Bacterial enzymes involved in lignin degradation

Jou nal o Bio echnology 236 (2016) 110–119 Con en s lis s a ailable a ScienceDi ec Jou nal o Bio echnology j ou na l ho me pa ge: www.else ie .com/loca e/jbio ec Re iew Bac e ial enzymes in ol ed in lignin deg ada ion Gonzalo de Gonzaloa, Dana I. Colpab, Mohamed H.M. Habibb,c, Ma co W. F aaijeb,∗ aDepa amen o de Química O gánica, Uni e sidad de Se illa, c/P o eso Ga cía González 1, 41012 Se illa, Spain bMolecula Enzymology, G oningen Biomolecula Sciences and Bio echnology Ins i u e, Uni e si y o G oningen, Nijenbo gh 4, 9747 AG G oningen, The Ne he lands cDepa men o Mic obiology and Immunology, Facul y o Pha macy, Cai o Uni e si y, 11562 Kas El-Aini S ee , Cai o, Egyp a i c l e i n o A icle his o y: Recei ed 21 July 2016 Accep ed 16 Augus 2016 A ailable online 17 Augus 2016 Keywo ds: Lignin deg ada ion DyP Pe oxidases Laccases Dioxygenases a b s a c Lignin o ms a la ge pa o plan biomass. I is a highly he e ogeneous polyme o 4- hyd oxyphenylp opanoid uni s and is embedded wi hin polysaccha ide polyme s o ming lignocellulose. Lignin p o ides s eng h and igidi y o plan s and is a he esilien owa ds deg ada ion. To imp o e he (bio)p ocessing o lignocellulosic eeds ocks, mo e e ec i e deg ada ion me hods o lignin a e in demand. Na u e has ound ways o ully deg ade lignin h ough he p oduc ion o dedica ed ligninoly ic enzyme sys ems. While such enzymes ha e been well ho oughly s udied o ligninoly ic ungi, only in ecen yea s biochemical s udies on bac e ial enzymes capable o lignin modifica ion ha e in ensified. This has e ealed se e al ypes o enzymes a ailable o bac e ia ha enable hem o ac on lignin. Two majo classes o bac e ial lignin-modi ying enzymes a e DyP- ype pe oxidases and laccases. Ye , ecen ly also se e al o he bac e ial enzymes ha e been disco e ed ha seem o play a ole in lignin modifica- ions. In he p esen e iew, we p o ide an o e iew o ecen ad ances in he iden ifica ion and use o bac e ial enzymes ac ing on lignin o lignin-de i ed p oduc s. © 2016 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). Con en s 1. In oduc ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110 2. Bac e ial enzymes ac ing on lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 2.1. DyP- ype pe oxidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 2.2. Lignin-modi ying bac e ial laccases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 2.3. Glu a hione-dependen ␤-e he ases ac ing on lignin deg ada ion p oduc s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2.4. The ole o supe oxide dismu ases in bac e ial lignin modifica ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 2.5. Ca alase-pe oxidases a e associa ed wi h lignocellulose deg ada ion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .116 2.6. Bac e ial dioxygenases may play a ole in lignin deg ada ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 3. Ou look . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Acknowledgmen s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Re e ences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 1. In oduc ion Plan biomass is he mos abundan enewable biomass on ea h and is conside ed as an a ac i e sou ce o bioene gy and biobased chemicals. I is mainly composed o lignin, cellulose and hemicel- ∗Co esponding au ho . E-mail add esses: [email p o ec ed] (G. de Gonzalo), [email p o ec ed] (D.I. Colpa), [email p o ec ed] (M.H.M. Habib), [email p o ec ed] (M.W. F aaije). lulose. The lignin pe cen age in lignocellulosic biomass is a ound 10–30% and is he second mos abundan na u al o ganic poly- me . Lignin enables plan s o gene a e igid s uc u es and p o ides p o ec ion agains hyd olysis o cellulose and hemicellulose. The bio echnological con e sion o lignocellulose in o di e en ca - bohyd a es, including glucose, is he basis o he p oduc ion o e hanol, ca bohyd a es and a oma ic p oduc s (Asghe e al., 2014; Ragauskas e al., 2014; Kawaguchi e al., 2016). Such plan biomass de i ed p oduc s can be used as uel, polyme p ecu so s, ood and fla o compounds, and pha maceu ical building blocks. Fo op imizing he use o plan biomass h ough bio efining, lignin h p://dx.doi.o g/10.1016/j.jbio ec.2016.08.011 0168-1656/© 2016 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/). G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 111 deg ada ion has become a key a ge in he las ew yea s. E ficien and cos -e ec i e me hods o selec i e lignin deg ada ion a e in high demand. I is wo h no ing ha , while he ecen in ensified e o s in comple e alo iza ion o plan biomass, lignin was al eady conside ed as a majo indus ial by-p oduc in he fi s hal o he p e ious cen u y (Go lieb and Pelcza , 1951). While cellulose and hemicellulose a e buil om ca bohyd a es, lignin is a highly c oss-linked polyme o med by polyme iza ion o 4-hyd oxyphenylp opanoid monome s (monolignols) h ough a ious e he and ca bon–ca bon bonds. The phenolic moie ies o he monome ic uni s a e p-hyd oxyphenyl (H), guaiacyl (G) and sy ingyl (S) g oups and he pe cen age o each depends on he plan species and issue. The o ma ion o lignin is igge ed by plan pe - oxidases and/o laccases. By oxidizing he phenolic monolignols in o hei espec i e phenolic adical, o ma ion o dime s is ca - alyzed. Subsequen enzyme-ca alyzed single elec on oxida ions p omo e polyme iza ion. Monolignols can couple ia a ious bonds wi h a p e e ence o coupling h ough he ␤-ca bon. The mos occu ing linkages in ol e ␤ ␤, ␤ O 4, and ␤ 5 bonds (Vanholme e al., 2010), as shown in Fig. 1. Due o i s a oma ic na u e and highly b anched polyme ne - wo k, lignin is a he ine owa ds deg ada ion (Abdel-Hamid e al., 2013). Ye , o comple e global ca bon cycling, na u e has e ol ed ca abolic pa hways since he ime ha plan s s a ed o p oduce lignin (Nelsen