Natural products from Bacillus subtilis with an tiMicrobial properties☆

Tao W ang ,Yafei Liang ,M ianbin W u ,,*,Zhengjie Chen ,Jianping Lin ,Lirong Yang

1 Key Laboratory of Biomass Chemical Engineering of M inistry of Education,Hangzhou 310027,China

2 Department of Chemical and Biological Engineering,Zhejiang University,Hangzhou 310027,China

3 JinhuaPeople's Hospital,Jinhua 321000,China

4 Zhejiang Key Laboratory of Antifungal Drugs,Taizhou 318000,China

Keyw ords:Bacillus subtilis AntiMicrobial Bioseparation Drug design

ABSTRACT Bacillus subtilis produces many chemically-diverse secondary metabolites of interest to chem ists and biologists.Based on this,this review gives a detailed overview of the natural com ponen ts produced by B.subtilis including cyclic lipopep tides,polypeptides,proteins(enzym es),and non-peptide products.Their structures,bioactive activities and the relevant variants as novel lead structures for d rug discovery are also described.The challenging effects of ferm entation metabo lites,iso lation and purification,as w ell as the overp roduction of bioactive compounds from B.subtilis by metabolic engineering,were also high ligh ted.System atically exploring biosyn thetic routes and the functions of secondary metabolites from B.subtilis may not only be beneficial in im proving yields of the products,bu t also in helping them to be usedin foodindustry and pub lic medical service on a large-scale.

1.Introduction

with the increased em ergence of d rug-resistan t bacteria[1,2]and pub lic concerns over pesticide hazards,the need for novel d rugs and environmentally-friend ly alternatives to chemical pesticides is u rgen t.Bacillus subtilis is gram-positive,aerobic,endospore-form ing rod comm only foundin soil,water andin association with plants.Strains have a w ell-developed secretory system[3],p roducing diverse secondary metabolites with a wide spectrum of antibiotic activities valuable to medicine and agricu lture[4].Natural products especially from marine organism s,to a great exten t,con tinue to rep resen t an excellen t source of novel lead structures for d rug discovery or d rug design for their diverse chemical structures,novel action mechanism s,low toxicity and biodegradable.

The discovery of an increasing number of products from B.subtilis has further em phasized their potential industrial applications.The antiMicrobial com pounds produced by B.subtilis have been studied extensively as biological plant pathogen control agents,as food preservatives and as medicines in anim als[5].Also the use of bacteria as biological control agents has received more atten tion,as such strains can supp ress different diseases by diverse modes of action such as com petitive inhibition of glucose-6-phosphate syn thase.Serenade TM and Souata AS derived from B.subtilis QST 713[6]and B.subtilis QST 2808[7]are bactericides registered for use in the USA.Kodiak,Sub tilex and Taegro are o ther exam ples of agen ts produced from B.subtilis[8].

B.subtilis produces a variety of bioactive com ponen ts with chemically diverse structures[9,10].Studying the relationship between mo lecu lar structures and bio logical activities is im po rtan t in the developm en t of novel d rugs,and a significan t method of op tim izing lead com ponen t.In the fields of d rug design,its effect is irreplaceab le as the lead com ponen ts.In th is paper,an tibacterial com ponen ts produced by B.subtilis,as w e ll as their structures and activities,were explored,and perspectives for fu rther research and developm ent were presented.

2.Natural An tiMicrobial Products from B.subtilis

2.1.Non-ribosomally synthesized products

The non-ribosom al syn thesis of pep tide an tibiotics is widesp read am ong bacteria,usually invo lving large mu lti-enzym e com plexes which catalyzeall thenecessary steps in pep tide biosyn thesis,including the selection and ordered condensation of am ino acid residues.This fam ily con tains three main groups and several subclasses(chemical structures are show n in Fig.1).

