molecules Review Deconstruction of Lignin: From Enzymes to Microorganisms Jéssica P. Silva 1, Alonso R. P. Ticona 1 , Pedro R. V. Hamann 1, Betania F. Quirino 2 and Eliane F. Noronha 1,* 1 Enzymology Laboratory, Cell Biology Department, University of Brasilia, 70910-900 Brasília, Brazil; [email protected] (J.P.S.); [email protected] (A.R.P.T.); [email protected] (P.R.V.H.) 2 Genetics and Biotechnology Laboratory, Embrapa-Agroenergy, 70770-901 Brasília, Brazil; [email protected] * Correspondence: [email protected]; Tel.: +55-61-3307-2152 Abstract: Lignocellulosic residues are low-cost abundant feedstocks that can be used for industrial applications. However, their recalcitrance currently makes lignocellulose use limited. In natural environments, microbial communities can completely deconstruct lignocellulose by synergistic action of a set of enzymes and proteins. Microbial degradation of lignin by fungi, important lignin degraders in nature, has been intensively studied. More recently, bacteria have also been described as able to break down lignin, and to have a central role in recycling this plant polymer. Nevertheless, bacterial deconstruction of lignin has not been fully elucidated yet. Direct analysis of environmental samples using metagenomics, metatranscriptomics, and metaproteomics approaches is a powerful strategy to describe/discover enzymes, metabolic pathways, and microorganisms involved in lignin breakdown. Indeed, the use of these complementary techniques leads to a better understanding of the composition, function, and dynamics of microbial communities involved in lignin deconstruction. We focus on omics approaches and their contribution to the discovery of new enzymes and reactions that impact the development of lignin-based bioprocesses. Citation: Silva, J.P.; Ticona, A.R.P.; Hamann, P.R.V.; Quirino, B.F.; Keywords: lignin; bacteria; biodegradation; auxiliary activities; metagenomics; metaproteomics; Noronha, E.F. Deconstruction of metatranscriptomics Lignin: From Enzymes to Microorganisms. Molecules 2021, 26, 2299. https://doi.org/10.3390/ molecules26082299 1. Introduction Academic Editor: Claudio Gioia The conversion of lignocellulosic biomass into biofuels and chemicals has gained inter- est because of its potential application in biorefineries as a green platform. Lignocellulosic Received: 26 February 2021 biomass is mainly composed of lignin and polysaccharides (i.e., cellulose, hemicellulose, Accepted: 26 March 2021 and pectin), arranged in plant cell walls. Cellulose is a homopolymer, composed of D- Published: 15 April 2021 glucose monomers joined by linear β (1–4) linkages. Hemicellulose is also a sugar polymer, but it is composed of different monosaccharide molecules mainly joined by β-1,4 glyco- Publisher’s Note: MDPI stays neutral sidic linkages. Among the sugars that compose hemicellulose, D-xylose, D-mannose, and with regard to jurisdictional claims in arabinose are present. Hemicellulose polymers are strongly interlinked through covalent published maps and institutional affil- and non-covalent bonds, and also linked to lignin, which together with cellulose will form iations. the recalcitrant lignocellulosic matrix [1,2]. In the plant cell wall, lignin is linked to the carbohydrate moiety via the ester linkage. This association gives the plant cell wall greater strength and impermeability [3]. Lignin is formed by radical coupling reactions involving the three main phenylpropane units: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S), Copyright: © 2021 by the authors. linked by C–C and C–O linkages [4,5]. Licensee MDPI, Basel, Switzerland. Lignin is the main plant cell wall component responsible for recalcitrance [6]. Thus, This article is an open access article pretreatment is an essential step for removing lignin in the process of lignocellulosic distributed under the terms and biomass conversion into biofuels [7,8]. The high carbon/oxygen ratio and the natural conditions of the Creative Commons abundance of lignin make it a promising feedstock material for biological conversion into Attribution (CC BY) license (https:// value-added products [9]. In addition to the biofuel industry, lignin may also be found as creativecommons.org/licenses/by/ a by-product from wood-biomass in industrial processes for paper and pulp production. 