e al., 2016). Whi e- o ungi ha e de eloped a ich collec ion o ex acellula oxida i e enzymes o a ack and deg ade lignin. They employ di e en ypes o heme-con aining pe oxidases, which include he so-called lignin pe oxidases (LiP), manganese pe oxidases (MnP), e sa ile pe oxidases (VP), and dye- decolo izing pe oxidases (DyP) (Lambe z e al., 2016). While some o hese pe oxidase a e capable o a acking lignin o lignin ag- men s, pe oxidases also a ack lignin om a dis ance. By oxidizing media o s, small oxidizing agen s a e gene a ed ha can pene a e he b anched lignin polyme o igge depolyme iza ion ia adi- cal chemis y (Nousiainen e al., 2014; Baciocchi e al., 2002; Glenn and Gold, 1999). Known media o s a e lignin de i ed a oma ic compounds (e.g. o ma ion o e a yl alcohol ca ion adical) and manganese ions (Hun e al., 2013). Fo e ec i e pe oxidase-based lignin deg ada ion, also a ious ungal oxidases a e sec e ed o p oduce he equi ed hyd ogen pe oxide. Candida es o he ex a- cellula p oduc ion o hyd ogen pe oxide a e a yl alcohol oxidases, glyoxal oxidases, and a ious ca bohyd a e oxidases. Excep o pe oxidases, ungi also sec e e a ious coppe -con aining oxida- i e laccases ha assis in lignin deg ada ion. In iguingly, i seems ha he same ypes o enzymes used o lignin syn hesis in plan s (pe oxidases and laccases) a e used by ungi o ecycle he a o- ma ic polyme . Genome sequence analysis o ligninoly ic ungi has e ealed ha he e is no one defined se o enzymes o lignin deg ada ion (Floudas e al., 2012). The composi ion o he se o oxida i e enzymes being p oduced depends on he ungus. While a weal h o biochemical knowledge has been ob ained on ungal deg ada ion o lignin, he ligninoly ic capaci y o bac e ia has been less well s udied. While i appea s ha whi e- o ungi a e e y well equipped o lignin deg ada ion, e idence is g owing ha also bac e ia a e capable o delignifica ion. Al eady in 1930 Phillips e al. epo ed on a ho ough s udy on lignin decomposi ion by “soil mic oo ganisms”, which p esumable we e bac e ia (Phillips e al., 1930). While many claims o bac e ial lignin deg ada ion ha e been epo ed since hen, only in he las ew decades some bac e ial enzymes in ol ed in delignifica ion ha e been iden ified. Wi h his e iew we aim a p o iding an o e iew o he bac e ial enzymes ha ha e been implica ed o be in ol ed in deg ading lignin o he oxida ion o lignin de i ed deg ada ion p oduc s. 2. Bac e ial enzymes ac ing on lignin 2.1. DyP- ype pe oxidases As desc ibed abo e, whi e- o ungi p oduce se e al di e en kinds o heme-con aining pe oxidases o igge lignin decomposi- ion. Howe e , homologs o he mos common ungal ligninoly ic pe oxidases, LiPs MnPs and VPs, ha e no been encoun e ed in biochemical s udies on ligninoly ic bac e ia. Also when analysing sequenced genomes (Da is e al., 2013) o p o eomes (B own e al., 2011) o ligninoly ic bac e ia, no homologs eme ge. I seems ha hese lignin-deg ading pe oxidases, belonging o he supe amily o plan pe oxidase (Class II) (Welinde , 1992), a e es ic ed o ungi. Ye , ecen ly i has become clea ha bac e ia a e ela i ely ich in ano he ype o pe oxidase, he so-called dye-decolo izing pe oxidases (DyPs, EC 1.11.1.19) (Van Bloois e al., 2010). DyPs ep- esen a newly disco e ed amily o heme-con aining pe oxidases, which has ecen ly ecei ed a en ion due hei abili y o deg ade lignin and o he compounds (Sugano, 2009; Colpa e al., 2014; Singh and El is, 2015; Yoshida and Sugano, 2015). The fi s disco e ed membe o his enzyme amily, DyP om Bje kande a adus a, was isola ed and cha ac e ized in 1999 (Kim and Shoda, 1999). S ud- ies on he ac i i y o his enzyme on syn he ic an h aquinone and azo-dyes ha e se ed o name his amily o pe oxidases (Sugano e al., 2007). In ecen yea s a la ge numbe o bac e ial DyPs ha e been desc ibed in li e a u e (Lambe z e al., 2016) which is in line wi h he obse a ion ha pu a i e DyP-encoding genes a e abundan ly p esen in bac e ial genomes (Table 1) (Van Bloois e al., 2010). In ac , al eady in 1988 a bac e ial ‘lignin pe oxidase’ was desc ibed om S ep omyces i idospo us. Un o una ely, no sequence has e e been deposi ed o his p o ein o he espec i e gene while se e al pape s ha e appea ed on cloning o he espec- i e gene (Ramachand a e al., 1998; Wang e al., 1990; Thomas and C aw o d, 1998). Ye , when analysing he ecen ly sequenced genome o his S ep omyces isola e, a gene encoding a pu a i e Ta - sec e ed DyP can be iden ified (Da is e al., 2013). This may well be he enzyme ha was desc ibed long be o e he fi s ungal DyP was desc ibed. DyPs ha e a p o ome weigh o a ound 40–60 kDa and a - ious oligome ic s a es ha e been obse ed (Colpa e al., 2014). They belong o he pe oxidase-chlo i e dismu ase supe amily o p o eins and con ain a non-co alen ly bound heme b co ac- o (Zámock´ y e al., 2015). DyPs show a dime ic e edoxin-like old consis ing o a ou -s anded an i-pa allel ␤-shee su ounded by ␣-helices. DyP- ype pe oxidases con ain a highly conse ed GXXDG-mo i and a conse ed p oximal his idine, which ac s as he fi h ligand o he heme i on. Ye , while DyPs a e s uc u ally un ela ed o he common ungal pe oxidases, hey exhibi simi- la ca aly ic p ope ies wi h ha ing simila edox po en ials and eac i i ies (Lie s e al., 2014). Fu he mo e, some o he bac e ial DyPs a e sec e ed ia he Ta sec e ion machine y which adds o he analogy wi h he sec e ed ungal pe oxidases. Based on sequence cha ac e is ics, DyPs ha e been di ided in ou classes in he Pe oxiBase da abase (Fawal e al., 2013). P o eins belonging o classes A–C a e mainly ound in bac e ia, while class D DyPs a e ex acellula ungal ep esen a i es (Yoshida and Sugano, 2015). Class A DyPs ypically ha e a Ta -signal sequence and a e he e o e sec e ed. In con as , he DyP p o ein sequences o class B and C DyPs do no disclose any sec e ion signal pep