Identifying and Creating Pathways to Improve Biological Lignin Valorization T ⁎ Zhi-Hua Liua,B,1, Rosemary K

Renewable and Sustainable Energy Reviews 105 (2019) 349–362

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Renewable and Sustainable Energy Reviews

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Identifying and creating pathways to improve biological lignin valorization T ⁎ Zhi-Hua Liua,b,1, Rosemary K. Lec,1, Matyas Kosad, Bin Yange, Joshua Yuana,b, , ⁎⁎ Arthur J. Ragauskasc,f,g, a Synthetic and Systems Biology Innovation Hub (SSBiH), Texas A&M University, College Station, TX 77843, United States b Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, United States c Department of Chemical & Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996, United States d Fortress Advanced Bioproducts Inc., Vancouver, BC, Canada V6T 1W5 e Bioproducts, Sciences, and Engineering Laboratory, Department of Biological Systems Engineering, Washington State University, Richland, WA 99354, United States f Joint Institute for Biological Sciences, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, United States g Center for Renewable Carbon, Department of Forestry, Wildlife, and Fisheries, University Tennessee, Knoxville, TN 37996, United States

ARTICLE INFO ABSTRACT

Keywords: Biological lignin valorization to fuels and value-added chemicals enables sustainable and economic biorefineries. Biological lignin valorization While significant progress has been made, several major challenges arose due to high recalcitrance and het- Ligninolytic microorganism erogeneity of lignin, which needs to be addressed to improve lignin processing. This work provides an overview Bioprospecting of biological lignin conversion and its regulation from a metabolic engineering and systems biology viewpoint. Metabolic engineering Biological lignin valorization includes three stages: lignin depolymerization, aromatics degradation, and target Systems biology product biosynthesis. Ligninolytic microorganisms have an extensive enzymatic toolbox to break down the lignin Synthetic biology Techno-economic analysis and convert heterogeneous lignin derivatives to central intermediates, such as protocatechuate or catechol, through a peripheral pathway. These intermediates undergo ring cleavage via the β-ketoadipate pathway and are ultimately transformed into metabolites by yielding acetyl-CoA for internal product biosynthesis, such as triacylglycerols, polyhydroxyalkanoates, etc. Bioprospecting will expand the knowledge base of ligninolytic microbial communities, strains, and enzymes to facilitate the understanding of aromatics metabolism. Systems biology analyses achieve an understanding of molecular and systems-level degradation mechanisms and metabolic pathways of lignin and aromatics. By identifying these mechanisms, synthetic biology provides promising approaches to create the lignin conversion pathways and engineer ligninolytic strains suitable as potential hosts for lignin conversion. Techno-economic analysis of biological lignin upgrading to coproducts in biorefineries will guide the implementation of lignin valorization by mitigating technical risk for scale-up and improving the profitability of biorefinery. By improving the understanding of biological lignin valorization, it should be possible to create biological lignin valorization route to effectively produce value-added products from lignin.

1. Introduction considered to be suitable for the production of biofuels as well as bulk and ﬁne chemicals, potentially addressing energy and environmental Demand for renewable resource based fuels and chemicals is on the concerns [1,3]. With the intensive development of bioreﬁneries for rise due to the dramatic increase in greenhouse gas emissions [1,2]. The fuels and chemicals from carbohydrates, the amount of lignin-rich re- lignin polymer in plant biomass is the most abundant renewable source sidues has increased, warranting new strategies for upgrading lignin of aromatic carbon and the second most abundant terrestrial polymer [2,4–6]. For instance, in the cellulosic ethanol and pulp and paper in- after cellulose. Aromatic compounds derived from lignin have been dustries, about 112 million tons of lignin are annually produced as

Abbreviations: CDW, Cell dry weight; DyP, Dye peroxidase; LiP, Lignin peroxidase; MnP, Manganese peroxidase; mcl-PHAs, Medium chain-length polyhydroxyalkanoates; O2-KL, Oxygen pretreatment Kraft lignin; PAHs, Polycyclic aromatic hydrocarbons; PHA, Polyhydroxyalkanoates; TAG, Triacylglycerol; TCA, Tricarboxylic acid; TEA, Techno-economic analysis; US-EOL, Ultrasonicated ethanol organosolv lignin; WT, Wild type ⁎ Corresponding author at: Synthetic and Systems Biology Innovation Hub (SSBiH), Texas A&M University, College Station, TX 77843, United States. ⁎⁎ Corresponding author at: Department of Chemical & Biomolecular Engineering, University of Tennessee Knoxville, Knoxville, TN 37996, United States. E-mail addresses: [email protected] (Z.-H. Liu), [email protected] (R.K. Le), [email protected] (M. Kosa), [email protected] (B. Yang), [email protected] (J. Yuan), [email protected] (A.J. Ragauskas). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.rser.2019.02.009 Received 3 June 2018; Received in revised form 18 January 2019; Accepted 12 February 2019 Available online 16 February 2019 1364-0321/ © 2019 Elsevier Ltd. All rights reserved. Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362

