Characterization of the keratinolytic activity of three streptomyces strains and impact of their co-cultivation on this activity

Ci a ion: Ma ín-González, D.; Bo del, S.; San os-Benei , F. Cha ac e iza ion o he Ke a inoly ic Ac i i y o Th ee S ep omyces S ains and Impac o Thei Co-Cul i a ion on This Ac i i y. Mic oo ganisms 2023, 11, 1109. h ps://doi.o g/10.3390/ mic oo ganisms11051109 Academic Edi o : Myung-Ji Seo Recei ed: 22 Ma ch 2023 Re ised: 20 Ap il 2023 Accep ed: 23 Ap il 2023 Published: 24 Ap il 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). mic oo ganisms A icle Cha ac e iza ion o he Ke a inoly ic Ac i i y o Th ee S ep omyces S ains and Impac o Thei Co-Cul i a ion on This Ac i i y Diego Ma ín-González 1,2, Se gio Bo del 1,2 and Fe nando San os-Benei 1,2,3,* 1Ins i u e o Sus ainable P ocesses, D . Me gelina, s/n, 47011 Valladolid, Spain 2Depa men o Chemical Enginee ing and En i onmen al Technology, School o Indus ial Enginee ing, Uni e si y o Valladolid, D . Me gelina, s/n, 47011 Valladolid, Spain 3Depa men o Func ional Biology, Medical School, Uni e si y o O iedo, A . Julián Cla e ía, 6, 33006 O iedo, Spain *Co espondence: [email p o ec ed] Abs ac : In his s udy, we desc ibe he cha ac e iza ion o h ee e icien chicken ea he -deg ading S ep omyces bac e ia isola ed om honeybee samples and assess he impac o hei co-cul i a ion on his ac i i y and an is aphylococcal ac i i y. S ep omyces g iseoau an iacus AD2 was he s ain showing he highes ke a inoly ic ac i i y (4000 U × mL −1 ), ollowed by S ep omyces albido la us AN1 and S ep omyces d ozdowiczii AD1, which bo h gene a ed app oxima ely 3000 U × mL −1 . Mo eo e , a conso ium cons i u ed o hese h ee s ains was able o use chicken ea he s as i s sole nu ien sou ce and g ow h in such condi ions led o a signi ican inc ease in an ibio ic p oduc ion. S. g iseoau an iacus AD2 was he only s ain ha exhibi ed weak an imic obial ac i i y agains S aphylococcus au eus. UPLC analyses e ealed ha a signi ican numbe o peaks de ec ed in he ex ac s o co-cul u es o he h ee s ains we e missing in he ex ac s o indi idual cul u es. In addi ion, he p oduc ion o specialized me aboli es, such as undecylp odigiosin and manumycin A, was clea ly enhanced in co-cul u e condi ions, in ag eemen wi h he esul s o he an imic obial bioassays agains S. au eus. Ou esul s e ealed he bene i s o co-cul i a ion o hese bac e ial species in e ms o me abolic weal h and an ibio ic p oduc ion. Ou wo k could hus con ibu e o he de elopmen o no el mic obial-based s a egies o alo ize ke a in was e. Keywo ds: S ep omyces; undecylp odigiosin; S aphylococcus au eus; ke a inase; an ibio ic; ea he s; bio e ilize s; co-cul u e 1. In oduc ion P omo ing he ci cula economy is pa o he Uni ed Na ions Sus ainable De elopmen Goals, and is one o he pilla s o he so-called “New G een Deal” o he Eu opean Union. The e o e, i is e y impo an o cha ac e ize he po en ial o all li ing beings, including ha o mic oo ganisms o use and/o alo ize na u al esou ces ha a e conside ed was e. Fo example, globally, poul y a ming p oduces housands o ons o chicken ea he s each yea [ 1 ] and chicken ea he s a e conside ed was e since hey canno be used o eed animals, e en i hey ha e a po en ial nu i ional alue, o canno be used in any o he p o i able applica ion. The e o e, ea he s a e no mally dumped in land ills o incine a ed [ 2 ]. Howe e , some mic oo ganisms p oduce special enzymes, known as ke a inases, ha can deg ade hese ke a in- ich was es [ 3 ]. P e iously, he isola ion o s ains om soil and honeybee p oduc s led o he cha ac e iza ion o h ee Bacillus s ains wi h high ke a inoly ic ac i i y [ 4 , 5 ]. These s ains in addi ion o showing ke a inoly ic ac i i y also exhibi ed o he in e es ing p ope ies, such as he capabili y o deg ade plas ics (Bacillus al i udinis B12 [ 4 ]) o o inhibi he g ow h o G am-posi i e bac e ia (Bacillus licheni o mis CG1 and Bacillus sono ensis AB7, bo h isola ed om honeybee p oduc s [ 5 ]). Mic oo ganisms 2023,11, 1109. h ps://doi.o g/10.3390/mic oo ganisms11051109 h ps://www.mdpi.com/jou nal/mic oo ganisms Mic oo ganisms 2023,11, 1109 2 o 10 Among he h ee s ains, he highes ke a inoly ic ac i i y was achie ed by B. licheni o mis CG1 (3800 U × mL −1 ), ollowed by B. al i udinis B12 (1500 U × mL −1 ) and B. sono ensis AB7 (1450 U ×mL−1). S ep omyces species s and ou as he mos impo an p oduce s o an ibio ics among all gene a o bac e ia disco e ed o da e [ 6 ]. In addi ion o an ibio ics, mos o hese bac e ia can p oduce o he use ul bioac i e compounds, including an icance , immunosupp essan s, he bicides, insec icides, pigmen s and a wide a ie y o enzymes ha can hyd olyze a huge numbe o di