2.1.1.Iturin group

M em bers of the iturin group are cyclic lipopep tide an tibiotics produced by various strains of B.subtilis.Fou r itu rin an tibiotics(itu rins A,C,D,E)[11-13],five bacillom ycins[14,15]and mycosubtilin[16]are know n,and their structures have been describedin detail.These antibiotics are characterized by an invarian t LDDLLDL am ino acid sequence and a variable hydrophobic tail— β-am inofatty acid with 14 to 17 carbon atom s linked at the C3in to a cyclic pep tide through a β-pep tide bond[17].Activity is enhanced with an increasing number of carbon atom s in the fatty acid chains[18].Eigh t different itu rin hom ologues(iturins A1 to A8)have been reported,based upon their different side chains[13,19].The differences between iturin A,iturin D anditurin E are:one Asn or Gln residue in iturin A is replaced by an Asp or Glu residue in iturin D,but by an Asp-ΟCH3or Glu-ΟHSin iturin E respectively.Com pared with iturin A,itu rin C has no an tibiotic activity[20]which may be due to the change in an Asn residue.Bacillom ycins and M ycosubtilin differ from iturins in the com position of the pep tide chains at positions L4,L5,D6and L7.M em bers of the iturin fam ily were though t to be restricted to B.subtilis,how ever iturins are also produced by other Bacillus strains[21,22].

Iturins display strong in vitro inhibitory effects on a wide range of fungi such as yeasts[16,23],bu t are less active against bacteria[24-26]and possess no an tiviral activity[21-23].The M IC of itu rin A is as lowas 4 μg/disk,which strongly supp ressed Penicillium italicum,Penicillium viridicatum,Aspergillus ochraceus and Aspergillus versicolor,while Penicitllum citrinurn and Aspergillus parasiticus were least sensitive[27].The an tifungal spectrum,low toxicity and allergic effects[28,29]m ake itu rins valuab le d rugs and promising long-term biocon tro l agents[19,30,31],and their structures make them more biodegradable than other chemical agen ts.The structure of itu rins determines function,which isaffected by the position and the length of the free carboxyl group,indicating their im portance to the bio logical activity of iturin antibiotics[24].

2.1.2.Fengycin

Fengycins(plipastatin if Tyr9is D-configured[32])are produced by B.subtilis and other Bacillus strains such as Bacillus am yloliquefaciens[33,34]and Bacillus thuringiensis[35].They are cyclic decapep tides,eight am inooni acids form ing a cyclic structure.A β-hyd roxy fatty acid chain is attached to the other two am ino acids.The configu ration of am ino acid sequence is LDLDLDDLLDL,with a lactone bond connecting L-Tyr to L-Ile[36].This lipopep tide class includes fengycins A and B and relevant hom ologues,which differ in the am ino acid at position 6 of the pep tide moiety(D-Ala for fengycin A and D-Val for fengycin B)and the length of the fatty acid chain(13 to 17 carbon atom s).Fengycins are macrolactone rings,in which the Tyr side chain at position 3 of the pep tide sequence form san ester bond with theC-terminalresidue,yielding an internal ring.

Fig.1.Chemical structures of non-ribosom ally synthesized products.(A)Iturin A,iturin C(L-Asn=L-Asp);(B)m ycosubtilin;(C)bacillom ycins;(D)fengycin A;(E)m altacines;and(F)surfactin.

Fig.1(continued).

Fengycins are less haem olytic than itu rins and have strong antifungal activity,specifically against filam en tous fungi[37].Fengycins and the strains producing them are usefu l in protecting plants against fungal pathogens including Fusarium gram inearum,Rhizoctonia solani,Pythium irregulare and Cladosporium fulvum,phytopathogenic fungi responsib le for severe agricu ltural losses[33,38,39].similar to other lipopep tides,fengycins act in a synergistic manner making them more effective,w hen itu rin and fengycin are used together[40].

2.1.3.Surfactin and related com pounds

The most thorough ly studied fam ily of lipopep tides are surfactins.Th is fam ily con tains～20 different lipopep tides such as esperin,lichenysin,pum ilacidin and surfactins.The surfactin group(and several variants)are cyclic hep tapep tides with a chiral LLDLLDL configu ration form ing a cyclic lactone ring structure linked to a β-hyd roxy fatty acid tail with 13-15 carbon atom s[41].A D-Leu at positions 3 and 6,and an L-Asp attached to the ring at position 4 except esperin[42].Earlier studies indicated that differences between surfactins were due to the length of the β-hyd roxy acid chains.How ever,recent studies show ed that surfactins are a mixture of pep tidolipids differing in the hom ology of the lipid portions[43]and terminal am ino acid residues[20,44-46].Based on these characteristics,surfactins are dividedin to three types:surfactin A with L-leucine,surfactin B with L-valine and surfactin C with L-iso leucine at the position invo lvedin lactone ring form ation with the β-hyd roxy fatty acid[47].Surfactins also have macro lactone rings similar tofengycin,but the ring closure is catalyzed between the β-hyd roxyl fatty acid and the C-terminal pep tide for surfactin(which differs from fengycin).