Fig. 1. Schematic structure of lignin biopolymer in plant biomass. inexpensive and readily available residue in the United States alone [7]. exactly controlled to synthesize the desired product in microorganisms. Recently, lignin valorization has attracted more attention and been Among available state-of-the-art technologies, biological lignin va- considered in plant biomass upgrading strategies for economic and lorization that involved lignin depolymerization and conversion by sustainable biorefineries. Even so, the commercial application of lignin microorganisms holds great potential. A desirable lignin bio-depoly- is limited to only a few examples (e.g. vanillin and lignosulfonates) merization protocol would be a series of reactions with high reaction because of the challenging approaches for lignin depolymerization. rates that also allow for bioconversion to value-added compounds. Lignin is biosynthesized in oxidative coupling reactions using cin- Despite significant progress, biological lignin valorization is still highly namyl alcohol derivatives: p-coumaryl alcohol, coniferyl alcohol, and challenging with low levels of useful products generated. Several major sinapyl alcohol (Fig. 1). It is an arguably random three-dimensional issues need to be addressed to further improve biological lignin valor- biopolymer composing of phenylpropanoid units linked together by ization. Herein this paper overviews the biological lignin biorefinery various linkages, including β-O-4-aryl ether, β-5-phenylcoumaran, 5–5- from a metabolic engineering and systems biology viewpoint, and biphenyl, β-β-resinol and 4-O-5-diaryl ether linkage [8–10]. β-O-4-aryl points out future prospects for microbial production of lignin-derived ether is the most abundant linkage in lignin polymer [3]. Lignin carries biofuels and chemical. a variety of functional groups, for example, aliphatic hydroxyl, car- bonyl, phenolic hydroxyl, methoxyl, and benzyl alcohol groups, which 2. Biological processing of lignin by ligninolytic microorganisms determine the polarity and quality of both native and technical lignin. These chemistries are responsible for its heterogeneity and high re- 2.1. Ligninolytic microorganisms calcitrance, imposing a unique challenge for its depolymerization. Numerous studies have been conducted in the past few decades into Biological processing of lignin has emerged as a potentially effective valorizing lignin, in which the resulting monomers and oligmers from approach to gain lignin-derived products. The benefits of using biolo- lignin depolymerization by biological, thermal, or chemical pretreat- gical processing include the ability to use renewable carbon sources as – ments are converted to fuels and chemicals [6,11 15]. Most pretreat- feedstock to sustain cellular growth for generating various inter- ment approaches for lignin depolymerization produce a heterogeneous mediates and the ability for target molecules to be expressed with a array of aromatic species from lignin, depending on the feedstock types specificity that surpasses synthetic chemistry [25–28]. The capability of – used and the pretreatment approaches employed [16 18]. A hetero- microorganisms to selectively create target chemicals with special ffi geneous mixture of aromatic compounds may be su cient to produce features can be harnessed to generate new molecules for unique biofuels; however, high purity of aromatic compounds is necessary for the fuels or chemicals. fi production of ne chemicals [11,12,17,19]. Therefore, heterogeneity of Both fungi and bacteria have been found capable of degrading lignin lignin or aromatics generated from these depolymerization processes in vitro and/or in vivo with multiple enzymes and associated small fi presents a major obstacle for upgrading lignin to ne chemicals. molecule co-factors. The degradation mechanisms of lignin and aro- Research of biological lignin processing has been ramped up and matics have also been studied in different natural biomass utilization screening of microorganisms in ligninolytic environments has led to the systems [23,29]. Fungi have relatively powerful enzyme systems for discovery of bacteria and fungi that exhibit highly diverse enzymatic lignin depolymerization, based on research dating from as far as the – activities for lignin degradation and conversion [3,20 23]. These ad- mid-1980s. The most frequently characterized wood-rotting organism vances have improved understanding of lignin degradation and also are basidiomycota white-rot fungi, e.g. Phanerochaete chrysosporium demonstrated that biological processing of plant biomass and specifi- that is known for its capability of converting lignin into chitin, CO2 and cally lignin can someday become feasible. In theory, if processing of H2O thoroughly [30]. Basidiomycete fungi are the primary organisms lignin could generate more uniform lignin-derived intermediates, such that can metabolize lignin in nature. Other fungi, such as Ceriporiopsis as aromatics and phenolics, these in turn could be transformed into subvermispora, Echinodontium taxodii, Pleurotus ostreatus, Cyathus ster- platform chemicals, which could act as sustainable replacements for coeus, [31–34] and so forth, have also been observed to metabolize petroleum-based plastics, fuels and commodity-, or specialized-chemi- lignin [27,35]. Bacteria that are promising lignin metabolizers include: cals [11,12,24]. Consequently, biological lignin valorization should be Pseudomonas putida [36–38], Rhodococcus jostii [39,40], Rhodococcus fi pro table if the depolymerization and metabolism of lignin can be opacus [9,41], Acinetobacter baylyi [42], Amycolatopsis sp. [39], and

