/8 11 Anthranilamide -/19 Catalysts 2019, 9, 1030 15 of 20

Table 2. Cont.

Scale Halogen Conversion Yield %/ Enzyme Substrate Ref. (mg) Insertion Isolated Product Yield % 1000 L-tryptophan 100/- RebH (combiCLEAs) 40 D-tryptophan Br 57 / [49] 40 L-5-hydroxytryptophan 53 / RebH (purified) 10 L-tryptophan Cl 94/74 Br 97/85 RebH (purified) 10 D-tryptophan 99/78 Tryptamine 98/53 N-Ω-methyltryptamine 65/65 Tryptophol 95/95 2-methyltryptamine Cl 96/60 [52] 5-methyltryptamine 99/58 2,3-disubstituted indole tryptoline 58/54 Indole 64/53 2-aminonaphthalene 98/93 1-naphthol 95/59

RebH (lysate) 100 L-tryptophan Cl 74/69 L-tryptophan -/69 2-methyltryptamine -/56 RebH (purified) 10 Cl [53] 2-aminonaphthalene -/62 Tryptoline -/67 PyrH 50 L-tryptophan -/76 (combiCLEAs)

Thal [54] 50 L-tryptophan Br -/61 (combiCLEAs)

RebH 150 L-tryptophan -/84 (combiCLEAs) SttH (combiCLEAs) 100 Anthranilamide Cl -/25 [60] RebH variants 0S/8F/10S 10 Tryptamine Cl 98/73/78 [b] [61] (purified) Cl 30/- Thal-RebH5 variant (lysate) 30 L-tryptophan [62] Br 15/- RebH variant 4-V (purified) 10 t-Bu dianiline methane Cl 80/-[63] Tryptoline -/78 Eleagnine -/83 RebH variant 3-SS 10 Pinoline Cl -/80 (purified) Tetrahydroharmine -/79 [58] Debromodeformylflustrabromine -/68 Yohimbine -/54 RebH variant 4-V 10 Pindolol Cl -/93 (purified) Carazolol -/79 Carvedilol -/69 RebH variant 3-SS (purified) 10 Tryptoline Br RebH variant 4-V (purified) 10 Carvedilol Cl n.d. [a] [71] Pindolol Br Thenalidine RebH/ThaI/PyrH 150 L-tryptophan Br 100 [c] [72] (combiCLEAs) [a] Enzymatic halogenation yields not determined for these chemoenzymatic cascade reactions. [b] Isolated yields % for the three different variants studied (0S/8F/10S). [c] Conversion yield % was 100 for all cases of immobilized Fl-Hals studied. Catalysts 2019, 9, 1030 16 of 20

Following the discovery of promising wildtype enzymes, engineering strategies that should equally consider enzyme stability and activity will, at least to some extent, inform about the structure-function relationship in flavin-dependent halogenases. In this respect, in silico design based on energy functions might be a very valuable tool, as methods such as Rosetta_ddg and FireProt have been proven to optimize protein structures successfully [74–78]. The obtained knowledge may then lead to the development of tailored enzyme variants, which through their increased stability, activity and, robustness may facilitate industrial usage. Looking forward, engineering flavin-dependent halogenases towards the acceptance of more electron poor substrates [79] through increasing the electrophilicity of the halogenating species will be a way to further enhance their applicability. As proposed by Latham et al., reaction engineering aspects, such as optimizing oxygen concentration in the reaction medium and the management of the level of reactive oxygen species could be further levers to increase flavin-dependent halogenase productivity [2].

Author Contributions: Conceptualization: J.B., A.P., and R.B; writing: J.B, A.P and R.B. Funding: This research was funded by the Swiss State Secretariat for Education, Research and Innovation (Federal project contribution 2017–2020, P-14: Innovation in Biocatalysis). Acknowledgments: We gratefully acknowledge financial support by the Swiss State Secretariat for Education, Research and Innovation (Federal project contribution 2017–2020, P-14: Innovation in Biocatalysis). Conflicts of Interest: The authors declare no conflict of interest. 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; nor in the decision to publish the results.

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