The reason w hy surfactins have attracted so much in terest in recent years is their excep tionalsurfactantpower,which low ers the surface tension of water from 72 to 27 mN·m-1at a concentration as lowas 20 μm ol·L-1[41].These am phiphilic com ponen ts serve as biosurfactants which are more effective,biodegradable,less toxic and even effective at extrem e temperatures or pH values[5,48]than chemical surfactants know n to have detrimental environmental effects.M ore recently,these molecu les have been reported to possess new activities including em u lsification,foam ing[49],antiviral[50,51],an ti-m ycoplasm ic,an tiphytopathogenic fungi,larvicidal and enzym e inhibition[52,53].Ghribi etal.[54]reported thatabiosurfactin isolated from SPB1 killed four different lepidop teran larvae.Sabate and Audisio[55]discovered that a surfactin syn thesized by B.subtilis C4 inhibited Listeria monocytogenes,a gram-positive pathogen of hum ans and anim als at 0.125 mg·m l-1.Surfactin is also active against Sem liki Forest virus(SFV),herpes sim plex virus(HSV-1,HSV-2),suid herpes virus(SHV-1),vesicu lar stom atitis virus(VSV),sim ian imm unodeficiency virus(SIV),feline calicivirus(FCV)and murine encephalom yocarditis virus(EMCV)at 25 mm ol·L-1in a medium containing 5%fetal calf serum(FCS)[50].All these indicate that surfactin may be used as an anti-viral agent as w ell.

2.1.4.Maltacines

Hagelin etal.[56]reported a new method for the separation and purification of the maltacine com plex from B.subtilis fermentation broth using preparative high-performance liquid ch rom atography.Their molecu lar w eights(1447.7 and 1519.8 Da)suggest that they are novel com ponents.Fu rther study reveals the linear am ino acid sequences of maltacines similar tofengycins,which con tains 10 am ino acids but differing in the last two N-terminal am ino acids,indicating they are novel lipopep tide an tibiotics[57].This group con tains four different am ino acid sequences(B,C,D and E),where the differences between them are due to the pyrrolysine and lysine am ino acids at position 2,though pyrro lysine is rarely seen,which is unusual in lipopep tides.The am ino acids in maltacines are in the L-and D-configu rations.Fermentation broth displays strong activity against fungi such as Candida albicans,Trichophyton mentagrophytes,Aspergillus fum igatus and Staphylococcus aureus[56].How ever,less in form ation is available on their antiMicrobialactivity than thatof their structures,whichm igh tpu ta brakeon their developm ent as potential drugs.

Fig.2.Chemical structures of other non-ribosom ally synthesized products.(A)Bacilysin;(B)rhizocticin;(C-G)am icoum acins;(H-J)L-Pro-L-Leu,D-Pro-L-L D-Pro-L-Tyr;(K)m ycobacillin;and(L)TL-119.

2.1.5.Other non-ribosom ally synthesized peptide antibiotics

There are also severalpep tidean tibioticswith special structures and different features com pared to the an tibiotics listed above.These include bacilysin,rh izocticin,am icoum acin,TL-119,m ycobacillin and diketopiperazines(DKPs)(chemical structures are show n in Fig.2).

Bacilysin is a hydrophilic dipep tide produced by B.subtilis A14[57]with a molecu lar w eigh t of 270,an N-terminal L-alan ine and L-an ticapsin at its C-terminus[58,59].An ticapsin is 3-epoxycyclo hexanonyl-alanine bearing the reactive epoxyketone w arhead which plays an im portant role in antiMicrobial activity[60,61].Bacilysin is active against a wide range of bacteria such as S.au reus[62]and the fungus C.albicans[28],while the activity of an ticapsin is low er than bacilysin,and also show s activity against Escherichia coli and C.albicans.