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Sphingomonas [43]. Among these strains, P. putida and R. opacus, have to degrade Kraft lignin, both cell growth and product yields were sig- been considered as potential strains for engineering lignin bioconver- nificantly improved, demonstrating the synergistic effects of a bacterial sion, due to the strong capacity for the accumulation of poly- and fungal enzymatic systems [41]. Although the activity of lignin- hydroxyalkanoates (PHA) and triglycerides. degrading bacteria is much less than that of fungi, more attention has Although these wild-type strains have shown potential to degrade been paid to lignin-degrading bacteria due to its relatively simple and convert lignin, the understanding of degradation pathways are far protein expression and genetic modification modes. Current know-how from complete [3,38]. Lignin depolymerization pathways in fungi have suggests that lignin-degrading bacteria could use similar types of en- been investigated, but the ability to overexpress ligninolytic enzymes is zymes as fungi for lignin depolymerization, however, in vitro lignin much more challenging due to obstacles in genetic manipulation and depolymerization is still inefficient and the bacterial enzymology of the recombinant expression of functionally active proteins. Bacteria lignin degradation is poorly understood. Accordingly, the development also have the ability to catabolize lignin and phenolic compounds, of novel ligninolytic enzymes with enhanced stability and superior however, ligninolytic bacteria's capacity for lignin degradation is much catalytic properties are necessary and timely. lower than that of fungal systems [3,44]. Even though genetic engineering of bacteria is much easier, genetic tools for these strains are 2.3. Metabolic engineering challenges of lignin and aromatic compound still lack and deficient. Taken together, these challenges hinder the catabolism development of an efficient lignin conversion system of bacteria and make these strains unsuitable as industrially-useful hosts [3,6]. There- Soil contains a plethora of lignin-degrading bacteria and fungi, fore, the molecular mechanisms for microbial lignin degradation should where the accumulating plant detritus results in lignin- and aromatic- be systematically revealed in order to improve biological lignin valor- rich resources that are utilized by these strains via degradation path- ization and enhance the economic feasibility of biorefineries. ways [3] The depolymerization of lignin by ligninolytic microorganisms yield a heterogeneous mixture of low molecular weight compounds that 2.2. Enzymatic toolbox for lignin degradation varies depending on the plant biomass types and strains employed. For example, Chen et al. investigated the breakdown of spruce wood using Some microorganisms in nature have an extensive enzymatic Phanerochaete chrysosporium fungi and detected 28 compounds, some of toolbox for breaking down the lignin polymer, including laccase (Lac), which originated from lignin interunit cleavage and resulted in a lignin peroxidase (LiP), manganese peroxidase (MnP), versatile perox- variety of carboxylic aromatics while acyclic 2,4-hexadiene-1,6-dioic idase (VP), aryl alcohol oxidase (AAO), and H2O2-generating enzymes, acids from Cα-Cβ oxidative cleavage were also observed [55,56]. Using as well as others. Biological lignin valorization requires a variety of P. putida, R. jostii RHA1, and S. paucimobilis for lignin depolymerization ligninolytic enzymes. A subset of these enzymes are capable of produ- also yielded a similar range of phenolic compounds [43,57]. S. pauci- cing aromatic radicals, which in turn could cleave a variety of linkages mobilis SYK-6, a gram-negative proteobacterium, has exhibited cata- via non-enzymatic reactions as well as other oxygenated aromatic bolism of aromatic compounds and served as an initial model for aro- compounds [13,22,35,45]. Among these lignin-degrading enzymes, the matics catabolism. Ligninolytic pathways for metabolism of β-aryl degradation mechanisms of laccases and peroxidases have been ex- ethers, biphenyls, diarylpropanes, phenylcoumarane, pinoresinol, and tensively studied. Laccases have been confirmed to oxidize lignin de- ferulic acid model compounds have been characterized in S. paucimo- rived phenolic compounds and reduce molecular oxygen to water. They bilis SYK-6 [43,58–60]. R. jostii RHA1, an engineered soil bacterium, has also catalyze the formation of phenoxyl radicals from phenolic com- been shown to accumulate vanillin while growing on Kraft lignin or pounds, leading to Cα-hydroxyl oxidation to ketone, alkyl-aryl cleavage, plant biomass as carbon sources when the vanillin dehydrogenase gene demethoxylation, and Cα-Cβ cleavage [46]. MnP has been confirmed to was knocked out [61]. oxidize non-phenolic lignin-related components, during which the Transporters play a significant role in the metabolism of aromatics oxidizing potential of MnP can be transferred to Mn3+ and then dif- and the availability of transporter function in bacterial cell membranes fused into the lignified cell wall to attack it from the inside of lignin has been confirmed as a major limiting factor for the efficient catabo- [47]. LiP can directly oxidize non-phenolic units of the lignin polymer, lism of aromatics [62]. Therefore, it is important to consider that to such as Cα-Cβ bonds, through removing one electron and creating ca- obtain efficient utilization of lignin these organisms should exhibit the tion radicals [48]. ability to metabolize a broad range of aromatic compounds and have Besides ligninolytic enzymes mentioned above, microbial β- pathways for lignin or aromatic compound uptake. However, the etherases has also emerged as promising and more specific alternatives knowledge of bacterial lignin catabolism pathways is inadequate, and it for lignin degradation [49]. These non-radical ligninolytic enzymes is based on studies of aromatic monomer and oligomer usage in bacteria cleave the β-O-4 aryl ether linkage selectively [49,50]. The scarcely not lignin itself [43]. reported natural occurrence of β-etherases is mainly concentrated in Aromatic compounds derived from the depolymerization of lignin genus Chaetomium, Novosphingobium sp. PP1Y, Novosphingobium ar- may act as inhibitors to the growth of microorganisms, which hinder omaticivorans DSM1244 and soil-proteobacterium Sphingobium pauci- biological lignin valorization. To address aromatic compound inhibi- mobilis SYK-6 [50–52]. Guaiacylglycerol β-guaiacyl ether is generally tion, metabolic engineering and adaptation strategies can be used to used as the lignin model dimer substrate to detect β-etherases activity. generate strains that have an increased tolerance to lignin-derived in- Rosini et al. found that LigG from Sphingobium sp. SYK-6 catalyze the hibitors, while the ability to degrade aromatic compounds is balanced. glutathione-dependent thioether cleavage of both β(R)- and β(S)-isomer Cupriavidus basilensis B-8 is an example of a strong catabolizer of aro- intermediates produced by LigE and LigF β-etherases, which resulted in matics. The genetic information of C. basilensis B-8 can be screened for almost 100% conversion of guaiacylglycerol β-guaiacyl ether [53]. characteristics that allows it to consume a diverse range of aromatic Recently, the additions of other lignin-degrading enzymes to the compounds and their respective degradation pathways [63]. Adaptive existing biological process have been attempted to accelerate the lignin evolution has been used on R. opacus PD630 to generate a strain that is degradation. Using a lignin-degrading enzyme from P. chrysosporium enabled to tolerate increased phenol concentrations by the up-regula- and 4-O-methyltransferase from the plant C. breweri have been con- tion of its phenol degradation pathway and putative transporters [64]. firmed to increase the oxidation activity of veratryl alcohol [54]. The These results imply that through genetic engineering, inhibitor toler- present of engineered 4-O-methyltransferase is the cause of this phe- ance can be enhanced by overexpression of specific transporter genes in nomenon due to the reduction of the inhibiting effect of free-hydroxyl degradation pathways. Accordingly, understanding the mechanisms of phenolic groups on the lignin-degrading enzyme activity. By the com- lignin- and its derived aromatics-degradation and tolerance to asso- bination of R. opacus and an exogenous laccase from Trametes versicolor ciated inhibitors is crucial to design efficient metabolic pathways.

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Fig. 2. Schematic lignin fragmentation routes to produce vanillin and vanillic acid intermediates from lignin polymer.

3. Lignin fragmentation and primary pathway identiﬁed down various linkages of lignin and converting its heterogeneous derivatives. Aromatic catabolism generally occurs through “upper path- Due to the abundant presence of lignin and aromatic compounds in ways” acting as a “biological funnel” that convert heterogeneous lignin the environment microorganisms have adapted to utilize these aro- derivatives to central intermediates, such as protocatechuate or ca- matics by developing catabolic pathways that can be used for energy techol (peripheral pathway) (Figs. 2 and 3) [36,68]. It is well docu- production [65–67]. Lignin valorization mainly involves three steps, mented that in some cases these intermediates are further degraded via including lignin depolymerization, aromatics degradation, and target the β-ketoadipate pathway [69]. Protocatechuic acid and catechol are product biosynthesis [24]. Each step has its signiﬁcance in breaking typical substrates for both extradiol and intradiol cleavages in bacteria,