e en subs a es [ 7 ]. We ha e also isola ed S ep omyces species om honeybee samples and cha ac e ized hei an imic obial p ope ies [ 8 ]. These species we e di ec ly collec ed ei he om aw honey (i.e., S. d ozdowiczii AD1 and S. g iseoau an iacus AD2) o om bee pollen (i.e., S. albido la us AN1). Using Liquid Ch oma og aphy-Mass Spec ome y (LC-MS) and de eplica ion analyses (LC-DAD-HRMS), we we e able o cha ac e ize wo me aboli es, he ed undecylp odigiosin and he colo less manumycin A, ha we e esponsible o he an ibio ic ac i i y o S. g iseoau an iacus AD2 agains G am- posi i e bac e ia such as S aphylococcus au eus,En e ococcus aecalis and Bacillus sub ilis [ 8 ]. Since hese S ep omyces s ains ha e been isola ed om he same ype o samples as he p e iously men ioned Bacillus isola es [ 5 ], which showed bo h ke a inoly ic p ope ies and an imic obial ac i i y, in his s udy, we cha ac e ize he ke a inoly ic po en ial o hese S ep omyces species ha we e p e iously cha ac e ized in e ms o hei an imic obial po en ial bu no on hei capabili y o deg ade ke a in. We also assessed he impac o co- cul i a ion o hese h ee S ep omyces s ains on hei abili y o use chicken ea he s as hei sole nu ien sou ce and o p oduce undecylp odigiosin and manumycin A using bioassays as well as LC-DAD-HRMS and LC-MS analyses. Ou esul s sugges ha mic obial-based s a egies o ecycle and alo ize ke a in- ich was es could be de eloped o yield use ul an ibio ic mix u es o no el bio e ilize s. 2. Ma e ials and Me hods 2.1. Mic obial G ow h Condi ions and G ow h De e mina ion The S ep omyces s ains used in his s udy we e isola ed om samples o honey and pollen ha we e collec ed om beehi es loca ed in Sou h Eas England [ 8 ]. R5A medium [ 9 ] inocula ed wi h 10 6 spo es pe mL [ 10 , 11 ] was used o ou ine g ow h o he S ep omyces s ains. O he wise, S ep omyces cells we e g own in a mine al sal medium wi h chicken ea he s ha we named CFMS ( C hicken F ea he s M ine al S al s). This medium con ains glucose (1 g × L −1 ), pep one (0.5 g × L −1 ), KH 2 PO 4 (0.7 g × L −1 ), K 2 HPO 4 (1.4 g × L −1 ), MgSO 4 (0.1 g × L −1 ), NaCl (0.5 g × L −1 ), ZnSO 4· 7H 2 0 (0.05 g × L −1 ), FeSO 4· 7H 2 0 (0.015 g × L −1 ) and chicken ea he s (10 g × L −1 ) and had pH = 8; o on a medium ha we named CFTW ( C hicken F ea he s T ap W a e ). This medium con ains solely chicken ea he s (10 g × L −1 ) and ap wa e (no ca bon, ni ogen, o phospha e sou ces no ace elemen s we e added o his medium whose pH was also no adjus ed). Fo he expe imen on he e ec o pH on ke a inoly ic ac i i y, he pH o he CFMS medium was adjus ed o 7 (neu al) o 6 (acid) ins ead o 8 (basic). In all cases, chicken ea he s we e p o ided by a poul y a m in Valladolid, Spain. The ea he s we e p ocessed as ollows: (i) washing wi h ap wa e and T i on-X o ge id o any deb is, (ii) emo al o T i on-X wi h dis illed wa e , (iii) d ied a 60 ◦ C o e nigh , (i ) cu in o small pieces o p ope addi ion o he cul u es. Fo he chicken ea he s deg ada ion expe imen on CFTW medium, cul u es we e pe o med in 2.2 L bo les wi h 200 mL o medium and 1 mL o an R5A p ecul u e wi h each o he S ep omyces species g own sepa a ely o oge he in a co-cul u e du ing an iden ical pe iod o ime (i.e., 48 h). The ini ial R5A p e-cul u es o each indi idual S ep omyces s ain we e achie ed by inocula ion wi h 10 6 spo es pe mL whe eas ha o he co-cul u e was inocula ed wi h 10 2 spo es pe mL o each s ain yielding a o al amoun o 10 6 spo es pe mL. To quan i y bac e ial g ow h and moni o he ke a in deg ada ion p ocess du ing ime-se ies expe imen s o se e al weeks, he cul u es we e pe o med in 2.2 L glass bo les he me ically closed wi h an isop ene ubbe and aluminum c imp seal. Since he mycelial g ow h o he S ep omyces cells and he u bidi y Mic oo ganisms 2023,11, 1109 3 o 10 gene a ed by he deg ada ion o he ea he s hampe s he use o op ical densi y o moni o g ow h, CO 2 and O 2 concen a ions we e quan i ied in he a mosphe e o he he me ically closed bo les h oughou he ime-se ies expe imen s. Oxida ion o ca bohyd a es h ough ae obic espi a ion consumed O 2 and eleased CO 2 a an equi alen a io. O 2 and CO 2 concen a ions we e measu ed using a gas ch oma og aph (Agilen 8860, The Ne he lands) equipped wi h a he mal conduc i i y de ec o (GC-TCD) ollowing p ocedu es desc ibed elsewhe e [12]. 