Rh izocticins are hydrophilic phosphono-o ligopep tides from B.subtilis ATCC 6633 first discoveredin 1949,bu t structural details were not reported un til 1988[63].Th is group con tains four variants[64]which are di-and tripep tide antibiotics having a variable am ino acid at the N-terminus follow ed by arginine and the non-p roteinogenic am ino acid(Z)-L-2-am ino-5-phosphono-3-pen tenoic acid(APPA).The general structures of these four variants can be summ arized as Arg-APPA,Val-Arg-APPA,Ile-Arg-APPA and Leu-Arg-APPA[65].It is believed that the am ino acids at the N-terminus are necessary for their an tifungal activity through inh ibition of th reonine syn thase activity by APPA[66].

Am icoum acins were isolated from Bacillus pum ilus[67]and B.subtilis[68],and 12 different variants have been reported con taining dihyd roisocoum arin with a hyd roxy am ino acid side chain[69].Their bio logical activities include an tibacterial,an ti-inflamm atory,an tiu lcerative,gastroprotective and anti-Helicobacter pylori.The C12am ide functionalgroupsplay a critical role in the cytotoxicity and antibacterial activities[68,70,71].

TL-119(or A-3302-B)is a pep tide antibiotic isolated from a strain of B.subtili s[72,73].Itsstructurewas initially reported asAc-D-Phe-D-Leu-LPhe-L-Thr-L-Val-L-Ala-(Z)-σAba,but differences in the antibacterial activity of chemically-synthesized TL-119 resultedin the revised structure Ac-D-Phe-D-Leu-D-aPhe-L-Thr-L-Val-L-Ala-(Z)-σAba[74].The antiMicrobial spectrum of TL-119 includes gram-positive bacteria such as Streptococcus pyogenes and E.coli,but it is preferentially active against S.aureus[72].

M ycobacillin is a polypep tide an tibiotic[75]with a cyclic structure of 13 am ino acids without an N-terminal residue[76]and the sequence is determined as s-L-Ala-D-Asp-L-Pro-D-Asp-D-Glu-L-Tyr-L-Asp-L-Tyr-LSer-D-Asp-L-Leu-D-Glu-D-Asp[77].It has no activity against bacteria such as E.coli and S.aureus,bu t displays an tifungal activity at 0.02 mg/m l against Aspergillus niger and other fungi[75].

Diketopiperazines(DKPs)[78]are heterocyclic com pounds which exist in several stereoisom eric form s(DD,DL and LL)[79].The headto-tail dipep tide structure is a comm on naturally-occurring skeleton that leads to different pep tide derivatives such as cyclo(L-His-L-Leu)[80],cyclo(L-Pro-L-Leu),cyclo(D-Pro-L-Leu)and cyclo(D-Pro-L-Tyr)[81].Their pecu liar chiral,rigid and functionalized structures make them a rich source of new biologically-active com pounds[79,82]with diverse biological activities such as anti-tum or[83-85],antifungal[81]and antibacterial[82,86]activities.

2.2.Products synthesized by ribosomes

Am ong thebioactive com ponen ts from B.subtilis,m any antiMicrobial com pounds are produced through gene-encoded pathw ays or ribosom ally-syn thesized.The predom inan t mem bers of this fam ily are lantibiotics and several an tifungal proteins.

Lan tibiotics have a long history of use as food preservatives since their discovery in the 1920s[87,88].Lantibiotics with inter-residual thioether bondsare usually form ed by the reaction ofdehyd rated serine and threonine residues with the su lfhyd ryl group of cysteine[89-91].Usually they are dividedin to Type A and Type B[92].Flexib le,elongated pep tides with a net positive charge such as subtilin(p roduced by B.subtilis ATCC 6633[93])are includedin the Type A group.Type B lantibioticssuch asm ersacidin[94,95]exhibitam oreglobu lar structure.Lan tibiotics such as sublancin[96]have also been discovered recently[97].

Com pared with other lipopep tides,lan tibiotics con tain a high proportion of unusual am ino acids such as dehyd roalanine(Dha),dehyd robu tyrine(Dhb),lan thionine(Ln),and methyllanthionine[92].Lantibioticsproduced by B.subtilis are discussed below(chemical structures are show n in Fig.3).