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Fig. 3. The aromatic ring cleavage and the biosynthesis of triacylglycerols (TAG) and polyhydroxyalkanoate (PHA) in biological lignin valorization [24]. and these compounds are key nodes in aromatic degradation [70]. protocatechuic acid that is observed in soil bacteria that degrade lignin These intermediates undergo ring cleavage and then further trans- compounds via non-heme iron-dependent catechol dioxygenase en- formed into metabolites for the tricarboxylic acid (TCA) cycle (central zymes. The cleavage of aromatic compounds highly depends on the pathway) via the β-ketoadipate pathway, ultimately resulting in acetyl- redox state of ligninolytic enzymes [3,74]. As the dominant linkage, the CoA [71]. Internal products from microorganisms cultivated on lignin- degradation of the β-aryl ether bond is known to occur predominantly derived substances include fatty acids, triacylglycerols (TAG), PHA, etc. via Cα-Cβ oxidative cleavage by lignin peroxidase in white-rot fungi (such as P. chrysosporium) and bacteria (such as S. paucimobilis), re- 3.1. Lignin fragmentation routes and common intermediates sulting in vanillin as a product [75]. Another catabolic pathway of the β-aryl ether bond in bacteria is to initially oxidize the α-hydroxyl group In lignin valorization, lignin polymers in plant biomass need to be to a ketone followed by the glutathione-dependent β-etherase enzyme primarily broken down via thermal, chemical, physical and biological dependent reductive ether cleavage to form another ketone product routes [1,17,19,72,73]. In the biological route lignin is fragmented into that in ﬁnally converted to vanillic acid through the oxidation of the G- oligomeric lignin-derived compounds or directly depolymerized into a lignin hydroxyl group [43]. large slate of oxygenated aromatic compounds through non-enzymatic Soil bacteria are capable of degrading biphenyl components via reactions by multiple enzymes, such as laccases, peroxidases, and ad- oxidation to 2,3-dihydroxybiphenyl, producing 2,2′-dihydroxy-3,3′-di- ditional oxidative enzymes from fungi and bacteria. As a result of the methoxy,-5,5′-dicarboxybiphenyl. Following the demethylation of one abundance of aromatic compounds, ligninolytic microorganisms have methoxy group in aromatics via a non-heme demethylase enzyme LigX, evolved various catabolic pathways to utilize these carbon sources. the catacholic product is subjected to an oxidative meta-cleavage via Some lignin fragmentation routes produce intermediates like va- the extradiol dioxygenase LigZ, and cleaved by C-C hydrolase LigY to nillin and vanillic acid from the lignin polymer which are then transform 4-carboxy-2-hydroxypentadienoic acid and 5-carboxyvanillic acid, formed to protocatechuate (peripheral pathway) and eventually con- which would be converted into the central intermediate vanillic acid verted into metabolites for the TCA cycle (central pathway) by yielding through two decarboxylase enzymes LigW and LigW2 [76–79]. acetyl-CoA via the β-ketoadipate pathway (Fig. 2). Degradation of Diarylpropane degradation pathway in fungi is similar to the me- lignin and/or aromatic compounds is shown to converge at vanillin and tabolism of the β-aryl ether bond. P. chysosporium have the ability to vanillic acid in the pathway. Vanillic acid results from carboxylation to secrete speciﬁc lignin peroxidases to cleave the Cα-Cβ bond, yielding

353 Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362 aromatic aldehyde products [80,81]. Bacteria catabolizing a diaryl- Frimicutes: Bacillus sp. and Paenibacillus sp. All of these organisms have propane model lignin compound was also identified in P. paucimobilis been shown to mineralize milled wood and changes in the lignin con- TMY1009 [82]. Breakdown starts with a fragmentation reaction; tent have been observed through colorimetric assays. In addition, namely, Cγ-elimination to generate a stilbene-type intermediate. After Gram-negative Protobacteria, such as: Sphingobium SYK-6, Ochrobactum this, a unique dioxygenase designated as lignostilbene-α, β-dioxygenase sp., Cupriavidus basiliiensis, Comamonas sp. B-9, P. putida mt-2, P. putida can catabolize its Cα-Cβ linkage to produce two equivalents of vanillin. KT2440, Acinetoacter sp., Enterobacter lignolyticus, Citrobacter freundii There are different pathways to break down the phenylcoumarane and Bacteroidetes Shingobacterium sp. T2 have similarly shown evidence component of lignin. Fungi such as Fusarium solani M-13-1 have the of lignin degradation [65]. Although initial investigation has identified ability to break down the Cα-Cβ bond to give 5-acetylvanillone, and a large diversity of lignin and aromatic degradation pathways, it is then yield an epoxide intermediate and cleave the Cα-Cβ bond [83]. S. likely that there is still a plethora of pathways and enzymes that remain paucimobilis SYK-6 have been reported to consume phenylcoumarane unknown. With continued screening analyses, useful genotypes and lignin model compounds [78,84]. There is some speculation that this phenotypes can be discovered to improve biological valorization of bacterium may use lignostillbene dioxygenase for the break-down of lignin, in the effort to generate value-added products. The expansion of phenylcoumarane, finally generating the diaryl propane skeleton. Both bioprospecting will also increase knowledge of lignin-degrading en- of these two pathways may convert G-phenylcoumarane compounds vironments and microbial communities to improve genetic databases into vanillic acid [84,85]. and biochemical assays for studying metabolism of lignin and aromatic compounds. Several organisms exist that may be proved to be potential 3.2. Aromatic ring cleave and product biosynthesis pathways powerhouse cell factories to generate biofuels and chemicals from lignin. After a series of fragmentation pathways by “upper pathways” [36,86], aromatic compounds are converted into several common 4.1. Rhodococcus central intermediates, such as protocatechuate and catechol, which are the archetypal substrates for aromatic ring opening enzymes (Fig. 3) The Gram-positive and oleaginous Rhodococcus genus has been ex- [3,24,70,87]. Ring-opening dioxygenases have the ability to open the tensively investigated due to its ability to accumulate intracellular li- aromatic rings either via intra- or extra-diol (ortho or meta) cleavages. pids > 20% based on cell dry weight (CDW) (Table 1) [9,92–94]. For The products from the ring-cleavage of aromatics are then converted example, R. opacus PD 630 exhibits oleaginicity above 80% based on via ‘lower pathways’ to central carbon metabolism intermediates [86]. CDW using glucose as a carbon source with limited nitrogen [95,96]. Aromatic compounds are degraded via the β-ketoadipate pathway and When lignin related compounds are utilized, the wild-type species have converted into acetyl-CoA, which is a key metabolite connecting aro- also shown the ability to effectively convert aromatic compounds. The matic catabolism with many anabolic pathways. Acetyl-CoA is the degradation of aryl units observed in lignin is made possible by the β- starting compound of the TCA cycle, and it is also a key precursor for ketoadipate pathway [97]. When benzoate derivatives are employed as the biosynthesis of fatty acids, lipids, polyketides, and amino acids. P. analogues of lignin depolymerization products, R. jostii strain demon- putida and Acinetobacter calcoaceticus convert protocatechuic acid into strate the ability to accumulate over 55% TAG based on CDW [98]. 3-oxoadipate using 3,4-dioxygenase in the ortho-cleavage pathway. 3- Furthermore, R. opacus DSM 1069 and PD 630 strains have been shown oxoadipate can eventually be utilized in the TCA cycle at the end of the to use the β-ketoadipate pathway to convert ethanol organosolv lignin β-ketoadipate catabolism as acetyl-CoA and succinyl-CoA [70].InS. with or without ultrasonicated treatment to lipids. However, the con- paucimobilis SYK-6, protocatechuic acid is degraded by meta-cleavage version is limited to < 5.0% CDW, which is suggested to be the result of using 4,5-dioxygenase enzyme LigAB eventually yielding oxaloacetate the recalcitrant nature of lignin [9]. R. opacus DSM 1069 is also re- and pyruvate [88]. Various attempts have been made to increase acetyl- ported to utilize organosolv pretreated pine as a sole carbon source for CoA synthesis. Over-expression of a heterologous-acetyl-CoA synthetase producing lipids [99]. Under the conditions of 120 h fermentation at gene and endogenous ALD genes (Salmonella enterica ACS L641P) has 1.5% (w/v) solid concentration, R. opacus DSM 1069 accumulates improved the production of a wide range of acetyl-CoA dependent 26.9% of its cellular dry weight in lipids mainly containing oleic, pal- products by four-fold [89,90]. mitic, and stearic fatty acids [99]. The bioconversion of lignin-rich All the aforementioned aromatic catabolism has been studied in biorefinery wastes such as Kraft lignin and oxygen pretreatment Kraft aerobic bacteria; while the degradation mechanism of lignin and aro- lignin (O2-KL) were compared for lipid production using R opacus DSM matic compounds in anaerobic bacteria is still relatively unexplored. 1069 [93]. R. opacus DSM 1069 was shown to be capable of consuming