2.2. Analysis o Me aboli es by Ul a Pe o mance Liquid Ch oma og aphy (UPLC) Me aboli es om he S ep omyces R5A cul u es (inocula ed wi h 10 6 li e spo es) g own du ing 96 h (i.e., s a iona y phase) we e ex ac ed using he ollowing p ocedu e. E hyl ace a e was added o he samples (con aining bo h supe na an s and biomass) a a 1:1 a io (20 mL:20 mL) and main ained in cons an mixing o 2 h. The o ganic phase ( ≤ 20 mL) was sepa a ed om he aqueous phase by cen i uga ion and collec ed o u he e apo a ion using a SpeedVac. The esul ing ex ac s we e e-suspended in 200 µ L o me hanol. The cell ex ac s we e analyzed by e e sed-phase ch oma og aphy using ace oni ile and wa e con aining 0.1% i luo oace ic acid (TFA) as sol en s in an Acqui y UPLC ins umen i ed wi h a BEH C18 column (1.7 mm, 2.1 × 100 mm; Wa e s). Samples we e elu ed wi h 10% ace oni ile o 1 min, ollowed by a linea g adien om 10 o 100% ace oni ile o e 7 min, a a low a e o 0.5 mL min −1 and a column empe a u e o 35 ◦ C. De ec ion and spec al cha ac e iza ion o he peaks ob ained om UPLC was pe o med by pho odiode a ay de ec ion using Empowe so wa e .3 (Wa e s) o ex ac bi-dimensional ch oma og ams a di e en wa eleng hs. 2.3. Analysis o Me aboli es by Liquid Ch oma og aphy-Mass Spec ome y (LC-MS) LC-MS analysis was pe o med using an Alliance ch oma og aphic sys em coupled o a ZQ4000 mass spec ome e and a SunFi e C18 column (3.5 mm, 2.1 × 150 mm; Wa e s). Sol en s we e he same as hose used in he UPLC analyses and elu ion was pe o med wi h an ini ial isoc a ic hold wi h 10% ace oni ile o 4 min ollowed by a linea g adien om 10 o 88% ace oni ile o e 26 min, a 0.25 mL min −1 . A e he LC phase, MS was ca ied ou by elec osp ay ioniza ion in he posi i e mode, wi h a capilla y ol age o 3 kV and a cone ol age o 20 and 50 V. De ec ion and spec al cha ac e iza ion o he peaks was pe o med by pho odiode a ay de ec ion using Empowe so wa e .3 (Wa e s) o ex ac bi-dimensional ch oma og ams a di e en wa eleng hs, no mally wi hin he ange be ween 200 and 500 nm depending on he spec al cha ac e is ics o he desi ed compound. 2.4. De eplica ion S udies (LC-DAD-HRMS) The o ganic ex ac s o he co-cul u e we e subjec ed o de eplica ion using a combi- na ion o LC-DAD-HRMS analysis wi h an Agilen 1200 Rapid Resolu ion HPLC sys em coupled o an ESI mode B uke maXis mass spec ome e as desc ibed in [ 8 ]. The sepa a ion o compounds was conduc ed using ace oni ile and wa e con aining ammonium o ma e 13 mM and 0.01% TFA as sol en s in a Zo bax SB-C8 column (3.5 mm, 2.1 × 30 mm). The esul ing ch oma og aphic uns we e p ocessed using a B uke in-house componen ex ac ion algo i hm o iden i y To al Ion Ch oma og am (TIC) posi i e peaks a 210 nm. Fo molecula o mula in e p e a ion, we used e en ion ime and exac mass as sea ch c i e ia on he high- esolu ion mass spec ome y da abase o he Medina Founda ion (G anada, Spain). We look speci ically o compounds wi h known an ibio ic ac i i y, such as undecylp odigiosin and manumycin A. 2.5. G ow h Inhibi ion Bioassays Cell ex ac s om he S ep omyces cul u es we e assessed o g ow h inhibi ion agains S aphylococcus au eus. Fo he bioassays, 10 µ L o cell ex ac s we e deposi ed on s e ile disks ha had been p e iously placed on op o TSA ( yp one soya aga ) pla es inocula ed wi h he co esponding indica o mic oo ganism as ollows. To g ow he p einoculum o S. Mic oo ganisms 2023,11, 1109 4 o 10 au eus, he cells we e aken di ec ly om a glyce ol s ock a − 80 ◦ C and inocula ed in 20 mL o TSB ( yp one soya b o h) medium and hen g own un il hey each an OD 600 ~1. Then, 200 µ L o he S. au eus cells we e homogenized in o 5 mL o empe ed TSB (con aining 0.7% o aga ) and pou ed as a hin laye (5 mL) o e a solid laye (20 mL) o TSB (con aining 1.4% o aga ) placed on he Pe i dishes p e iously. A e he solidi ica ion o he hin laye , he s e ile disks we e placed on he pla es. In all cases, he same amoun o e hyl ace a e ex ac ed sample (10 µ L) was added o he disks. An imic obial ac i i y was examined as isible inhibi ion halos a e incuba ion o he aga pla es o 24 h a 30 ◦C. 2.6. Ke a inoly ic Ac i i y De ec ion Assay Ke a inoly ic ac i i y was de e mined ollowing p e ious exis ing me hods [ 13 , 14 ]. Liquid samples o 0.7 mL o each cul u e we e aken e e y day and cen i uged o 20 min a 13,000 pm o ge a inal 0.5 mL aliquo o supe na an . Wi h he cen i uga ion s ep, cells and ea he deb is we e emo ed. The 0.5 mL aliquo was mixed wi h 0.5 mL o 100 mM glycine-NaOH (pH 10) con aining 1% casein and incuba ed a 37 ◦ C o 20 min. In his eac ion, y osine is eleased a e he b eaking o casein by he ke a inases, among o he enzymes. The eac ion was s opped by he addi ion o 0.5 mL o ichlo oace ic acid (20% w/ ) and incuba ion o 15 min a oom empe a u e. A e he inac i a ion o he enzymes, he samples we e cen i uged o 15 min a 13,000 pm and he OD 280nm o he supe na an was measu ed wi h a spec opho ome e . A s anda d cu e was pe o med using solu ions o 0–700 mg L −1 o y osine. One ke a inase uni (U) was de ined as he amoun o enzyme equi ed o inc ease he abso bance by 0.01 uni (OD 280nm ) in one minu e unde he assay condi ions employed. 