2.2.1.Type A lantibiotics:Subtilin and ericin

Type A lantibiotics are usually com posed of 21-38 am ino acids resides(bu t may be 19-38 am ino acids)with mo lecu lar w eigh ts of＜5 kDa[88].Subtilin and subtilin B(p roduced by B.subtilis ATCC 6633[93,94])are 32-am ino-acid pen tacyclic lantibiotics with～60%sequence hom o logy to the widely-utilized biop reservative—Nisin[92].Am ong the 32 am ino acids,13 are post-translationallym odified by dehydration ofspecific serinesand th reoninesand the form ation of lanthioninebridges.Five lanthionine ringsoccur in subtilin,thefirstat positions3 and 7,the second at positions 8 and 11,the third at positions 13 and 19,the fourth at positions 23 and 26 and the last at positions 25 and 28.NMR show s that this structure contains two am phipathic dom ains,that is the first three lanthionine ring regions and the last lanthionine ring regions.Very recently,a novel subtilin-like pep tide produced by B.subtilis DSM 15029Tnam ed en tianin was reported w hose structure was identical to subtilin excep t three am ino acid changes:Leu6to Val6,Ala15to Leu15and Leu24to Ile24[97].

Lantibiotics have high an tiMicrobial activity against various grampositive bacteria but little antifungal activity.They are used as food preservatives[98]due to their activity against Listeria monocytogenes and Clostridium botulinum,pathogens of concern to the foodindustry[99].Subtilin[100]and the recently discovered variants of subtilins[99]also display strong inhibitory activity against Listeria monocytogenes and Clostridium botulinum[101].

Ericin[102]is another Type A lan tibiotic produced by B.subtilis strain A1/3.Recently two lan tibiotics-like pep tides ericin A(2986 Da)and ericin S(3442 Da)were discovered[103].Ericin S is quite similar to subtilin,differing in four am ino acids:Val6to Leu6,Val15to Ala15,Ile24to Leu24,and His29to Lys29,suggesting that erincin S is a varian t of subtilin.Οn the con trary,ericin A has 13 am ino acid differences and a different ring organization at the C-terminus com pared with ericin S.

Their variations in structure resultin differentantiMicrobialactivities.Ericin S show s activity similar to subtilin against B.am yloliquefaciens and L.lactis,S.aureus[102],while ericin A has minim al an tibiotic activity equivalent to a 100-fo ld-diluted sam ple of ericin S.

2.2.2.Type B lantibiotics:Mersacidin

M ersacidin[104](1825 Da)is another lanthionine-con tain ing peptide an tibiotic consisting of 20 am ino acids produced by B.subtilis HIL Y-85,54728.Itbelongs to the type B lantibioticwith a globular structure due tofour interm olecular thioether bridges at positions 1 and 2,4 and 12,13 and 18,and 15 and 20[105].The unusual α,β-didehyd roalanine am ino acidis alsofound within the structure.Usually mersacidins are either uncharged or negatively-charged at neu tral pH and have overall hydrophobic properties[95].

M ersacidinsareactiveagainstgram-positivebacteria including streptococci,bacilli,and staphylococci(similar to subtilin and ericin)[106,107].S.aureus[94,108],one of them ost intensively-investigated bacterial pathogen of hum ans and anim als[109],is am ong the most sensitive organism s tested to date.The M IC of mersacidins(25 μg·m l-1against E.faecium BM 4147)is low er than vancom ycin(500μg·m l-1),andis further reduced(to 12.5 μg·m l-1)w hen mersacidin and vancom ycin are mixed[110].

2.2.3.Unusual lantibiotics:Sublancin and subtilosin A

The novel lan tibiotic sub lancin(164.48 Da)[96]is produced by B.subtilis 168.It has a macrocyclic structure of 37 am ino acid residues with three inter-residual linkages.Οne lanthionine(Cys22linked with Abu19by a thioether bond)and two disu lfides(Cys7-Cys36and Cys14-Cys29)instead of three lanthionines,but none of the disu lfides present in other lan tibio tics(such as subtilin).A special feature of sub lancin is that it con tains a disu lfide bridge in addition to a βmethyllan thionine bridge com pared with other lan tibiotics[111].Recent studies have show n that sublancin is an S-linked glycopep tide containing a glucose attached to a cysteine residue,indicating a new post-translational modification[112].It is believed to be the first naturally-occurring bacterially-derived S-linked glycopeptide.Several studies indicate that glycosylation is essen tial for an tiMicrobial activity[113-115].

Fig.3.Chemical structures of ribosom ally-synthesized products.(A)Subtilin;(B)ericin S;(C)ericin A;(D)sublancin;(E)revised sublancin;(F)subtilosin A;and(G)m ersacidin.Ab indicates α-am inobutyri,ΔA indicates a 2,3-didehydroalanine and ΔB indicates 2,3-didehydrobutyrine.