ides, sugges ing ha hey a e in acellula enzymes. The In e P o da abase cu - en ly con ains 8318 DyP sequences. App oxima ely hi y o hese enzymes ha e been isola ed and cha ac e ized (Colpa e al., 2014; Yoshida and Sugano, 2015). DyPs a e mainly ac i e a acidic pH and show a e y b oad subs a e p ofile, including se e al classes o syn he ic dyes, monophenolic compounds, e a yl alcohol, ␤-ca o enes, Mn+2 and lignin model compounds, bu hei physio- 112 G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 Fig. 1. Example o a lignin s uc u e con aining he mos equen bonds as well as he co esponding monome s ha ake pa o i s s uc u e: 4-hyd oxyphenyl (H), guaiacyl (G) and sy ingyl (S). Table 1 Occu ence o DyPs in bac e ial genomes. By pe o ming a BLASTP analysis o he p edic ed p o eomes, homologs o known DyPs we e iden ified. O ganism DyP ype A B C Esche ichia coli K-12 1 1 The mobifida usca YX 1 Rhodococcus jos ii RHA1 1 1 S ep omyces i idospo us s ain T7A 1 S ep omyces coelicolo A3(2) 2 1 Amycola opsis sp. 75i 2 1 2 Pseudomonas sp. s ain YS-1p 2 logical subs a es s ill emain unknown. DyP-pe oxidases can also ca alyse in e es ing syn he ic eac ions such as enan ioselec i e sul oxida ions (Van Bloois e al., 2010), heme de e ochela a ions (Lé o é e al., 2009) and e en ca bonyl olefina ion p ocesses in he absence o hyd ogen pe oxide (Weissenbo n e al., 2016). A ungal DyP has ecen ly been ound o enhance lignocellulose deg ada ion (Linde e al., 2015). In he las yea s, se e al bac e ial DyP- ype pe oxidases ha e been implica ed in he deg ada ion o lignin and lignin model compounds. DyP-media ed oxida ion o e a yl alcohol and he lignin model dime s guaiacylglyce ol-␤-guaiacol e he and e a ylglyce ol-␤-guaiacol e he has been epo ed. The bac e- ial DyPs in es iga ed o da e appea o ha e a lowe oxidizing powe han he ungal coun e pa s, and seem o be limi ed o he oxida ion o less ecalci an phenolic lignin models. DyP- ype pe oxidases a e gene ally ac i e on monophenolic subs a es, bu se e al bac e ial DyPs ha e shown significan ac i i y owa ds he nonphenolic e a yl alcohol: BsDyP om Bacillus sub ilis KCTC2023 (Min e al., 2015), PpDyP om Pseudomonas pu ida MET94 (San os e al., 2014), S iDyP om Saccha omonospo a i idis DSM 43017 (Yu e al., 2014) and T uDyP om The mobifida usca (Van Bloois e al., 2010). Bo h S iDyP and T uDyP a e class A DyPs and a e sec e ed ia he Ta -sys em. This would be in line wi h ex acellula deg ada ion on lignin. The A- ype TcDyP om The momonospo a cu a a, al hough showing a elaxed subs a e specifici y, was inac i e owa ds e a yl alcohol. None heless, i was able o deca boxyla e he nonphenolic lignin- ela ed subs a e 4-me hoxymandelic acid, yielding p-anisaldehyde as final p oduc (Chen e al., 2015). In e es ingly, he C- ype DyP2 om Amycola op- sis sp. 75i 2 was also able o deca boxyla e 4-me hoxymandelic acid in he p esence o Mn2+ and O2, wi h no need o H2O2(B own e al., 2012). This hin s o he abili y o DyPs o ac as oxidases. Se e al bac e ial DyPs a e able o oxidize he phenolic lignin dime guaiacylglyce ol-␤-guaiacol e he . Fo ins ance, T uDyP has been es ed o he oxida ion o his lignin-model compound. I was ound ha T uDyP does no clea e he e he bond in he model compound bu oxidizes he phenolic moie y esul ing in oxida i e coupling o he guaiacylglyce ol-␤-guaiacyl e he , mainly yielding in dime ic and ime ic p oduc s (Lonˇ ca e al., 2016; Rahmanpou e al., 2016). This is in line wi h he obse a ion ha T uDyP e fi- cien ly dime izes se e al monophenolic compounds (e.g anillin, anillin alcohol and anillin ke one) (Fig. 2a). This beha iou is di - e en om TcDyP and DyPB, a B- ype DyP om Rhodococcus jos ii RHA1. The use o he la e wo enzymes esul ed in a mo e di e se p oduc p ofile, which could be explained by he deg ada ion o he C␣-C␤ linkages o he model subs a e and subsequen adical cou- pling o he p oduc s o med (Chen e al., 2015; Ahmad e al., 2011). The main oxida ion p oduc s o DyPB- ea ed guaiacylglyce ol-␤- guaiacol e he we e guaiacol, guaiacol ime s and anillin (Ahmad e al., 2011) (Fig. 2b). Some o he compounds eco e ed a e ea - men o he lignin model subs a e wi h TcDyP could be iden ified as hyd oxyla ed guaiacol pen ame s and c esol dime s, as shown in Fig. 2c (Chen e al., 2015). DyP2 has also shown ac i i y on his phenolic lignin dime , bu he p oduc s o med ha e no been cha - ac e ized, so i s deg ada ion pa hway emains unclea (B own e al., 2012). Ve a ylglyce ol-␤-guaiacol e he has been also used as lignin model o in es iga ing he oxida i e po en ial o DyPs. This compound does no con ain a phenolic moie y and is mo e ecalci- an o oxida ion by DyP- ype pe oxidases. None o he enzymes men ioned abo e we e able o oxidize his lignin model dime . Rema kably, BsDyP, which was inac i e owa ds he phenolic lignin dime men ioned abo e, showed ac i i y owa ds bo h e a yl alcohol and he e a ylglyce ol-␤-guaiacol e he (Min e al., 2015), as indica ed in Fig. 2d. The decomposi ion o he lignin dime was measu ed based on he elease o he p oduc e a aldehyde, he same p oduc o med when using LiP (Ki k e al., 1986). The deg a- da ion o he dime occu ed h ough he b eakage o he C␣-C␤ bond. Thus, BsDyP is he fi s bac e ial DyP showing ac i i y owa ds his compound. This ac i i y was also desc ibed o a ew ungal DyP (Lie s e al., 2013). Se e al bac e ial DyPs, including DyPB, T uDyP and he wo DyPs ob ained om Pseudomonas fluo escence P -5: DyP1B and G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 113 Fig. 2. Some o he deg ada ion eac ions ca alyzed by DyP- ype pe oxidases: (a) T uDyP-ca alyzed dime iza ion o anillin; (b) oxida ion o guaiacylglyce ol-␤-guaiacol e he by DyPB leading o guaiacol, guaiacol ime s and anillin; (c) TcDyP-ca alyzed deg ada ion o guaiacylglyce ol-␤-guaiacol o hyd oxyla ed guaiacol pen ame s and c