3. Resul s 3.1. Cha ac e iza ion o Ke a inoly ic Ac i i y in S ep omyces Albido la us AN1, S ep omyces D ozdowiczii AD1 and S ep omyces G iseoau an iacus AD2 In ou ecen wo k, we ocused on he cha ac e iza ion o s ains ha can deg ade chicken ea he s [ 4 , 5 ]. Chicken ea he s a e conside ed was e and a e no mally dumped in land ills o incine a ed [ 2 ]. The e o e, s ains wi h he abili y o hyd olyze such was e a e e y in e es ing om a bio echnological poin o iew. To ollow up wi h his app oach we decided o es he ke a inoly ic ac i i y o he h ee S ep omyces species analyzed in his s udy. In o de o cha ac e ize hei ke a inoly ic ac i i y, each s ain was g own on CFMS ( C hicken F ea he s M ine al S al s) medium un il eaching a s a iona y phase. Figu e 1shows he ke a inoly ic ac i i y alues ha each o he s ains displayed du ing he ime cou se o he expe imen . S. g iseoau an iacus AD2 showed he highes ac i i y (~4000 U × mL −1 ); ollowed by he wo o he s ains, which displayed a 25% lowe ac i i y (~3000 U × mL −1 ). In conclusion, he h ee s ains ha e ke a inoly ic ac i i y, which makes hem p omising candida es o bio echnological applica ions on ea he alo iza ion. Mic oo ganisms2023,11,xFORPEERREVIEW5o 11    Figu e1.Ke a inoly icac i i yo S ep omycesalbido la usAN1(g eensqua es),S ep omyces d ozdowicziiAD1(blue iangles)andS ep omycesg iseoau an iacusAD2( edci cles)g ownon CFMS(ChickenFea he sMine alSal s)medium(pH=8).Sampleswe ecollec ede e y24hun il cells eached hes a iona yphase ode e mineke a inoly icac i i y.Ve icale o ba sco espond o hes anda de o o  hemeano  h ee eplica es. 3.2.Effec o pHon heKe a inoly icAc i i yo  heS ains pHplaysamajo  oleinsomecellula ac i i ies.Inp e iouswo k,usingsimila as- saycondi ions,wede e mined ha  hehighes ke a inoly icac i i ywasachie edbyB. licheni o misCG1when hepHo  hemediumwas8[5].Fo  his eason, hepHo  he CFMSmedium ha weha ede eloped o  hes udyo  heS ep omycess ainswasse  o 8(seeMa e ialsandMe hodssec ion).Howe e , oha eabe e insigh in o hecondi- ions equi edby hes ains odeg adeke a in,we es ed heeffec o acid(pH=6),neu- al(pH=7)andbasic(pH=8)pHon heke a inoly icac i i yo  he h eeS ep omyces s ains.Theke a inoly icac i i yo eacho  hes ainswasde e minedin hesecondi ions in hesamewayasinSec ion3.1. Thehighes ke a inoly icac i i ywasde ec eda pH=8inall hes ains,as o  he B.licheni o misCG1s ain.Indeed, heke a inoly icac i i ydec easedas ollows:basic> neu al>acid(seeFigu es1and2).Asimila  endwasalsono ed o  heke a inaseac- i i yo  heB.al i udinisB12s ain,whichwasshown obe a o edinbasiccondi ions (i.e.,pH om8 o10)[4].Wealsochecked hee olu iono  hepHdue ochicken ea he  deg ada ionandmic obialme abolism om hes a o  heexpe imen ( =0h) o heend o  heexpe imen ( =96h).AsshowninTable1, he inalpHdec eased oa alueo ~6 inall hes ains es ed,independen lyo  heini ialpH(no e ha when heini ialpH alue was6, he inalpHwassligh lydec easedby~0.2uni s;seeTable1).Inconclusion,acidi- ica iono  hemediummigh dec ease heke a inoly icac i i yo  hes ains,bu only sligh lysinceallshoweda a he highke a inoly icac i i yin he angeo pH es ed. Table1.pHe olu ion om heini ial alue o he inal alue eacheda e 4dayso g ow ho  he s ainsS.d ozdowicziiAD1,S.g iseoau an iacusAD2andS.albido la usAN1. Ini ialpH =0h FinalpH =96h Ini ialpH =0h FinalpH =96h Ini ialph =0h FinalpH =96h AD18.06.2±0.27.06.2±0.16.05.9±0.1 AD28.06.0±0.17.06.0±0.16.05.8±0.1 AN18.06.0±0.17.06.0±0.16.05.7±0.2 Figu e 1. Ke a inoly ic ac i i y o S ep omyces albido la us AN1 (g een squa es), S ep omyces d oz- dowiczii AD1 (blue iangles) and S ep omyces g iseoau an iacus AD2 ( ed ci cles) g own on CFMS (Chicken Fea he s Mine al Sal s) medium (pH = 8). Samples we e collec ed e e y 24 h un il cells eached he s a iona y phase o de e mine ke a inoly ic ac i i y. Ve ical e o ba s co espond o he s anda d e o o he mean o h ee eplica es. Mic oo ganisms 2023,11, 1109 5 o 10 3.2. E ec o pH on he Ke a inoly ic Ac i i y o he S ains pH plays a majo ole in some cellula ac i i ies. In p e ious wo k, using simila assay condi ions, we de e mined ha he highes ke a inoly ic ac i i y was achie ed by B. licheni o mis CG1 when he pH o he medium was 8 [ 5 ]. Fo his eason, he pH o he CFMS medium ha we ha e de eloped o he s udy o he S ep omyces s ains was se o 8 (see Ma e ials and Me hods sec ion). Howe e , o ha e a be e insigh in o he condi ions equi ed by he s ains o deg ade ke a in, we es ed he e ec o acid (pH = 6), neu al (pH = 7) and basic (pH = 8) pH on he ke a inoly ic ac i i y o he h ee S ep omyces s ains. The ke a inoly ic ac i i y o each o he s ains was de e mined in hese condi ions