Generally, aromatic catabolism in anaerobic bacteria generates ben- O2-KL for lipid accumulation. The maximum lipid yield is 0.067 mg/mL zoyl-CoA as a central intermediate, which is then degraded by ring- at 36 h fermentation, while these lipids mainly consist of 42.7% stearic reducing enzymes via central pathways. In anaerobic pathways, dear- and 46.9% palmitic acids [93]. The synergistic degradation of lignin by omatization proceeds by using reductive CoA thioesters [68,91]. In- the combination of a bacterial and ligninolytic enzymes have been re- termediates, e.g. benzoyl-CoA, can be further converted using ATP- ported [41]. The cell growth of R. opacus increases exponentially in dependent or ATP-independent reductases to enable aromatic ring- response to the level of laccase treatment. The combination of R. opacus opening and β-oxidation-like reactions to generate central inter- PD630 cell and laccase fermentation produces a 17-fold increase in mediates. lipid accumulation, suggesting the synergistic effects between laccase and bacterial cells in lignin degradation. Confirmed by NMR analysis, 4. Bioprospecting of ligninolytic microorganisms and enzymes R. opacus and laccase can degrade lignin synergistically due to the effective utilization of aromatic monomers produced by laccase to pro- Bioprospecting shows the greatest potential for identifying unique mote the depolymerization of lignin [41]. ligninolytic microorganisms and enzymes from different habitats to produce high yield of lignin-based products. Using bioinformatics to 4.2. Pseudomonas putida search for ligninolytic enzymes and aromatic degradation pathways in bacteria has showed that proteobacteria and actinobacteria exhibit high Previous studies confirmed that P. putida also has the ability to proportions of lignin-degrading genes [65]. These bacteria include the degrade aromatic compounds to produce target products by the aro- Gram-positive Actinobacteria: Streptomyces viridosporus, Streptomyces matic metabolic pathways [57]. Under nitrogen limited conditions, P. coelicolor, Amycolatoposis sp. 75iv2, R. jostii RHA1, R. erythropolis, No- putida can use alkaline pretreated lignin as its carbon source to accu- cardia autorphica, Microbacterium phyllospharae, Micrococcus sp. and mulate PHA [36]. P. putida KT2440 has been reported to consume both

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model compounds and lignin-rich biorefinery residues to generate medium chain-length polyhydroxyalkanoates (mcl-PHAs). To further exploit their utility, the resulting mcl-PHAs are catalytically trans- Reference [100] [100,101] [102] [41] [41] [9] [103] [24] [36] [38] [19] formed to fuel-range hydrocarbons and chemical precursors [36].Me- tabolic engineering has been applied to P. putida to produce cis,cis- muconic acid through aromatic degradation pathway, by blocking the degradation pathway of protocatechuate and re-routing to catechol degradation [37]. Using catalytic hydrogenation cis,cis-muconic acid can be transformed to adipic acid, a precursor of nylon, which presents a potential route to produce adipic acid from lignin [37]. Yield of pyruvate, an important precursor of the TCA cycle, can be increased in P. putida by exchanging genes between the catechol intradiol and ex- PHA yield 0.25 g PHA/L, 32% PHA (w/w 1.0 g PHA/L, 19% PHA (w/w 73% PHA (w/w CDW) CDW) CDW) tradiol pathways [104]. Protocatechuate 3,4-dioxygenase inhibitor has also been developed as a potential approach to directing lignin degradation [105]. A systems biology study analyzed the lignin degradation mechanisms in P. putida to further enhance the PHA production [38]. P. putida A514 strain was identified, after extensive screening, to contain a dye peroxidase-based enzymatic system for lignin depolymerization, which is overexpressed, facilitating the cell growth on the medium containing Kraft lignin. The dye peroxidase (DyP) class is shown to be associated with oxidative cleavage of Cα-Cβ 45.8% TAG (w/w CDW) 0.33 g lipid/g CDW Lipid yield 54% TAG (w/w CDW) 0.004 g lipid/g US-EOL, 8.5 mg lipid/L 145 mg lipid/L 20 mg/L 1.01 g lipid/L and capacity for β-O-4 cleavage. Furthermore, key enzymes are overexpressed to employ peripheral and central catabolism pathways for aromatic compound metabolism. β-Oxidation products are rechanneled by up-regulated enzymes, while the synthesis of fatty acid is down- regulated, resulting in 73% CDW of PHA content using lignin and vanillic acid as carbon sources [38]. erent pretreatments erent pretreatments 1.83 g lipid/L ff ff 4.3. Yeasts

Oleaginous yeasts have been shown to contain aromatic degradation pathways and as an added beneﬁt they are also able accumulate TAG or PHA [9,40], just like Rhodococci and other bacteria. Yeast is more effective than microalgae and bacteria in the ability to accumulate lipids [106,107]. Previous studies reported that the accumulation of lipids in Carbon source Corn stover hydrolysate Alkali-extracted corn stover lignin Kraft lignin Kraft lignin Ultrasonicated EOL Solubilized lignin from laccasetreatment -mediator Alkaline pretreated lignin Fractioned lignin with di Fractioned lignin with di Lignin, vanillic acid oleaginious yeast such as Cryptococcus curvatus, Cryptococcus albidus, Lipomyces tetrasporus, Lipomyces starkeyi, Trichosporon pullulans, and Rhodotorula glutinis can reach more than 65% of CDW, under speciﬁc conditions [108]. Y. lipolytica has been considered as a model oleagi-

gene library Kraft hardwood hydrolysate nous yeast strain that has been used extensively for studies in heterologous protein synthesis, lipid accumulation, and degradation steps [109]. It has the ability to consume a variety of substrates such as al- kanes, poly-alchols, carotenoids, and aromatics. In addition, it is lauded Streptomyces padanus for its superior ability to produce oils and fatty acid-derived com-

ciations ﬀ

from pounds. Extensive e orts have been put into making Y. lipolytica cap- ﬁ

Streptomyces padanus able of accumulating ~90% of CDW as fatty acids using glucose as a xylB carbon source, reaching 85% of the theoretical yield. Y. lipolytica shows

and the potential to be used as a platform for the single-cell oils production Clone from Genetic modi xylA expressed VanA- n/a n/a n/a and the fatty acids bioconversion [110]. Thus, the use of oleaginous yeasts presents a potential alternative route for biofuels and green chemical production. However, economic proﬁtability is hindered, due to high production costs, despite high titers, remains a challenge for the ﬁeld. Further engineering systems in yeast to utilize readily available and inexpensive aromatic carbon sources, such as lignin, need to be established.