esol dime s, and (d) BsDyP-ca alyzed deg ada ion o e a ylglyce ol-␤-guaiacol e he . DyPA, ha e been shown o ac on alkali K a lignin, a by-p oduc o he pape indus y (Rahmanpou and Bugg, 2015). S iDyP has shown in e es ing esul s in biobleaching p ocesses, which makes i a p omising candida e o u he indus ial applica ions. This he mos able bac e ial pe oxidase (60% emaining ac i i y a e incuba ing a 70 ◦C o 2 h) wi h a high alkali ole ance (>80% ac i - i y a e incuba ion a pH 5–10 a 37 ◦C o 1 h) has been employed success ully as bioca alys in he biobleaching o eucalyp us K a pulp (Yu e al., 2014). Mos DyP subs a es a e oo big o en e he ac i e si e and a e he e o e unable o in e ac di ec ly wi h he heme co ac o . S uc- u al analysis o DyPs (DyP2, he N246A mu an o DyPB, and a ungal DyP) ha e e ealed he p esence o su ace exposed sub- s a e binding si es (B own e al., 2012; Yoshida e al., 2012; Singh e al., 2013). Besides hese si es, a long- ange elec on ans e (LRET) pa hway be ween he heme co ac o and a su ace exposed y osine o yp ophan has been sugges ed, as p e iously desc ibed o LiPs and VPs (Doyle e al., 1998; Pé ez-Boada e al., 2005). In ac , simila o he ypical ungal LiPs, MnPs and VPs, DyPs also seem o be able o p omo e lignin deg ada ion by oxidizing edox media o s. Redox media o s as e a yl alcohol, monophenolic subs a es and Mn+2 ha e been es ed as DyP subs a es. Some DyP- ype pe oxi- dases we e shown o be ac i e on e a yl alcohol, monophenolic subs a es and Mn2+. Many DyPs a e ac i e on monophenolic sub- s a es. I is also wo h no ing ha AnaPX (a C- ype DyP) om Anabaena sp. s ain PCC 7120 showed a significan ly enhanced ac i i y owa ds se e al azo-dyes in he p esence o he na u al media o sy ingaldehyde (Ogola e al., 2009). The ac i i y owa ds Mn2+ and/o he use o Mn2+ as media o in DyP-ca alysed deg a- da ion o lignin has been widely s udied o se e al bac e ial DyPs. DyP2, DyP1B, DyPB, BsDyP and PpDyP om Pseudomonas pu ida MET94 (San os e al., 2014) ha e been es ed o ac i i y wi h Mn2+. DyP2 om Amycola opsis sp. 75i 2 showed he highes ac i i y on his ca ion, wi h a kca o 24 ± 1 s−1and a kca /KM alue only one o wo o de s o magni ude lowe han he ac i i ies om espec- i ely VP (Pleu o us e yngii) and LiP (Phane ochae e ch ysospo ium) (B own e al., 2012). F om he h ee DyP pe oxidases ob ained om Pseudomonas flu- o escence P -5 and o e exp essed in Esche ichia coli, only DyP1B showed ac i i y o he oxida ion o Mn2+ and o he deg ada ion o powde ed whea s aw lignocellulose. Using Mn2+, o ma ion o a lignin dime om his lignin ma e ial could be boos ed (Rahmanpou and Bugg, 2015). A mo e ex ensi e s udy on he po en ial lignin deg ada ion capaci y by a bac e ial DyP in he p esence o Mn2+ was pe o med using DyPB om Rhodococcus jos ii RHA1 (Ahmad e al., 2011). DyPB clea es he C␣-C␤ link- age o he phenolic lignin dime guaiacylglyce ol-␤-guaiacol e he (Fig. 2b) and is also able o ac on K a lignin. These ac i i ies we e enhanced by 23 and 6.2 imes, espec i ely, h ough he addi ion o 1.0–1.5 mM MnCl2. DyPB also showed ac i i y owa ds whea s aw lignocellulose and whea s aw milled wood lignin when incuba ed in he p esence o 1.0 mM MnCl2and in absence o 114 G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 H2O2. The ob ained p oduc s ha e no been cha ac e ized, bu HPLC analysis has e ealed a ious b eakdown p oduc s. Lignin deg a- da ion did no occu in he absence o Mn+2. Using pu ified DyPB i could be confi med ha i ca alyses he pe oxide-dependen oxida ion o Mn2+, albei less e ficien ly han ungal manganese pe oxidases. An enginee ed a ian o DyPB, con aining he N246A mu a ion, showed an 80- old inc eased ac i i y owa ds Mn2+ (kca = 39 ± 3 s−1) (Singh e al., 2013). This mu an has been es ed in he ans o ma ion o ha d wood K a lignin and on i s sol en ex ac ed ac ions. This esul ed in eco e y o sy ingaldehyde and 2,6-dime hoxybenzoquinone as majo p oduc s. These esul s highligh he po en ial o bac e ial enzymes as bioca alys s o ans- o m lignin. In con as o A- ype DyPs, B- and C- ype DyPs ypically lack a sec e ion signal. This may no exclude a ole as ex acellu- la enzyme. The ex acellula ac ion o he dypB mu an o Rhodococcus jos ii RHA1 showed a highly educed ac i i y owa ds ni a ed lignin, sugges ing ha he loca ion o DyPB is ex acellu- la . Thus, i has been p oposed ha his enzyme migh be expo ed h ough ano he mechanism, po en ially h ough encapsula ion and subsequen sec e ion o DyPB. App oxima ely 14% o he genes o B- ype DyPs a e loca ed in an ope on oge he wi h an encap- sulin gene. Su e e al. ha e shown ha hese DyPs o en con ain a 30–40 amino acid C- e minal ex ension. Enzymes con aining his C- e minal ex ension, o ins ance DyPB, BlDyP and M DyP, a e a ge s o encapsula ion by a p o ein-based cages, he so-called encap- sulins (Su e e al., 2008; Con e as e al., 2014). In e es ingly, DyPB, when being encapsula ed, showed an eigh - old enhanced ac i i y owa ds ni a ed lignin (Rahmanpou and Bugg, 2013), when compa ed wi h DyPB alone. This indica es ha in some way encapsula ion which enhances DyP-media ed lignin deg ada ion. 2.2. Lignin-modi ying bac e ial laccases Laccases (EC 1.10.3.2) a e mul i-coppe oxidases able o pe - o m he single elec on oxida ions o o ganic compounds o he co esponding adical species. Laccases employ a clus e o ou coppe ions o such oxida ions which use dioxygen as elec on accep o , gene a ing wa e as byp oduc . The o med adical p od- uc s can unde go u he oxida ion o unde go o he eac ions such as hyd a ion, disp opo iona ion o polyme iza ion eac ions. Laccases a e ubiqui ous in na u e, being ound in plan s, ungi, bac- e ia and insec s. They a e o en sec e ed as ex acellula ca alys s and ypically pe o m polyme iza ion o depolyme iza ion eac- ions (Ri a, 2006). Laccases a y la gely in hei molecula weigh , oligome ic s a e and s uc u e (San hanam e al., 2011). While mos laccases consis o h ee s uc u al domains, also laccases ha lack one o hese h ee domains exis . Simila o ungal laccases, many bac e ial laccases a e sec e ed. Fo he expo o bac e ial laccases he Ta sec e ion sys em is used, which acili a es he expo o olded and holop o eins ac oss he cell memb ane. Laccases a e indus ially a ac i e bioca alys s, as unlike many oxido educ ases, hey do no equi e he addi ion o co ac o s. Addi ionally, di e en om mos oxidases, hey do no p o- duce oxic hyd ogen pe oxide as byp oduc . Laccases ha e been employed in bio emedia ion p ocesses o decon amina e indus ial was ewa e s, in ood indus y o he s abiliza ion o be e ages and imp o emen o he o ganolep ic p ope ies o ood, in he syn- hesis o pha maceu icals and o he fine chemicals, in ex ile dye ans o ma ion, and in he delignifica ion o wood, especially du - ing he bleaching p ocess (Sh addha e al., 2011). Laccases can be also employed in he p e ea men o so wood samples wi h he aim o imp o ing he subsequen hyd olysis ea men (Palonen and Viika i, 2004). Mos o he laccases known, s udied and applied o da e a e om ungal o igin. F om hese s udies i has become clea ha laccases play a ole in lignin deg ada ion. Only in ecen yea s bac e ial lac- cases ha e gained conside able in e es conce ning hei possible ole in lignin deg ada ion and o he bio echnological applica ions. The fi s laccase was desc ibed in 1995 (Fau e e al., 1995). Recen ad ances in genome analysis and o he app oaches ha e allowed he iden ifica ion o nume ous laccases in bac e ia (Alexand e and Zhulin, 2000; San hanam e al., 2011; Ma ins e al., 2015). The ole and e ficacy o bac e ial laccases in lignin deg ada ion is nowadays hea ily s udied (Chand a and Chowdha y, 2015). In o de o pe o m he deg ada ion and depolyme iza ion o lignin, laccases equi e he p esence o small molecules, he so- called media o s, which ac as edox shu les be ween he laccase ac i e si e and he lignin s uc u e (Fig. 3). Se e al compounds ha e been iden ified as e ec i e media o s. The fi s one was ABTS (2,2- azino-bis(3-e hylbenoz hiazoline-6-sul onic acid) which is used in pulp deg ada ion (Bou bonnais and Paice, 1990). The mos e ec- i e media o in lignin ans o ma ion a e N-he e ocycles bea ing NOH g oups, o ins ance, N-hyd oxybenzo iazole (HBT). Simila o DyPs, he mos s udied bac e ial laccases in lignin deg ada ion a e om ac inomyce es, pa icula ly om S ep o- myces species (Fe nandes e al., 2014). Mos o he laccases p esen in his genus belong o he g oup o wo-domain laccases. The abil- i y o hese so-called small laccases o assis on lignin modifica ion was confi med by s udying knock-ou s ains. Fu he mo e, c ys- al s uc u es wi h lignin model compounds bound o laccases ha e been elucida ed (Majumda e al., 2014). Recen ly, a PCR p ime was de eloped in o de o iden i y he wo domains (Lu e al., 2014) o S ep omyces genes du ing compos ing o ag icul u al was e. Gene lib a ies ob ained we e clus e ed wi h S. coelicolo , S. iolaceusnige and S. g iseus. The obse ed inc ease in he S ep omyces small lac- case genes du ing he ini ial s ages o compos ing was due o he p esence o ela i ely high amoun s o deg adable o ganic com- pounds. A clea co ela ion was ound be ween he abundance o laccase-encoding genes and he lignocellulose deg ada ion a es. In 2009, a halo ole an laccase (SilA) om S ep omyces ipomoea CECT 3341 was isola ed and analyzed (Molina-Guija o e al., 2009). Depending on he subs a e, his enzyme showed an op imal ac i - i y a acid o basic condi ions. SilA was highly ac i e a alkaline pH o he oxida ion o he phenolic compound 2,6-dime hoxyphenol, which has only been desc ibed o a ew laccases. SilA was able o wo k a high saline concen a ions (100% o ac i i y e ained a 1.0 M NaCl, pH 8.0), which makes he enzyme eally a ac i e o indus ial pu poses. Thus, ecombinan SilA ob ained om cul- u es o E. coli BL21 has been applied in biobleaching p ocesses on Eucalyp us globulus K a pulps using ace osy ingone as media o (Eugenio e al., 2011). In o de o e alua e he enzyma ic sys em, a u he alkaline ex ac ion o he K a pulp ollowed by hyd o- gen pe oxide ea men was de eloped. The enzyma ically ea ed biobleached pulps esul ed in a significan educ ion in he kappa numbe (41.5%) due o he deg ada ion o bo h lignin and he hex- enu onic acids p esen in he eucalyp us pulp. Addi ion o oxygen du ing he delignifica ion p ocesses was equi ed in o de o ob ain sligh ly imp o ed esul s. The laccase-media o sys em has also an ad an ageous e ec on he pulp ea men , as i was obse ed ha less hyd ogen pe oxide was equi ed o he ea men a e he enzyma ic p ocedu e, which leads o lowe amoun s o pollu an s e fluen s. Laccase-media o deg ada ion led o a dec ease in he pulp iscosi y as well as a o ding a high dec ease in pulp b igh - ness. SilA was eco e ed om he biobleaching ea men s wi h only a sligh deac i a ion (36%) when compa ed wi h he emaining ac i i y o o he laccases a e his p ocess. Fou small-laccases om S ep omyces (S. coelicolo A3(2), S. li i- dans TK24, S. i idospo us T7A) and Amycola opsis sp. 75i 2 we e exp essed in E. coli and pu ified wi h high yields (15–20 mg/L cul- u e). The enzymes we e ound o be e y s able and ac i e o e a wide pH ange (3–10), which makes hem in e es ing candida es G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 115 Fig. 3. Laccase-ca alyzed edox cycle o lignin deg ada ion in he p esence o di e en chemical media o s (le ) and he s uc u es o he mos employed media o s N-hyd oxybenzo iazole (HBT), (2,2,6,6- e ame hylpipe idin-1-yl)oxyl (TEMPO) and 2,2-azino-bis(3-e hylbenzo hiazoline-6-sul onic acid) (ABTS). o indus ial applica ions (Majumda e al., 2014). The ou lac- cases we e able o deg ade a phenolic model compound in o a mix u e o di e en p