The antiMicrobial activity spectrum of sublancin is similar to other lantibiotics,exhibiting bactericidal activity against gram-positive but not gram-negative bacteria[96].Bacillus cereus[116],Streptococcus pyogenes,and Staphylococcus aureus are the very three im portant pathogens[117]and Listeria monocytogenes[118]are am ong its antiMicrobial activity spectrum.Bacillus megaterium and Streptococcus pyogenes are inhibited by sublancin with varying degrees of sensitivity.

Subtilosin A[119],with 35 am ino acids(3399.7 Da),is another unusual lan tibio tic produced by B.subtilis 168.The structure of sub tilosin A differs in that there is a cyclized pep tide backbone th io l linkage to the α-carbon of am ino acid residues[120].This undiscovered phenom enon makes it to be to a new class of bacteriocins[121,122].Three cross-links are form ed between the su lfurs of Cys13,Cys7,and Cys4and the α-positions of Phe22,Th r28,and Phe31[123],respectively.The stereochem istry of all residues(excep t for the three modified)is L,with an L isom er at Phe22but a disom er at Th r28and Phe31.Subtilosin A[124]is active against a wide range of bacteria including L.m onocytogenes,E.coli,S.aureus and K.pneum oniae.

2.3.Cell-wall-degrading enzym es(CWDEs)

Enzym es in this fam ily can degrade bacterial and fungal cell walls,m oreoverm uch atten tion has been paid to com ponents specifically acting on chitins and β-glucanase that do not affect hum an health[125,126].These can serve as poten tial biocon trol agen ts for many fungal pathogens through their degradation activity and have been used as food and seed preserving agents[127,128].As these com ponen ts have less potential for use as d rugs in hum ans,they will not be explored fu rther.

3.Non-pep tide Products

Non-pep tide com pounds such as po lyketides and phospholipid from B.subtilis are reported to be antiMicrobial.Polyketides and others such as difficidin,bacillaene,m acro lactins,bacilysocin and 3,3′-neotrehalosadiam ine(NTD)(chemical structures are show n in Fig.4 and Fig.5)account for most of the non-pep tide products.

Fig.4.Chemical structures of macrolactins.A-Q rep resent macrolactin A-N;S:m acrolactin S;Ο-Q rep resent 7-Ο-succiny lm acrolactin A,7-Ο-succiny lm acrolactin F and 7-Ο-m alonylm acro lactin A.

Fig.5.Chemical structures of other non-pep tide products.(A)Difficidin and oxydifficidin;(B)bacilysocin;(C)3,3′-neotrehalosadiam ine;and(D)bacillaene.

A total of 18 different macro lactins have already been described:m acrolactin A-N[129],7-Ο-succinylm acrolactin A,7-Ο-succinyl macro lactin F[130],7-Ο-m alonylm acro lactin A[131]and macro lactin S.They are a group of com ponents with 24-m em bered lactones,excep t macro lactin G-M[132]w h ich has a 22-m em bered lactone ring and three separate diene structure elements.Recently,three new variants were discoveredin the broth of B.subtilis which contained an oxetane,an epoxide and a tetrahydropyran ring[133].How ever the stability of these com ponen ts has not been determined.Mondol et al.[134]discovered a new glycosylated macro lide,which con tained a novel glycosyl.These com ponents have high inhibitory activity against both grampositive and gram-negative pathogenic bacteria(B.subtilis,E.coli and S.aureus),an tiviral activity(Herpes sim plex types I andiI and HIV virus)[135]and anticancer activity(inhibited B16-F10 murine melanom a cancer cells)[136].

Difficidin and oxidifficidin[137],p roduced by ATCC39320 and ATCC 39374,have unsaturated macro lide-like structures with a phosphate ester group attached to the 22-m em bered macrocyclic polyene lactone phosphate ester ring.Difficidin displays a broad antibacterial spectrum,especially against the en terobacterium E.am ylovar,a plan t pathogen causing necrotrophic fire b ligh t disease[138,139].