RHA1 VanA- 4.4. Synergistic metabolic activities on lignin-derived aromatic compounds through microbial consortium

)/R. jostii Considering the relatively weak ability of lignin degradation by select bacteria, the synergistic metabolic activities of microbial communities oﬀer considerable potential to depolymerize and degrade Xsp8 MITXM-61 PD630 (WT) PD630 (WT) + laccasse DSM 1069 (WT) A514 PD 630 (WT PD 630 PD 630 –

KT2440 KT2440 lignin for its valorization [111 113]. Microbial community shows a potential for solving the challenges in lignin valorization because of its cofermentation low cost and economic security. Biological valorization of lignin or R. opacus Strains R. opacus R.opacus R. opacus R. opacus R. opacus R.opacus R.opacus P.putida P. putida P.putida

Table 1 Genetically-engineered microorganism with improved lipid or polyhydroxyalkanoate (PHA) yield. Note: CDW represents cell dry weight; PHA represents polyhydroxyalkanoate; TAG represents triacylglycerol;US-EOL represents ultrasonicated ethanol organosolv lignin; WT represent wild type. aromatic compounds is a complex process with the consociation of a

355 Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362 variety of ligninolytic enzymes. It is assumed white rot fungi secrete pathways of lignin. An extracellular peroxidase is capable of the lignin phenol oxidases in response to environmental effects for first degrading degradation, while several other enzymes are closely related to the native lignin from plant biomass in soil, while bacteria play a major role degradation of β-aryl ether bond. Genes encoding enzymes are re- in the mineralization of lignin derivatives with low molecular weight. sponsible for the conversion of ferulic acid, which is a common inter- Due to the better environmental adaptability, bacteria possess ad- mediate of lignin degradation [121]. Masai et al. found that the com- vantages as compared to fungi. plete nucleotide sequence of Sphingobium sp. SYK-6 contains a A structurally stable consortium capable of efficiently degrading 4,199,332-bp-long chromosome and a 148,801-bp-long plasmid. This plant biomass substrates is established due to its lignocellulolytic en- strain is able to convert biaryls and monoaryls, and the corresponding zyme system [114]. By successive subcultivation on the medium con- catabolic genes are very helpful for producing valuable metabolites taining rice straw, a microbial consortium WCS-6 has been proven to from the lignin [122]. To understand the molecular and genomic me- degrade filter paper, cotton and rice straw by 99.0%, 76.9% and 81.3%, chanisms of the selective ligninolysis, the comparative genomic analysis respectively, after 3 days of cultivation [115]. Subsequently, the mi- of P. chrysosporium and C. subvermispora was carried out [123].C. crobial consortium is successfully screened to directly consume lignin, subvermispora genome encodes three times as many MnP genes as P. which converted 60.9% lignin at 30 °C after 15 days static culture chrysosporium. The genome of C. subvermispora contains more than 7 [116]. To obtain microbial consortia for effective lignin degradation, genes encoding laccases for lignin degradation, while the genome of P. the microbial consortium DM-1 has been isolated through successive chrysosporium contains none. The number of desaturase-encoding genes fermentation for over 5 generations [117]. High degradation selectivity is expanded and may be involved in lipid synthesis in C. subvermispora. by microbial consortium DM-1 is observed within 16-day fermentation. Furthermore, several upregulated desaturase and MnP genes are ob- The diversity analysis of the microbial consortium DM-1 indicates that served by complementary transcriptome analysis, indicating the im- Achromobacter, Cellulosimicrobium, Mesorhizobium, Stenotrophomones portant role of MnP in lignin degradation. These results also support a and Pandoraea are the predominant genera [117].Aneffective micro- certain mechanism of lignin degradation in which lipid peroxidation bial consortium that contains two inter-kingdom fusants (PE-9 and Xz6- products may mediate the cleavage of the dominant non-phenolics. 1) and two indigenous bacteria (Bacillus sp. (B) and P. putida (Pp)) was Results suggested that C. subvermispora has an genetic inventory and established for lignin degradation [118]. P. putida (Pp) and PE-9 and expression patterns with increased oxidoreductases for lignin de- Bacillus sp. (B) and PE-9 showed a significant interaction on the degradation [123]. Overall, genome analyses have already assessed many gradation of lignin. Results suggested that multiple microorganisms ligninolytic microorganisms and identified a catalog of genes and could generate laccase and exhibit excellent environmental adaptability pathways of lignin degradation. and capability of lignin degradation under alkaline conditions [118]. Transcriptomics analysis can provide a blueprint revealing the Bacterial consortium comprised of Citrobacter sp. Serratia marcescens, genes, pathways, and microorganisms for lignin degradation [124].To and Klebsiella pneumoniae was used to decolorize black liquor [86]. The get a far better understanding of the phenolic metabolism, over 40 ligninolytic activities of this consortium have been found to be growth generations of R. opacus were evolved using phenol as a substrate [64]. associated, reducing the color by about 85%. The decreased color in- These adapted R. opacus show ~2-fold higher lipid yields as compared tensity reveals the lignin depolymerization through the ligninolytic to the wild-type strain, while they also present higher phenol con- action of bacteria. The aromatic compounds in control are consumed by sumption rates. The upregulation of aromatic degradation pathways bacterial treatment, and the new metabolic products are generated in and putative transporters were observed by whole-genome sequencing the decolorized Kraft black liquor, reducing the toxicity by about 70% and comparative transcriptomics analysis of the adapted strains. The [86]. All of the above studies highlight the positive effects on lignin results revealed the systems-level mechanisms of the substrate uptake degradation by microbial consortium, providing a potential for over- and utilization regulation for evolved phenol tolerance [64]. These coming the challenges in lignin valorization. identified tolerance genes and pathways may be considered as potential candidates for metabolic engineering of R. opacus to improve lignin 5. Systems and synthetic biology approaches to lignin bioconversion. valorization Proteomics analysis of ligninolytic microorganisms plays a key role in revealing protein abundance and post-translational modification, Although aforementioned microorganisms have potential to be both of which are important for protein activity and functionality hosts for lignin bioconversion, the understanding of lignin degradation [125,126]. Proteomics analysis of Arthrobacter phenanthrenivorans pathways and targeted product biosynthesis pathways are far from Sphe3 has been conducted to investigate its metabolic adaptation on being complete. The systems biology analysis of ligninolytic micro- medium containing phthalate, phenanthrene and glucose as carbon organisms, the mechanisms of lignin degradation, and the identification sources [127]. Proteomics analysis identified about 33% of the total of lignin bioconversion pathway are still limited. Furthermore, lack of Sphe3 proteins predicted in the genome, which is consistent with an- synthetic biology tools render the aforementioned microorganisms notated genomic information and is helpful to explain the catabolic unsuitable as industrially-efficient hosts for lignin valorization cur- pathway of phenanthrene. Several proteins in aromatics degradation rently. Thus, there is an interesting opportunity for systems and syn- have been confirmed by identifying the ring hydroxylation and ring thetic biology in guiding microbial engineering for lignin valorization cleavage of phenanthrene to produce phthalate and further catabolism (Fig. 4). of phthalate and protocatechuate. The proteomic and transcriptional analyses also indicate the repression of catabolic genes by glucose. The 5.1. Omics analysis towards understanding the mechanisms of lignin abundance of proteins, which are related to substrate degradation, degradation stress response, and cell wall metabolism, are changed using aromatic compounds as the carbon source. The glyoxylate shunt may be acti- Systems biology using ‘omics’ technologies are emerging as essential vated by using aromatic compounds because of the up-regulated key approaches to reveal the molecular and systems-level mechanisms of enzymes [127]. lignin degradation to guide the synthetic biology design for lignin va- Polycyclic aromatic hydrocarbons (PAHs) are commonly present lorization [119,120]. The genomes analysis of ligninolytic micro- xenobiotics in contaminated soils. Proteomics analysis has been em- organisms can reveal potential mechanisms in lignin degradation. To- ployed to study the catabolism and associated proteins of three xeno- lumonas lignolytica BRL6-1T sp. nov. has been demonstrated to have the biotics (phenanthrene, naphthalene, and biphenyl) in S. chungbukense capacity of lignin utilization [121]. The genome analysis presents that DJ77 [128]. The identical changes in the intensity of 10 protein spots T. lignolytica BRL6-1T sp. nov encodes several putative degradation are observed upon exposure to three xenobiotics. Among the