oduc s including anillin, bu mos o he o he compounds ob ained we e no ully cha ac e ized. Con e - sion o a nonphenolic lignin model compound was only obse ed in he p esence o media o s such as ABTS and HBT while hese we e no needed o oxidizing a phenolic de i a i e. I was sugges ed ha he obse ed ac i i ies can explain he ole o bac e ial laccases in lignocellulosic deg ada ion by modi ying he lignin p ope ies in o de o allow he access o o he enzyma ic sys ems o cellulose and hemicellulose. Pu ified laccase om S. coelicolo A3(2) was es ed in he in i o deg ada ion o e hanolsol lignin. This lignin de i a i e is ob ained om he ea men o lignin wi h e hanol and sul u ic acid, p e- sen ing a lowe molecula weigh and a highe wa e -solubili y. A e a 16 h ea men a 37 ◦C, ch oma og aphy s udies showed a loss in ma e ial solubili y, due o he polyme iza ion o small lignin uni s. This p ocess can be explained by he laccase oxida ion o lignocellulosic subs a es o p oduce a yl ca ion adicals ha can ea ange and p omo e epolyme iza ion, which will be in compe- i ion wi h he enzyma ic depolyme a ion o he s a ing ma e ial. Unde in i o condi ions, he deg ada ion o lignin by depolyme - iza ion can be explained by he p esence o coope a i e enzymes ha p e en he o ma ion o adical species, he eby ac ing as quenche s. In addi ion o he “small” wo-domain laccases, some o he laccases p esen ing h ee-domains ha e been used o he deg ada- ion o lignin de i a i es. The endophy ic bac e ial s ain Pan oea anana is Sd-1 was isola ed om ice seeds using a su ace s e il- iza ion me hod (Xiong e al., 2013). This g am-nega i e bac e ium was able o deg ade lignin and ice s aw. The sequenced Pan oea anana is Sd-1 genome e ealed ha his mic oo ganism has he po en ial o p oduce in a- and ex acellula laccases (Shi e al., 2015). Fou pu a i e laccase-encoding genes we e disco e ed and s udied. F om sequence analyses i was concluded ha Lac4 had he highes homology o ypical bac e ial laccases. Thus, ecombi- nan Lac4 was cha ac e ized as being a dime ic enzyme wi h high ac i i y a low pH alues and a mode a e he mos abili y. In i o lignin deg ada ion by Lac4 in he p esence o ABTS as media o esul ed in 38% con e sion a e 12 h. The o med p oduc s con- ained se e al low molecula weigh a oma ic compounds such as 1,4-benzedica boxaldehyde, benzenep opanoic acid and phenol. Laccases om Bacillus species a e in gene al ole an o high empe a u es and alkaline condi ions, which makes hem sui able o lignin deg ada ion. Un o una ely, mos o hese laccases a e in acellula which complica es la ge scale p oduc ion. Recen ly, an ex acellula laccase was ob ained om Bacillus equilensis SN4 (Sondhi e al., 2015), an bac e ium isola ed om a pape mill e flu- en . The op imum empe a u e o his laccase was 80–90 ◦C, while i e en e ains pa o i s ac i i y a 100 ◦C. The SN4 laccase showed an op imal ac i i y a pH 8.0. Besides being an ex acellula enzyme, i s s abili y make SN4 laccase a use ul bioca alys o be exploi ed on indus ial scale. Laccase p oduc ion was op imized by using a medium con aining Mn+2 and Fe+2 sul a es as well as 3.5% −1 e hanol. SN4 laccase was employed in he biobleaching o so - wood pulp esul ing in a educ ion o 28% in he kappa numbe and a 7.6% inc ease in b igh ness. Addi ion o N-hyd oxybenzo iazole (HBT) as media o a low concen a ion (2.0 mM) led o a u he imp o emen in i s pe o mance. Se e al bac e ial s ains ob ained om soils o a biodi e si y- ich ain o es in Pe u ha e been es ed in he oxida ion o ABTS (Huang e al., 2013). This esul ed in he isola ion o wo Bacil- lus s ains: Bacillus a ophaeus (s ain B7) and Bacillus pumilus (s ain C6). Bo h mic oo ganisms exhibi in acellula and ex a- cellula laccase ac i i ies. Fu he mo e, K a lignin and he lignin model compound guaiacylglyce ol-␤-guaiacyl e he we e success- ully deg aded by bo h s ains. This sugges s ha hese s ains ha bo in e es ing laccases. A laccase om Bacillus pumilus (Co A) disco e ed by genome mining has been success ully cloned and o e exp essed in E. coli (Reiss e al., 2011). This enzyme showed a high he mos abili y wi h a maximum ac i i y a 70 ◦C. Co A was capable o oxidizing se e al phenolic compounds showing op imal alues a pH neu al o alkaline, which makes i an in e es ing bioca alys o u he bio echnological applica ions. The mus he mophilius HB27 is a he mophilic bac e ia which p oduces an in acellula laccase: T h-laccase (Miyazaki, 2005). This bac e ial laccase was success ully exp essed in E. coli. T ea - men o whea s aw pulp wi h his laccase was s udied (Zheng e al., 2012). Using op imized condi ions, he pulp b igh ness was inc eased while he kappa numbe was educed. These benefi- cial e ec s o laccase ac i i y can be u he imp o ed when he enzyma ic bleaching is combined wi h some o he chlo ine- ee chemical bleaching p ocess. The used o he laccase- ea ed whea pulp a o ds a 25% sa e in he use o hyd ogen pe oxide in subse- 116 G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 quen pulp ea men s. The use o ABTS o guaiacol as T h-laccase media o (5 mM) imp o ed he delignifica ion p ocess wi h an inc ease in he delignifica ion deg ee o 9% and 5.6%, espec i ely. Opposi e, HBT has no e ec on his p ocess, p obably due o he di e ences be ween he edox po en ial o he media o s. Whea s aw- ich soda pulp has ecen ly been ea ed wi h an alkalophilic laccase isola ed om ␥-p o eabac e ium JB (Bains e al., 2003). This mic oo ganism was only able o p oduce he laccase, bu no o he lignin deg ading enzymes. Th ee pa ame e s (laccase uni s, pH and ABTS concen a ion) we e op imized using esponse su ace me hodology based on cen al composi e design in o de o achie e he bes deg ada ion p ocess. This led o an inc ease in pulp b igh ness o a ound 6.0%, while he kappa numbe was educed in a 21% ex en (Singh e al., 2008). When