Bacillaene[140],with am olecu larw eigh tof580Da,isalso a polyene an tibiotic isolated from the fermentation broth of B.subtilis.Bacillaene is a linear mo lecu le con taining enam ine acid(unsatu rated)and two am ide bonds:the first links the α-hyd roxy carboxylic acid to aωam ino carboxylic acid containing a con jugated hexaen and the second links thehexane con taining carboxylic acid to an(ω-1)am ino carboxylic acid con taining a con jugated triene[141].similar to many other products in th is fam ily,bacillaeneis active against bacteria through the inhibition of protein syn thesis(instead of eukaryotic protein syn thesis).

Bacilysocin[142],1-(12-methyltetradecanoy l)-3-phosphoglyceroglycero l,is an an tiMicrobial phospho lipidiso lated from B.subtilis 168.Bacilysocin has an tiMicrobial activity against S.cerevisiae and S.aureus,as w ellas against certain fungi such as Candida pseudotropicalis and Cryptococcus neoformans.

The am ino-sugar antibiotic 3,3′-neotrehalosadiam ine(NTD)is produced by several Bacillus species and functions as an autoinducer by activating itsow n biosyn theticoperon(ntdABC)[143].NTdisuniqueasan am ino-sugar,due to the low frequency ofam ino-sugars in B.subtilis.It is the first exam ple of an α,β-glycoside with two am ino sugars com pared to other 1-1 glycosides,which are linked am ino disaccharide antibiotics with α,α-glycosides of an am ino sugar and a neu tral sugar[144].NTdinhibits the grow th of S.aureus and K.pneum onia[145],with little else reported.

4.Conclusions and Perspectives

Bacillus spp.are abundan t source of natural bioactive products with structurally diverse metabolites displaying strong antibacterial and antifungal activities.How ever,pu rification of secondary metabo lites from fermentation broths[146]can be a challenging task,so it is im perative to so lve the main problem of achieving higher yields.Com plications included,I think,are main ly due to these factors:(a)the com plexity of the medium and variations in pH,temperature and other factors in the process of fermentation[147-149],inheren tly resulting in the appearance of different products,poor iso lation/yield and loss of antibiotic activity[149].(b)Diversity of the bioactive products[133,150].It is not uncomm on that more than one bioactive componen t with similar an tiMicrobial activities exist in the broth cu lture,which poses challenges to isolation.(c)Unstable molecu lar structures or enzym es present in the fermentation broth may destroy bioactive structures,leading to a decrease or loss of bioactive activity.So how to main tain the activity in proper w ays is an im portan t issue to be solved.(d)The scale of the preparative process is another restraint.During the purification of the target products,procedures are alw ays on a laboratory scale,which will increase the cost inevitably.

Pub lished studies[61,151]have elucidated the biosyn thetic pathw ays of certain secondary metabo lites,including the gene clusters,enzym es and their relevan t functions.In other w ords,the mo lecu lar mechan ism s regu lating functional groups or genes invo lvedin the biosyn thesis of these com ponen ts have been exploredin detail.For exam ple,bacilysin is one of the sim plest pep tides antibiotics syn thesized non-ribosom ally by B.subtilis w hose syn thesis depends on the bacABCDEyw fG operon(bac operon)and the ad jacent yw fH gene[152-154].By means of genetic manipu lation,the knockout of certain useless gene invo lvedin branched metabo lic pathw ays,o r by strengthen ing the genes in the main metabo lic pathw ay,the yield and pu rity of the products of in terest may be increased.Th is has been accom plished for the overp roduction of NTD by B.subtilis by mutating the RNA polym erase(RNAP)and selecting for the rifam pin(Rif)-resistan t phenotype[143,155].Th is app roach cou ld be used to im prove large-scale production[156]of these mo lecu les in a mo re econom ical manner.

Up to now,antibiotics in therapeutic use are chemically syn thesized/m odified or derived from established com pound classes.Chemicalm odification has already been applied to substances of microbial origin,yielding sem isyn thetic products.Sim u ltaneously the modified com ponen ts,on am assive scalewillexhibita superior pharm acologicalbehavior and a broader spectrum of activity[157-160].In recent years,fragm en t-based d rug discovery(FBDD)has been used by the pharm aceu tical industry to reduce attrition and provide leads to previously intractable biological targets[161,162].Consequently,if the mechanism s of action for natural com ponen ts cou ld be determinedin detail,FBDD then cou ld be used to design chemically-syn thesized or sem isyn thetic molecules with similar modes of action[163].