in he same way as in Sec ion 3.1. The highes ke a inoly ic ac i i y was de ec ed a pH = 8 in all he s ains, as o he B. licheni o mis CG1 s ain. Indeed, he ke a inoly ic ac i i y dec eased as ollows: basic > neu al > acid (see Figu es 1and 2). A simila end was also no ed o he ke a inase ac i i y o he B. al i udinis B12 s ain, which was shown o be a o ed in basic condi ions (i.e., pH om 8 o 10) [ 4 ]. We also checked he e olu ion o he pH due o chicken ea he deg ada ion and mic obial me abolism om he s a o he expe imen ( = 0 h) o he end o he expe imen ( = 96 h). As shown in Table 1, he inal pH dec eased o a alue o ~6 in all he s ains es ed, independen ly o he ini ial pH (no e ha when he ini ial pH alue was 6, he inal pH was sligh ly dec eased by ~0.2 uni s; see Table 1). In conclusion, acidi ica ion o he medium migh dec ease he ke a inoly ic ac i i y o he s ains, bu only sligh ly since all showed a a he high ke a inoly ic ac i i y in he ange o pH es ed. Mic oo ganisms2023,11,xFORPEERREVIEW6o 11    Figu e2.Ke a inoly icac i i yo S ep omycesalbido la usAN1(g eensqua es),S ep omyces d ozdowicziiAD1(blue iangles)andS ep omycesg iseoau an iacusAD2( edci cles)g ownon CFMS(ChickenFea he sMine alSal s)mediuma  wodiffe en pH.(A)pH=6.(B)pH=7.Inall cases,sampleswe ecollec ede e y24hand o 4days ode e mine heke a inoly icac i i y.Ve - icale o ba sco espond o hes anda de o o  hemeano  h ee eplica edexpe imen s. 3.3.Tes ing heBene i so Co-Cul u ing heS ainsinTe mso An ibio icP oduc ion Weha ep e iouslyshown ha onlyS.g iseoau an iacusAD2sligh lyinhibi s he g ow ho someG am-posi i ebac e iawhe eas heo he  wodidno [8].The e o e,we decided oassess heimpac o co-cul u ingo S.albido la usAN1,S.d ozdowicziiAD1and S.g iseoau an iacusAD2on heabili y op oducean imic obialac i i yincompa ison wi h hean imic obialac i i yo eachs aing ownindi idually.Todoso, hes ains we ecul u ed(using hesameamoun o  o als a inginoculum)inR5Amediumei he  sepa a elyo  oge he  o 4days.Then,me aboli eswe eex ac edusinge hylace a e, and he esul ingcellex ac swe eanalyzedbyUPLCanalysesandan imic obialbioas- says(seeFigu e3). TheUPLCanalysis e ealed ha asigni ican numbe o peaks ha we ede ec edin heex ac so  heco-cul u ewe eabsen  om heex ac so  hesinglecul u es(Figu e 3A).I espec i eo whichme aboli esco espond oeacho  hesepeaks, he esul clea ly indica es ha co-cul u ingo  hes ainshighlyinc easesme aboli ep oduc ion.Among heseme aboli es,undecylp odigiosinandmanumycinA(wi h e en ion imesa ound7 min)ha ebeenp e iouslyshown ocon ibu e o hean ibio icpo en ialo S.g iseoau- an iacusAD2[8].These wopeaksa esigni ican lyinc easedin hecellex ac so  heco- cul u e,ingoodco ela ionwi h he esul so  hean imic obialbioassaysagains S.au eus showninFigu e3B.Theex ac s om heco-cul u eand hecul u eo S.g iseoau an iacus AD2exhibi edan ibio icac i i yagains  heS aphylococcusau euscon ols ain,bu  he sizeo  heinhibi ionhaloswasclea lybigge wi h heex ac so  heco-cul u e,which indica es ha  hean ibio icac i i yissigni ican lyinc easedwhen he h ees ainsa e cul u ed oge he (consis en lywi h he esul sobse edin heUPLCanalyses).The e- o e, hese esul shighligh  hebene i so co-cul i a iono  hesebac e ialspeciesin e ms o me abolicweal handan ibio icpo en ial. Figu e 2. Ke a inoly ic ac i i y o S ep omyces albido la us AN1 (g een squa es), S ep omyces d oz- dowiczii AD1 (blue iangles) and S ep omyces g iseoau an iacus AD2 ( ed ci cles) g own on CFMS (Chicken Fea he s Mine al Sal s) medium a wo di e en pH. ( A ) pH = 6. ( B ) pH = 7. In all cases, samples we e collec ed e e y 24 h and o 4 days o de e mine he ke a inoly ic ac i i y. Ve ical e o ba s co espond o he s anda d e o o he mean o h ee eplica ed expe imen s. Table 1. pH e olu ion om he ini ial alue o he inal alue eached a e 4 days o g ow h o he s ains S. d ozdowiczii AD1, S. g iseoau an iacus AD2 and S. albido la us AN1. Ini ial pH =0h Final pH =96h Ini ial pH =0h Final pH =96h Ini ial pH =0h Final pH =96h AD1 8.0 6.2 ±0.2 7.0 6.2 ±0.1 6.0 5.9 ±0.1 AD2 8.0 6.0 ±0.1 7.0 6.0 ±0.1 6.0 5.8 ±0.1 AN1 8.0 6.0 ±0.1 7.0 6.0 ±0.1 6.0 5.7 ±0.2 3.3. Tes ing he Bene i s o Co-Cul u ing he S ains in Te ms o An ibio ic P oduc ion We ha e p e iously shown ha only S. g iseoau an iacus AD2 sligh ly inhibi s he g ow h o some G am-posi i e bac e ia whe eas he o he wo did no [ 8 ]. The e o e, we decided o assess he impac o co-cul u ing o S. albido la us AN1, S. d ozdowiczii AD1 and S. g iseoau an iacus AD2 on he abili y o p oduce an imic obial ac i i y in compa ison wi h he an imic obial ac i i y o each s ain g own indi idually. To do so, he s ains we e cul u ed (using he same amoun o o al s a ing inoculum) in R5A medium ei he Mic oo ganisms 2023,11, 1109 6 o 10 sepa a ely o oge he o 4 days. Then, me aboli es we e ex ac ed using e hyl ace a e, and he esul ing cell ex ac s we e analyzed by UPLC analyses and an imic obial bioassays (see Figu e 3). Mic oo ganisms2023,11,xFORPEERREVIEW7o 11    Figu e3.Analysiso me aboli esand hean imic obialac i i yo S ep omycesalbido la usAN1, S ep omycesd ozdowicziiAD1,S ep omycesg iseoau an iacusAD2and heco-cul u eo  he h ee species.