356 Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362

Fig. 4. Systems biology technologies guiding the synthetic biology design to enhance the lignin conversion into biofuels, chemicals, and materials for lignin valorization. upregulated proteins, five protein spots are induced, including putative pathway. The induced ring-opening enzyme (PhaL) and β-ketoacyl CoA dehydrogenases, dioxygenases, and hydrolases. The identification of thiolase (PhaD) was observed while growing on phenylethylamine, and these major proteins facilitated mapping the multiple catabolic path- it was shown that these enzymes are related to the degradation pathway ways of phenanthrene, naphthalene, and biphenyl. A part of the initial of phenylacetate (pha). The induced homogentizate 1,2-dioxygenase diverse catabolism has been found to be converging into the catechol (HmgA) and 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) are key degradation branch. By detecting intermediates from 2,3-dihydroxy- enzymes in the degradation pathway of homogentizate and were also biphenyl degradation to pyruvate and acetyl-CoA, the study confirmed observed while growing on phenylalanine. All aromatics can induce that ring-cleavage products enter the tricarboxylic acid cycle and are alkyl hydroperoxide reductase (AphC). The aforementioned studies mineralized in S. chungbukense DJ77 [128]. These results indicate that highlighted that the proteomics analysis of ligninolytic microorganisms S. chungbukense DJ77 degrades a broad range of PAHs via multiple can complement and support the information predicted by genomic catabolic pathways. sequence analysis. Overall, proteomics analysis could effectively iden- Proteomics analysis of P. putida KT2440 was conducted by using tify a diverse set of proteins and enzymes in lignin degradation for monocyclic aromatic compounds as carbon source to analyze whether guiding the engineering of ligninolytic microorganisms. proteins related to the aromatics degradation are altered [129]. Eighty Metabolomics is used as an essential tool to analyze all metabolites unique proteins are identified in P. putida KT2440 using six organic and reveal the unique chemical fingerprints and system-level mechan- compounds as carbon sources. Benzoate induced catechol 1,2-dioxy- isms for lignin degradation. The metabolic traits of Sphingobium sp. genase (CatA) and benzoate dioxygenase (BenA, BenD). p-Hydro- SYK-6 has been investigated by using 13C metabolic flux analysis, 13C- xybenzoate and vanillin induced protocatechuate 3,4-dixoygenase fingerprinting, and RNA-sequencing differential expression analysis. (PcaGH). Benzoate, p-hydroxybenzoate and vanillin induced 3-ox- Sphingobium sp. SYK-6 grows well under alkaline conditions and shows oadipate enol-lactone hydrolase (PcaD) and β-ketoadipyl CoA thiolase the potential to be used as an efficient host for lignin consolidated (PcaF). Results suggested that these aromatic compounds are degraded bioprocessing [130]. The vanillin catabolic pathway has been con- by different dioxygenases as they then entered into the β-ketoadipate firmed to couple with the tetrahydrofolate-dependent C1 pathway. The