a esh chem- ically bleached pulp sample was subjec ed o laccase ea men , he final pulp p ope ies we e significan ly imp o ed in e ms o ch omopho es, educing suga s and hyd ophobic compounds. The applica ion o his laccase can educe he use o hypochlo i e by 10% in he pulp bleaching p ocedu e. Recen ly, a biosenso o he de ec ion o a oma ic lignin deg a- da ion p oduc s has been de eloped (S achan e al., 2014). This biosenso iden ified a no el mul icoppe oxidase by sc eening he me agenome o coal bed bac e ia. This enzyme has a high simila i y o CopA om Pseudomonas s u ze i ATCC14405 and Pseudomonas pu ida, wo s ains ha able o deg ade a wide ange o a o- ma ic compounds. The disco e ed oxidases we e employed in he deg ada ion o a lignin subs a e, leading o 2,6-dime hybenzene- 1,4-diol as he majo iden ified p oduc a e 3 h. 2.3. Glu a hione-dependen ˇ-e he ases ac ing on lignin deg ada ion p oduc s In he ‘80 s he fi s glu a hione-dependen ␤-e he ases we e disco e ed capable o ca alyzing he educ i e clea age o ␤-e he bonds in lignin- ela ed compounds (Masai e al., 1989). The fi s de ailed s udies we e epo ed o he ␤ O 4 a yl-e he clea - ing enzyme sys em om he ␣-p o eobac e ium Sphingobium sp. SYK-6. This sys em is composed o h ee sepa a e p o eins; LigD (aC␣-dehyd ogenase), LigF (a ␤-e he ase) and LigG (a glu a hione lyase), which ha e been success ully exp essed in E. coli. The mul i- enzyme sys em clea es he ␤-a yl e he s o a model lignin dime : guaiacylglyce ol-␤-guaiacyl. The ole o each o he h ee enzymes in he lignin deg ada ion is he ollowing: (1) LigD ca alyzed he NAD+-dependen oxida ion o he C␣ o lignin subs a e om he alcohol o he co esponding ke one; (2) LigF clea es he in e me- dia e wi h he a achmen o glu a hione a he C␤ posi ion, and finally (3) LigG oxidizes glu a hione and eleases he final p oduc (Fig. 4) (Sa o e al., 2009). Two o he glu a hione educ ases om Sphingobium sp. s ain SYK-6 (LigE and LigP) a e also able o ca alyze he same eac ion as LigF, bu hese enzymes a e ac i e on he oppo- si e enan iome o he ca bonyl compound o med by he oxida ion o guaiacylglyce ol-␤-guaiacyl wi h LigD (Tanamu a e al., 2011). Recen ly, he s e eop e e ence o LigG has been s udied oge he wi h wo no el glu a hione lyases (LigG-NS om No osphingob- ium sp. PP1Y and LigTD om Thiobacillus deni ificans ATC 25259) in he enzyma ic deg ada ion o a lignin model subs a e employ- ing an enzyma ic cascade o ␤-e he ases and glu a hione lyases. The es ed enzymes showed ␤-(R)-enan iop e e ence. This enan- iop e e ence can be inc eased o e en e e sed by mu agenesis (Pica e al., 2015). As hese enzyme sys ems a e inhe en in acel- lula (as NAD+is an in acellula me aboli e), hey a e no in ol ed in he ini ial deg ada ion o lignin. Ye , hey may play a ole in he deg ada ion o he small lignin oligome s o med in he fi s s eps o lignin deg ada ion and may de elop as use ul bioca alys s in he field o lignocellulose u iliza ion. 2.4. The ole o supe oxide dismu ases in bac e ial lignin modifica ion Recen ly wo bac e ial manganese-dependen supe oxide dis- mu ases (MnSODs) we e disco e ed o ha e lignin deg ading ac i i y (Rashid e al., 2015). Supe oxide dismu ases ypically ca alyze he disp opo iona ion o supe oxide anion adical in o molecula oxygen and hyd ogen pe oxide and play a key ole in cel- lula p o ec ion agains oxida i e s ess. Supe oxide dismu ases a e in gene al in acellula enzymes, bu some examples o ex acellu- la ac i i y ha e been epo ed. Recen ly wo ex acellula MnSODs om Sphingobac e ium sp. T2 (MnSOD1 and MnSOD2) we e iden- ified and pa ially pu ified (Rashid e al., 2015). In e es ingly, bo h enzymes a e able o pe o m he deg ada ion o O ganosol and K a lignin, as well as di e en lignin model subs a es, in o se - e al compounds. All he p oduc s o med esul ed om a yl-C␣ and C␣-C␤ bond oxida i e clea age eac ions as well as om O- deme hyla ion ac i i y. The lignin oxida ion eac i i y o hese MnSODs can be assigned o he o ma ion o a hyd oxyl adical, wi h a high oxidan e ficiency. I emains o be es ablished whe he such supe oxide dismu ases a e p omising candida es o bio ech- nological applica ion in he a ea o lignocellulose deg ada ion and whe he hey a e ypical bac e ial ligninoly ic enzymes. 2.5. Ca alase-pe oxidases a e associa ed wi h lignocellulose deg ada ion Recen ly, by a p o eomic app oach, a ca alase-pe oxidase (Amyco1) was iden ified as a heme-con aining enzyme sec e ed by Amycola opsis sp. 75i 2 when incuba ed wi h lignocellulosic ma e ial (B own e al., 2011). To p o ide u he e idence o i s in ol emen in lignin deg ada ion, he enzyme was ecombinan ly p oduced and pu ified. Amyco1 was ound o be able o con e a phenolic lignin model compound, while a me hyla ed de i a- i e was no a subs a e. Wi h expe imen al confi ma ion ha his ca alase-pe oxidase ac s on a lignin-like compound, a ole o Amyco1 in lignin modifica ion seem easible. Fu u e esea ch will ell whe he such ex acellula bac e ial ca alase-pe oxidases a e equen ly used by bac e ia o modi y lignin. 