(A)Compa a i eUPLCch oma og amso me aboli esex ac edwi he hylace a e om96 hcul u eso eacho  hes ainsand heco-cul u eo  he h eeS ep omycesspeciesinR5Amedium. (B)An imic obialbioassayagains S aphylococcusau euswi h10µLo  hecellex ac sused o  he UPLCanalyseso sec ionA ha we eex ac ed om hecul u esshownin he igu e. 3.4.Iden i ica iono Undecylp odigiosinandManumycinAin heCellEx ac so  heCo- Cul u e Tocon i m hesyn hesiso undecylp odigiosinandmanumycinAin heco-cul u e condi ionbo hLC-MSandLC-DAD-HRMSanalyseswe epe o med.In hela e e en ion imeso  heUVch oma og ams, heLC-MSanalysisallowed heiden i ica iono  wo me aboli eswi hmasseso m/z394.33[M+H] + andm/z551.32[M+H] + ,whichco espond o hemasseso undecylp odigiosin(C 25 H 35 N 3 O)andmanumycinA(C 31 H 38 N 2 O 7 ), espec- i ely(Figu e4A).ByLC-DAD-HRMSde eplica ionanalyses hese wome aboli eswe e u he con i med obeundecylp odigiosin(Figu e4B)andmanumycinAo D(depend- ingon henumbe o Hyd ogensin he o mula;i.e.,H 38 o H 40 , espec i ely. Figu e 3. Analysis o me aboli es and he an imic obial ac i i y o S ep omyces albido la us AN1, S ep omyces d ozdowiczii AD1, S ep omyces g iseoau an iacus AD2 and he co-cul u e o he h ee species. ( A ) Compa a i e UPLC ch oma og ams o me aboli es ex ac ed wi h e hyl ace a e om 96 h cul u es o each o he s ains and he co-cul u e o he h ee S ep omyces species in R5A medium. ( B ) An imic obial bioassay agains S aphylococcus au eus wi h 10 µ L o he cell ex ac s used o he UPLC analyses o sec ion A ha we e ex ac ed om he cul u es shown in he igu e. The UPLC analysis e ealed ha a signi ican numbe o peaks ha we e de ec ed in he ex ac s o he co-cul u e we e absen om he ex ac s o he single cul u es (Figu e 3A). I espec i e o which me aboli es co espond o each o hese peaks, he esul clea ly indi- ca es ha co-cul u ing o he s ains highly inc eases me aboli e p oduc ion. Among hese me aboli es, undecylp odigiosin and manumycin A (wi h e en ion imes a ound 7 min) ha e been p e iously shown o con ibu e o he an ibio ic po en ial o S. g iseoau an iacus AD2 [ 8 ]. These wo peaks a e signi ican ly inc eased in he cell ex ac s o he co-cul u e, in good co ela ion wi h he esul s o he an imic obial bioassays agains S. au eus shown in Figu e 3B. The ex ac s om he co-cul u e and he cul u e o S. g iseoau an iacus AD2 exhibi ed an ibio ic ac i i y agains he S aphylococcus au eus con ol s ain, bu he size o he inhibi ion halos was clea ly bigge wi h he ex ac s o he co-cul u e, which indica es ha he an ibio ic ac i i y is signi ican ly inc eased when he h ee s ains a e cul u ed oge he (consis en ly wi h he esul s obse ed in he UPLC analyses). The e o e, hese esul s highligh he bene i s o co-cul i a ion o hese bac e ial species in e ms o me abolic weal h and an ibio ic po en ial. 3.4. Iden i ica ion o Undecylp odigiosin and Manumycin A in he Cell Ex ac s o he Co-Cul u e To con i m he syn hesis o undecylp odigiosin and manumycin A in he co-cul u e condi ion bo h LC-MS and LC-DAD-HRMS analyses we e pe o med. In he la e e en ion imes o he UV ch oma og ams, he LC-MS analysis allowed he iden i ica ion o wo me aboli es wi h masses o m/z394.33 [M + H] + and m/z551.32 [M + H] + , which co e- spond o he masses o undecylp odigiosin (C 25 H 35 N 3 O) and manumycin A (C 31 H 38 N 2 O 7 ), espec i ely (Figu e 4A). By LC-DAD-HRMS de eplica ion analyses hese wo me aboli es we e u he con i med o be undecylp odigiosin (Figu e 4B) and manumycin A o D (depending on he numbe o Hyd ogens in he o mula; i.e., H38 o H40, espec i ely. Mic oo ganisms 2023,11, 1109 7 o 10 Figu e 4. Iden i ica ion o undecylp odigiosin and manumycin A me aboli es p esen in he co-cul u e o S ep omyces albido la us AN1, S ep omyces d ozdowiczii AD1 and S ep omyces g iseoau an iacus AD2 by LC-MS and LC-DAD-HRMS de eplica ion analyses. ( A ) UV ch oma og ams o bo h UPLC (g ey) and LC-MS (black) analyses and MS analyses o he ex ac ed UV peaks indica ed by a ows; esul ing in he iden i ica ion o he me aboli es manumycin A and undecylp odigiosin. ( B ) Iden i ica ion o undecylp odigiosin by LC-DAD-HRMS de eplica ion analyses. 