357 Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362

Fig. 5. Three scenarios of lignin utilization in the lignocellulosic ethanol biorefinery [132]. end products of aromatic compounds such as pyruvate and oxaloacetate two functional modules were designed to improve lignin degradation. A converge into the TCA cycle, and then the gluconeogenesis will be in- module for lignin depolymerization was developed by enhancing the itiated by using phosphoenolpyruvate carboxykinase. The catabolic secretion of a heterologous DyP2 enzyme, while a module for aromatic pathway of vanillin could contribute to the synthesis of NADPH and it is compound catabolism was built by over-expressing the first two en- crucial to achieve sufficient reducing equivalents for the stain growth zymes in the β-ketoadipate pathway. The functional modules promote a [130]. two-fold enhancement of cell growth and lead to more consumption of Overall, these studies have highlighted that ‘omics’ analyses in- lignin aromatics. Furthermore, using lignin and vanillic acid as carbon tegrating genomics, transcriptomics, proteomics, and metabolomics are source, the up-regulation of β-oxidation of fatty acids was observed. able to investigate all networks involved in lignin degradation in lig- Based on these results, the third functional module responsible for PHA ninolytic microorganisms. ‘Omics’ will be crucial to achieve exhaustive formation was then built by rechanneling β-oxidation products. A re- understanding of molecular and systems-level lignin degradation me- cord PHA yield of 0.73 g/g dried cell using aromatic compounds as a chanisms in microorganisms. carbon source was achieved. The incorporation of these functional modules significantly improved PHA production from lignin. The study 5.2. Synthetic biology creating the pathways to high value end products highlighted the effectiveness of systems biology-guided synthetic design of microorganisms to enhance lignin valorization [38]. Systems biology presents powerful tools to reveal the fundamental metabolic pathway of lignin. Applying this knowledge to engineer cells 6. The techno-economic analysis (TEA) perspectives using synthetic biology can in turn generate building block chemicals to be used in biofuels and materials (Fig. 4). Previous studies have con- Biological lignin valorization to bioproducts (e.g. lipids, PHA) has firmed that various value-added products can be produced and im- not been carried out at pilot, demonstration, or commercial scale. Thus, proved from lignin or aromatics using engineered lignin-degrading techno-economic analysis currently serves not only to identify primary microorganisms. Johnson and Beckham engineered P. putida to convert cost drivers of lignin valorization, but also to prioritize research di- aromatic compounds from lignin by investigating different degradation rections and project the economic impacts of the coproduction in- pathways [104]. Using an exogenous meta-cleavage pathway in P. pu- tegration of lignin-based products through process design in order to tida mt-2 to replace the endogenous catechol ortho catabolism pathway mitigate technical risk for scale-up and improve the cost competitive- in P. putida KT2440 increased the pyruvate yield by approximately 10% ness in the next generation biorefinery as a whole. from lignin aromatics. A five-fold increase in pyruvate yield was ob- Several scenarios of lignin, available for valorization to value-added served by replacing the endogenous protocatechuate ortho pathway to a products, were identified as shown in Fig. 5. In scenario 1 of the NREL meta-cleavage pathway in Sphingobium sp. SYK-6 [104]. By engineering 2011 design case [131], after the combustion of lignin-rich wastes a P. putida KT2440, the production of muconic acid from a lignin-en- 23.9% of all the energy is planned to be used for process steam or heat, riched stream has been confirmed [37]. The muconate was recovered while 52.5% would go to electricity consumption and 23.6% would be and catalytically upgraded to adipic acid. The studies confirmed that sold to the grid for revenue. In scenario 2, 23.6% of the lignin residues degradation pathways of aromatic compounds can be engineered ef- would be converted to lignin-based coproducts, while the rest lignin fectively to increase the target products yield using synthetic biology residues would be burned to produce plant steam/heat and electricity, approaches. and no extra electricity would be sold to the grid. In scenario 3, 76.1% Lin et al. investigated the degradation mechanism of lignin using of the lignin residues would be diverted to the production of lignin- multi-omics analysis technologies and then established three functional based coproducts, while the remaining 23.9% would be burned to modules for enabling a consolidated lignin utilization route in P. putida produce plant steam/heat. In scenario 3, plant electricity would be A514 [38]. P. putida A514 has been confirmed to employ an enzymatic purchased from the grid because no electricity would be produced from system based on a DyP and a variety of aromatics catabolic pathways to lignin. metabolize lignin aromatics. According to the systems biology analysis, When the coproduction plant is envisioned as a bolt-on process into

358 Z.-H. Liu, et al. Renewable and Sustainable Energy Reviews 105 (2019) 349–362 a lignocellulosic ethanol plant, additional operation units and proces- mitigating technical risk for scale-up, and thus guiding the im- sing steps in the integrated new biorefinery design will lead to higher plementation of lignin valorization and improving the cost competi- operating and overall capital costs [133]. Techno-economic analysis tiveness of biorefineries. results showed that high yields of lignin conversion to value-added Fifth, although some insights have been gained on lignin and aro- products and low costs of product separation were essential to eco- matic compound degradation in aerobic microorganisms, the catabo- nomic success of lignin valorization in biorefineries. The reduction of lism of lignin and aromatics in anaerobic bacteria is still relatively minimum ethanol selling price (MESP) was found directly affected by unknown. Many candidates of anaerobic bacteria as well as their de- the lignin-based coproduct selling price which was a direct function of gradation mechanism in lignin valorization should be further dis- plant productivity (i.e. lignin utilization and conversion). The projected covered and elaborated. minimum coproduct selling price will decrease as the technologies develop maturely over time. Thus, the advent of biorefineries that 8. Conclusions convert plant biomass into fuel ethanol will generate substantially more lignin, yet efforts are still needed to transform it into coproducts of This work provides a comprehensive summary on identifying and higher value to make a sustainable biorefinery. creating pathways of biological lignin valorization. Lignin valorization to generate bioproducts can reduce the reliance on petroleum-based 7. Future perspectives products, which will be necessary due to the limited reserves of non- renewable fossil energy. Biological lignin processing possesses the The desire to use microorganisms to convert plant biomass into ability for strain growth to generate various intermediates and the biofuels and chemicals is generating a need for microbial “biorefinery” ability for specific product biosynthesis. Ligninolytic microorganisms processes that can fuel future economies and help create sustainable have an extensive enzymatic toolbox for breaking down various lin- industries by utilizing renewable and carbon-neutral resources. Lignin kages of lignin polymer and possess peripheral pathways for converting in plant biomass has the potential to act as a renewable source for al- heterogeneous lignin derivatives to central intermediates and target ternative fuels and can thus be a source for value-added chemicals. products. Bioprospecting have expanded the knowledge of ligninolytic Recent studies have indicated that biological lignin processing is a enzymes, microorganisms and microbial communities and also im- potential approach for achieving higher lignin conversion efficiency proved databases to investigate lignin metabolism. Systems biology and can improve biorefinery sustainability by reducing the chemical analyses by integrating genomics, transcriptomics, proteomics, and use for lignin breakdown and the process costs in biorefineries. Despite metabolomics have identified more networks and metabolic pathways some progress, researches during the last decade have implied that involved in lignin degradation and will guide synthetic biology design challenges of biological lignin valorization still remain due to in- of ligninolytic microorganisms to enhance lignin valorization. The sufficient information of the lignin degradation pathway and low pro- discussion of techno-economic analysis has emphasized the essentials of duct yield in microorganisms. Based on the development of recent lignin conversion to value-added products with high yields for im- biological techniques, the following aspects can be improved for the proving the cost competitiveness in a biorefinery. Therefore, the find- implementation of biological lignin valorization. ings in the present study highlight that biological lignin valorization First, bioprospecting still needs to be expanded to exploit effective holds great potential for lignin degradation and conversion to valuable ligninolytic microorganisms and enzymes to improve biological lignin products and is also expected to hold significant market expansion processing for generating value-added products. Bioprospecting should potential, which will be crucial to make a profitable and sustainable also be necessary to discover and identify a large diversity of lignin and biorefinery in the future. aromatic degradation pathways and provide more genetic databases and biochemical assays for investigating lignin metabolism. Acknowledgements Second, the genomics of ligninolytic microorganisms and metabolic pathways for lignin degradation need to be understood in depth. This work was supported by the U.S. DOE (Department of Energy) Ligninolytic enzymatic systems and the key intermediate metabolites EERE (Energy Efficiency and Renewable Energy) BETO (Bioenergy need to be systematically characterized. Systems biology analysis can Technology Office) (grant no. 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