2.6. Bac e ial dioxygenases may play a ole in lignin deg ada ion In 2013 a peculia bac e ial enzyme was desc ibed ha is composed o a dioxygenase domain and a lignin-binding domain (Bianche i e al., 2013). Again, his enzyme was isola ed om a S ep omyces isola e and was ound o be associa ed wi h a wood- wasp which lays i s eggs in wood. The e o e i was an icipa ed ha he isola ed bac e ium would exhibi plan biomass deg ada ion capabili ies. Indeed, a de ailed cha ac e iza ion e ealed a sui e o sec e ed enzymes in ol ed in hyd oly ic and oxida i e a ack o lignocellulose. As pa o he iden ifica ion o sec e ed p o eins, he SACTE 2871 p o ein eme ged. Based on he p o ein sequence, i appea ed o be he esul o a usion o a in adiol dioxyge- nase and a ca bohyd a e binding module. The p o ein also con ains an N- e minal Ta - ansloca ion signal pep ide which confi ms i s ex acellula loca ion. Recombinan exp ession o he enzyme in E. coli was success ul and allowed he elucida ion o he c ys al s uc u e o he dioxygenase domain. Fu he mo e, i could be con- fi med ha he enzyme was ac i e as dioxygenase wi h se e al ca echol de i a i es. In iguingly, i was disco e ed ha he ca bo- hyd a e binding module displayed a fini y owa ds syn he ic lignin polyme s. The in ol emen o a dioxygenase in a bac e ial deg ada- ion pa hway o lignin- ela ed compound is no unp eceden ed. One o he bac e ia om which a ␤-e he ase was iden ified, Sphingomonas paucimobilis SYK-6, was ound o ha bo ano he G. de Gonzalo e al. / Jou nal o Bio echnology 236 (2016) 110–119 117 Fig. 4. Deg ada ion o guaiacylglyce ol-␤-guaiacyl by Sphingobium sp. SYK-6: (1) LigD ca alyzes he alcohol oxida ion o he lignin subs a e C␣; (2) LigF ca alyzes he inco po a ion o glu a hione a he C␤ posi ion, and finally (3) LigG oxidizes glu a hione, leading o he final p oduc . Fig. 5. Mul ienzyma ic deg ada ion o he biphenyl lignin de i a i e 2,2-dihyd oxy-3,3-dime hoxy-5,5-dica boxybiphenyl by Sphingomonas paucimobilis SYK-6. Fou enzymes a e in ol ed: (1) LigX ca alyzes he deme hyla ion o he me hoxy g oup; (2) ing fission is ca alyzed by he dioxygenase LigZ; (3) C C hyd olysis is ca alyzed by LigY, and (4) con e sion o 5-ca boxy anillic acid in o anillic acid is pe o med by wo deca boxylases (LigW and LigW2). in e es ing deg ada ion pa hway ela ed o lignin deg ada ion, which includes a dioxygenase. A biphenyl compound (2,2- dihyd oxy-3,3-dime hoxy-5,5-dica boxybiphenyl) was iden ified as a g ow h subs a e o Sphingomonas paucimobilis SYK-6 (Sonoki e al., 2009). Fou di e en ypes o enzymes a e in ol ed in he ini- ial s eps o deg ading his biphenyl: LigW/LigW2 (deca boxylases), LigY (a C C hyd olase), LigX (an i on-dependen deme hylase) and LigZ (an ex adiol dioxygenase) (Fig. 5). The deme hyla ion o he me hoxy g oup akes place by LigX. The p oduc om his clea - age s ep is he subs a e o he dioxygenase LigZ, which leads o a ing fission p oduc yielding 5-ca boxy anillic acid and 4-ca boxy- 2-hyd oxypen adienoic acid. The p oduc 5-ca boxy anillic acid is con e ed in o anillic acid in a p ocess ca alyzed by wo deca - boxylase enzymes (LigW and LigW2). These findings sugges ha dioxygenases ep esen s ano he ool used by bac e ia o assis in lignin deg ada ion. 3. Ou look Bac e ia do no possess he egula pe oxidases ha ungi employ o lignin deg ada ion. This may be due o in insic di ficul- ies in exp essing hese a he complex p o eins ha a e ypically glycosyla ed, con ain se e al disulphide bonds, and inco po a e se e al calcium ions and a heme co ac o . Folding and p ocessing may equi e special condi ions ha a e no compa ible wi h he bac e ial machine y o p o ein p oduc ion. In line wi h his, i is wo h no ing ha mos o he a emp s o p oduce pe oxidases om he plan pe oxidase supe amily ailed. Ye , he DyP- ype pe oxidases a e somewha less complex conce ning hei p o ein s uc u e (no calcium binding si es, glycosyla ion o disulphide bonds needed) and a e wide sp ead among bac e ia. Recombinan exp ession o a ious DyPs in E. coli ypically yielded high le els o exp ession which is in sha p con as wi h he exp ession o ungal pe oxidases (Lambe z e al., 2016). This makes hese bac e ial pe - oxidases in e es ing a ge s o enzyme de elopmen . The bac e ial laccases also seem o be sui ed o la ge scale ecombinan enzyme p oduc ion. A ecen de ailed s udy e ealed ha by op imizing exp ession condi ions, a ious bac e ial laccases can be p oduced in E. coli (Ihssen e al., 2015). While he numbe o known bac e ial pe oxidases and laccases ha may be in ol ed in lignin deg ada ion has g own ema kably in he las wo decades, he e may s ill be undisco e ed enzyme ypes a ound ha play a ole in bac e ial lignin deg ada ion. Se - e al new candida es ha e su aced in he las ew yea s, ide sup a. Fu he mo e, one class o enzymes ha has been o e looked a e he enzymes ha need o p o ide he bac e ial pe oxidases he equi ed hyd ogen pe oxide. Such oxidases ha e been iden ified o he ungal pe oxidases, and simila ly i is expec ed ha bac e ia sec e e oxidases. Ye , wha ype o oxidases and hei espec i e subs a es ha e s ill o be es ablished. I is in e es ing o no e ha only ecen ly, also o he deg a- da ion o he cellulose pa o plan biomass oxida i e enzymes a e ecognized as majo playe s in he deg ada ion p ocess. The coppe -con aining so-called ly ic polysaccha ide monooxygenases (LPMOs) ha e been ound o be essen ial in he a ack o he a he ine pa s o cellulose and o he polysaccha ides. The ecen find- ing o he Eijsink g oup ha he LPMOs seques e ed he equi ed elec ons om he lignin pa o plan biomass sugges s a s ong link be ween he deg ada ion o he di e en biomass moie ies (Wes e eng e al., 2015). I will be in e es ing o see whe he bac- e ial LPMOs can be linked o o he edox enzymes sec e ed by bac e ia and whe he bac e ial lignin deg ada ion is in e wined wi h plan polysaccha ide deg ada ion. Wi h he ealiza ion o he bac e ial abili y o modi y he lignin pa o plan biomass, biochemical s udies on bac e ial lignin deg a- da ion pa hways and hei espec i e enzymes has been e i alised. Wi h new app oaches o iden i y new lignin-deg ading bac e ia (S achan e al., 2014; Pica e al., 2016) in combina ion wi h de ailed genomic, p o eomic and biochemical s udies, he iden i- ies and oles o bac e ial ligninoly ic enzymes will be unco e ed in he coming yea s. Acknowledgmen s This wo k was suppo ed by he NWO g adua e p og am: syn- he ic biology o ad anced me abolic enginee ing, p ojec numbe 022.004.006, The Ne he lands. Gonzalo de Gonzalo (Ramón y Cajal P og am) hanks MINECO o pe sonal unding. 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