4.0/). Molecules 2021, 26, 2299. https://doi.org/10.3390/molecules26082299 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x FOR PEER REVIEW 2 of 22 Molecules 2021, 26, 2299 2 of 21 At least 75,000 tons of kraft-lignin were commercialized in 2018, and for the year 2025, At250,000 least 75,000 tons are tons expected of kraft-lignin [10]. were commercialized in 2018, and for the year 2025, 250,000The tons efficient are expected degradation [10]. of lignin by white-rot basidiomycete fungi has been exten- sivelyThe efficientstudied [11,12]. degradation Bacteria of lignin are also by white-rotcapable of basidiomycete deconstructing fungi lignin has [5,13], been exten-but they sivelyare studiedless studied [11,12 in]. Bacteriacomparison are alsoto fungi capable [12,14]. of deconstructing Recently, bacteria lignin [have5,13], been but they attracting are lessgreat studied attention in comparison due to their to fungihigh [adaptability12,14]. Recently, and bacteriabiochemical have versat beenility. attracting Furthermore, great attentionbacteria due have to theirmetabolic high adaptabilitypathways that and conver biochemicalt lignin versatility. and its derivates Furthermore, into products bacteria of havebiotechnological metabolic pathways interest that such convert as lactic lignin acid, and pyruvate, its derivates vanillin, into productslipids, polyhydroxyalka- of biotechno- logicalnoates interest (PHA), such and as cis lactic-muconic acid, pyruvate, acid (cis, cis vanillin,-MA) [15,16]. lipids, polyhydroxyalkanoates (PHA), and cis-muconicIn nature, acid lignin (cis is, cis deconstructed-MA) [15,16]. by the concerted efforts of microbial communities, ratherIn nature, than isolate lignin microorganisms. is deconstructed Therefore, by the concerted the strategy efforts of of studying microbial the communities, microbial com- rathermunity than may isolate provide microorganisms. a broader comprehension Therefore, theof the strategy process of of studying lignin deconstruction. the microbial In community may provide a broader comprehension of the process of lignin deconstruction. natural environments, different microorganisms in the microbial community operate syn- In natural environments, different microorganisms in the microbial community operate ergistically through the secretion of a variety of biocatalysts. Although in nature coopera- synergistically through the secretion of a variety of biocatalysts. Although in nature coop- tion between different microorganisms is common, in the laboratory, not all organisms eration between different microorganisms is common, in the laboratory, not all organisms are easily cultured, which is an obstacle to adapting the natural synergistic lignin decon- are easily cultured, which is an obstacle to adapting the natural synergistic lignin decon- struction to its bioconversion in industrial processes. However, investigation of natural struction to its bioconversion in industrial processes. However, investigation of natural microbial communities using culture-independent approaches (e.g., metagenomics, meta- microbial communities using culture-independent approaches (e.g., metagenomics, meta- transcriptomics, and metaproteomics) may deliver a detailed description of the processes transcriptomics, and metaproteomics) may deliver a detailed description of the processes and enzymes involved. Thus, the study of lignin-degrading microbiomes (e.g., forests, and enzymes involved. Thus, the study of lignin-degrading microbiomes (e.g., forests, animals’ digestive tract, and sewage [17,18]) may be crucial to the development of efficient animals’ digestive tract, and sewage [17,18]) may be crucial to the development of efficient industrialindustrial processes processes for for the the use use of ligninolyticof ligninolytic feedstocks. feedstocks. InIn the the present present review, review, we we explore explore the the contribution contribution of omicsof omics approaches approaches in in discover- discover- ing/describinging/describing new new enzymes enzymes and and microorganisms microorganisms inin microbiomes adapted adapted to to degrade degrade lig- lignin.nin. Primarily, Primarily, we we focus focus on on ligninolytic ligninolytic enzymes enzymes belonging belonging to tothe the auxiliary auxiliary activities activities (AA) (AA)family, family, with with emphasis emphasis on ontheir their impact impact on onlignin lignin valorization valorization in inindustrial industrial processes. processes. 2. Lignin2. Lignin 2.1.2.1. Structure Structure and and Composition Composition of Naturalof Natural Lignin Lignin LigninLignin is ais phenolic a phenolic macromolecule macromolecule of high of molecularhigh molecular weight, weight, composed composed of three of main three unitsmain of phenylpropaneunits of phenylpropane (monolignols): (monolignols): coniferyl alcohol,coniferyl sinapyl alcohol, alcohol, sinapyl and alcohol,p-coumaryl and p- alcoholcoumaryl (Figure alcohol1). When (Figure incorporated 1). When intoincorporated the lignin into structure, the lignin these structure, monomers these are monomers termed p-hydroxyphenylare termed p-hydroxyphenyl (H), guaiacyl (G), (H), and guaiacyl syringyl (G), (S)