3.5. Using Chicken Fea he s as Sole Subs a e o G owing he S ep omyces S ains Finally, we checked he capabili y o he indi idual s ains and he co-cul u e (since i p o ides an ad an age in e ms o me abolic weal h) o use chicken ea he s as he sole nu ien sou ce o hei g ow h. To do so, we de eloped a medium ha we named CFTW ( C hicken F ea he s T ap W a e medium). This medium con ains only ap wa e and chicken ea he s, which makes i s p oduc ion cheap and hus well adap ed o he de elopmen o a mic obial ecycling pla o m o he alo iza ion o ke a in- ich was e. In p elimina y analyses, we checked ha each o he s ains was able o g ow on chicken ea he s as hei sole nu ien sou ce (ei he using ap wa e o dis illed wa e ), bu none o he cul u es (including he co-cul u e) p oduced he cha ac e ized eddish colo o he S. g iseoau an iacus AD2 cul u es in R5A (Supplemen a y Figu e S1. This esul indica es ha bo h ap wa e and dis illed wa e can be employed o g owing he s ains (howe e , o a bio echnological applica ion ap wa e would p o ide an economic ad an age). In ag eemen wi h he ob ained pheno ypes (no ed an ibio ic is p oduced), we obse ed ha he an ibio ic ac i i y o bo h he co-cul u e and he S. g iseoau an iacus AD2 cul u e was dec eased in compa ison wi h he R5A cul u es (see Figu es 3B and S1). Supplemen a y Ma e ials Figu e S1 also shows ha he speed o g ow h o he co-cul u e was signi ican ly highe han ha o he mos e icien ke a in-deg ading s ain (i.e., S. g iseoau an iacus AD2), Mic oo ganisms 2023,11, 1109 8 o 10 which indica es ha , no only an imic obial ac i i y is inc eased in he co-cul u e, bu also he e iciency in he chicken ea he s deg ada ion. As a inal s age, we decided o measu e he ke a inoly ic ac i i y o he co-cul u e du ing a ime-cou se expe imen . Figu e 5shows he g ow h and ke a inoly ic ac i i y o he co-cul u e du ing he ime-cou se expe imen . Al hough he s ains g ew slowe and p oduced lowe ke a inoly ic ac i i y alues han when cul u ed in CFSM medium (compa e Figu e 5wi h Figu es 1and 2), hei abili y o g ow on chicken ea he s as hei sole nu ien sou ce is e y p omising o u u e mic obial ecycling s a egies. Mic oo ganisms2023,11,xFORPEERREVIEW9o 11   Asa inals age,wedecided omeasu e heke a inoly icac i i yo  heco-cul u e du inga ime-cou seexpe imen .Figu e5shows heg ow handke a inoly icac i i yo  heco-cul u edu ing he ime-cou seexpe imen .Al hough hes ainsg ewslowe and p oducedlowe ke a inoly icac i i y alues hanwhencul u edinCFSMmedium(com- pa eFigu e5wi hFigu es1and2), hei abili y og owonchicken ea he sas hei sole nu ien sou ceis e yp omising o  u u emic obial ecyclings a egies.  Figu e5.G ow handke a inoly icac i i yo S ep omycesalbido la usAN1,S ep omycesd ozdowi- cziiAD1andS ep omycesg iseoau an iacusAD2co-cul u eonchicken ea he sas hesolesubs a e. Samples o g ow h(CO 2 p oduc ionandO 2 consump ion)andke a inoly icac i i ywe ecollec ed e e y24hun ilO 2 wasdeple ed om hebo les.Blue iangles(ke a inoly icac i i y),G eysqua es (CO 2 p oduc ion)andBlackci cles(O 2 consump ion).Ve icale o ba sco espond o hes anda d e o o  hemeano  wo eplica edexpe imen s. 4.Discussion In hiss udy,wecha ac e ized h eeS ep omycesspecieswi hhighke a inoly icac- i i y.Oneo  heses ains(S.g iseoau an iacusAD2)showedamaximalke a inoly icac- i i yo 4000U×mL −1 ,whe eas heac i i ydisplayedby heo he  wo(S.albido la us AN1andS.d ozdowicziiAD1)wasali lelowe (~3000U×mL −1 ).Ve y ews ainswi h ke a inoly icac i i yhighe  han4000U×mL −1 we e epo edin heli e a u e[5], he e- o eS.g iseoau an iacusAD2couldbeconside edoneo  hemos efficien ke a in-deg ad- ingmic oo ganismsdisco e ed oda e.A  hesame ime,S ep omycesspeciesa e hemos  impo an an ibio icp oduce sdisco e ed oda e[15–17].S.g iseoau an iacusAD2isalso able oinhibi  heg ow ho G am-posi i ebac e ia,whichiden i ies hisspeciesasa e y p omisingcandida e o  he alo iza iono ke a in- ichwas es. Mos impo an ly,weha eshown ha aco-cul u eo  he h eeS ep omycess ains led og ea e ke a inoly icandan ibio icac i i ies hanmono-cul u eso eachindi idual s ain.All hes ainswe eable og owindi iduallyonchicken ea he sas hesolenu i- en andene gysou ce,bu  heco-cul u eo  heses ainsp oducedhighe an imic obial ac i i yand as e g ow hon hissubs a e.Many epo sin heli e a u edesc ibe he in e es ingeffec so in e -o in a-speciesco-cul i a ionondiffe en me abolicp ope ies bu  hecausesbehind heseimpac s emainun o una elyunknown.Ne e heless, his app oachcouldp o euse ul o alo izeke a in- ichwas es(suchaschicken ea he s)as acheapway op oducean ibio ics.Theabili y og owonamediummadejus wi hwa e  andchicken ea he s(wi hnoo he  ypeso  equi emen s)wouldmake heg ow ho  hesebac e ialspecies e ycheap o indus ialp oduc ionpla o ms.Ano he possible u iliza iono  hesespeciesisdi ec lyasbio e ilize sand/o biopes icidesinsus ainable ag icul u e.Indeed,mos S ep omycesspeciesa esoil-bo nebac e iaandli einsymbiosis wi hplan sando he mic oo ganisms[6].Theya eknown oha eaplan g ow h-p o- mo ingeffec [18]and ocon ibu e odisease-supp essi esoils[6].Al houghmuchiss ill Figu e 5. G ow h and ke a inoly ic ac i i y o S ep omyces albido la us AN1, S ep omyces d ozdowiczii AD1 and S ep omyces g iseoau an iacus AD2 co-cul u e on chicken ea he s as he sole subs a e. Samples o g ow h (CO 2 p oduc ion and O 2 consump ion) and ke a inoly ic ac i i y we e collec ed e e y 24 h un il O 2 was deple ed om he bo les. Blue iangles (ke a inoly ic ac i i y), G ey squa es (CO 2 p oduc ion) and Black ci cles (O 2 consump ion). Ve ical e o ba s co espond o he s anda d e o o he mean o wo eplica ed expe imen s. 4. Discussion In his s udy, we cha ac e ized h ee S ep omyces species wi h high ke a inoly ic ac i i y. One o hese s ains (S. g iseoau an iacus AD2) showed a maximal ke a inoly ic ac i i y o 4000 U × mL −1 , whe eas he ac i i y displayed by he o he wo (S. albido la us AN1 and S. d ozdowiczii AD1) was a li le lowe (~3000 U × mL −1 ). Ve y ew s ains wi h ke a inoly ic ac i i y highe han 4000 U × mL −1 we e epo ed in he li e a u e [ 5 ], he e o e S. g iseoau an iacus AD2 could be conside ed one o he mos e icien ke a in- deg ading mic oo ganisms disco e ed o da e. A he same ime, S ep omyces species a e he mos impo an an ibio ic p oduce s disco e ed o da e [ 15 – 17 ]. S. g iseoau an iacus AD2 is also able o inhibi he g ow h o G am-posi i e bac e ia, which iden i ies his species as a e y p omising candida e o he alo iza ion o ke a in- ich was es. Mos impo an ly, we ha e shown ha a co-cul u e o he h ee S ep omyces s ains led o g ea e ke a inoly ic and an ibio ic ac i i ies han mono-cul u es o each indi idual s ain. All he s ains we e able o g ow indi idually on chicken ea he s as he sole nu ien and ene gy sou ce, bu he co-cul u e o hese s ains p oduced highe an imic obial ac i i y and as e g ow h on his subs a e. Many epo s in he li e a u e desc ibe he in e es ing e ec s o in e - o in a-species co-cul i a ion on di e en me abolic p ope ies bu he causes behind hese impac s emain un o una ely unknown. Ne e heless, his app oach could p o e use ul o alo ize ke a in- ich was es (such as chicken ea he s) as a cheap way o p oduce an ibio ics. The abili y o g ow on a medium made jus wi h wa e and chicken ea he s (wi h no o he ypes o equi emen s) would make he g ow h o hese bac e ial species e y cheap o indus ial p oduc ion pla o ms. Ano he possible u iliza ion o hese species is di ec ly as bio e ilize s and/o biopes icides in sus ainable ag icul u e. Indeed, mos S ep omyces species a e soil-bo ne bac e ia and li e in symbiosis wi h plan s and o he mic oo ganisms [ 6 ]. They a e known o ha e a plan g ow h-p omo ing e ec [ 18 ] Mic oo ganisms 2023,11, 1109 9 o 10 and o con ibu e o disease-supp essi e soils [ 6 ]. Al hough much is s ill o be disco e ed conce ning he coloniza ion s a egies and molecula in e ac ions be ween plan oo s and hese bac e ia, hey a e des ined o become impo an playe s in mode n sus ainable ag icul u e [19,20]. In conclusion, his wo k cons i u es a i s s ep in he de elopmen o mic obial s a egies use ul o he de elopmen o a ious bio echnological applica ions ela ed o he u iliza ion o ke a in was es as bio e ilize s o as a cheap subs a e o p oduce an ibio ics. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps: //www.mdpi.com/a icle/10.3390/mic oo ganisms11051109/s1, Figu e S1: Compa ison o g ow h and an imic obial ac i i y be ween S ep omyces g iseoau an iacus AD2 and he co-cul u e on chicken ea he s as sole nu ien sou ce. Au ho Con ibu ions: F.S.-B. concep ualized he s udy, analyzed and in e p e ed he esul s, pe - o med he an imic obial bioassays and he UPLC and LC-MS analyses, w o e and edi ed he manusc ip . D.M.-G. and S.B. pe o med he ke a inoly ic expe imen s and co-cul u ing expe i- men s. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: We hank he FEDER p og am (TCUE 2021–2023) o allowing Fe nando San os-Benei o ob ain he p ojec wi h e e ence numbe : 067/229111. We also hank he Eu opean Union o suppo ing he wo k wi h an H2020-MSCA-IF-2016 Fellowship (UE-18-VANRESTREP-740080). Da a A ailabili y S a emen : The au ho s decla e ha all da a ob ained ha e been included in he manusc ip , i s addi ional iles and/o eposi o ies. Acknowledgmen s: We hank he g oup o Jose An onio Salas (Uni e si y o O iedo) o helping wi h he UPLC and LC-MS analyses. We also hank he Medina Founda ion (Spain) o pe o ming he de eplica ion analyses. Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Zoua i-Fakh akh, N.; Hadda , A.; Hmide , N.; F ikha, F.; Nas i, M. 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