From Waste to Value: Recent Insights into Producing Vanillin from Lignin.

Vanillin, one of the most widely used and appreciated flavoring agents worldwide, is the main constituent of vanilla bean extract, obtained from the seed pods of various members belonging to the Orchidaceae family. Due to the great demand in the food confectionery industry, as well as in the perfume industry, medicine, and more, the majority of vanillin used today is produced synthetically, and only less than one percent of the world's vanilla flavoring market comes directly from the traditional natural sources. The increasing global demand for vanillin requires alternative and overall sustainable new production methods, and the recovery from biobased polymers, like lignin, is an environmentally friendly alternative to chemical synthesis. The present review provides firstly an overview of the different types of vanillin, followed by a description of the main differences between natural and synthetic vanillin, their preparation, the market of interest, and the authentication issues and the related analytical techniques. Then, the review explores the real potentialities of lignin for vanillin production, presenting firstly the well-assessed classical methods and moving towards the most recent promising approaches through chemical, biotechnological and photocatalytic methodologies, together with the challenges and the principal issues associated with each technique.

One-Pot Catalytic Cascade for the Depolymerization of the Lignin ß-O-4 Motif to Non-phenolic Dealkylated Aromatics.

Aromatic monomers obtained by selective depolymerization of the lignin ß-O-4 motif are typically phenolic and contain (oxygenated) alkyl substitutions. This work reveals the potential of a one-pot catalytic lignin ß-O-4 depolymerization cascade strategy that yields a uniform set of methoxylated aromatics without alkyl side-chains. This cascade consists of selective acceptorless dehydrogenation of the γ-hydroxy group, subsequent retro-aldol reaction cleaving the Cα-Cß bond followed by in situ acceptorless decarbonylation of the formed aldehydes. This three-step cascade reaction, catalyzed by an iridium(I)-BINAP complex, resulted in 75% 1,2-dimethoxybenzene from G-type lignin dimers alongside syngas (CO:H2 ≈ 1.4:1). Applying this method to a synthetic G-type polymer, 11 wt% 1,2-dimethoxybenzene was obtained. This versatile compound can be easily transformed into 3,4-dimethoxyphenol, a valuable precursor for pharmaceutical synthesis, through enzymatic catalytic approach. Moreover, the hydrodeoxygenation potential of 1,2-dimethoxybenzene offers a pathway to produce valuable cyclohexane or benzene derivatives, presenting enticing opportunities for sustainable chemical transformations without the necessity for phenolic mixture upgrading via dealkylation.

CsPbBr3 Quantum Dots Promoted Depolymerization of Oxidized Lignin via Photocatalytic Semi-Hydrogenation/Reduction Strategy.

Due to the demanding depolymerization conditions and limited catalytic efficiency, enhancing lignin valorization remains challenging. Therefore, lowering the bond dissociation energy (BDE) has emerged as a viable strategy for achieving mild yet highly effective cleavage of bonds. In this study, a photocatalytic semi-hydrogenation/reduction strategy utilizing CsPbBr3 quantum dots (CPB-QDs) and Hantzsch ester (HEH2 ) as a synergistic catalytic system was introduced to reduce the BDE of Cß -O-Ar, achieving effective cleavage of the Cß -O-Ar bond. This strategy offers a wide substrate scope encompassing various ß-O-4 model lignin dimers, preoxidized ß-O-4 polymers, and native oxidized lignin, resulting in the production of corresponding ketones and phenols. Notably, this approach attained a turnover frequency (TOF) that is 17 times higher than that of the reported Ir-catalytic system in the photocatalytic depolymerization of the lignin model dimers. It has been observed via meticulous experimentation that HEH2 can be activated by CPB-QDs via single electron transfer (SET), generating HEH2 ⋅+ as a hydrogen donor while also serving as a hole quencher. Moreover, HEH2 ⋅+ readily forms an active transition state with the substrates via hydrogen bonding. Subsequently, the proton-coupled electron transfer (PCET) from HEH2 ⋅+ to the carbonyl group of the substrate generates a Cα ⋅ intermediate.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

RB-TnSeq identifies genetic targets for improved tolerance of Pseudomonas putida towards compounds relevant to lignin conversion.

Lignin-derived mixtures intended for bioconversion commonly contain high concentrations of aromatic acids, aliphatic acids, and salts. The inherent toxicity of these chemicals places a significant bottleneck upon the effective use of microbial systems for the valorization of these mixtures. Pseudomonas putida KT2440 can tolerate stressful quantities of several lignin-related compounds, making this bacterium a promising host for converting these chemicals to valuable bioproducts. Nonetheless, further increasing P. putida tolerance to chemicals in lignin-rich substrates has the potential to improve bioprocess performance. Accordingly, we employed random barcoded transposon insertion sequencing (RB-TnSeq) to reveal genetic determinants in P. putida KT2440 that influence stress outcomes during exposure to representative constituents found in lignin-rich process streams. The fitness information obtained from the RB-TnSeq experiments informed engineering of strains via deletion or constitutive expression of several genes. Namely, ΔgacAS, ΔfleQ, ΔlapAB, ΔttgR::Ptac:ttgABC, Ptac:PP_1150:PP_1152, ΔrelA, and ΔPP_1430 mutants showed growth improvement in the presence of single compounds, and some also exhibited greater tolerance when grown using a complex chemical mixture representative of a lignin-rich chemical stream. Overall, this work demonstrates the successful implementation of a genome-scale screening tool for the identification of genes influencing stress tolerance against notable compounds within lignin-enriched chemical streams, and the genetic targets identified herein offer promising engineering targets for improving feedstock tolerance in lignin valorization strains of P. putida KT2440.

Outer membrane vesicles catabolize lignin-derived aromatic compounds in Pseudomonas putida KT2440.

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications.

Characterization of alkylguaiacol-degrading cytochromes P450 for the biocatalytic valorization of lignin.

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O-demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA, encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta-cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC, encoding the meta-cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL, encoding a CYP255A2 family P450 and an ortho-cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O-demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities (kcat/KM): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoAEP4's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin.

Hydrodeoxygenation of Oxygenates Derived from Biomass Pyrolysis Using Titanium Dioxide-Supported Cobalt Catalysts.

Bio-oil upgrading to produce biofuels and chemicals has become an attractive topic over the past decade. However, the design of cost- and performance-effective catalysts for commercial-scale production remains a challenge. Herein, commercial titania (TiO2) was used as the support of cobalt (Co)-based catalysts (Co/TiO2) due to its low cost, high availability, and practicability for commercialization in the future. The Co/TiO2 catalysts were made with two different forms of TiO2 (anatase [TiO2-A] and rutile [TiO2-R]) and comparatively evaluated in the hydrodeoxygenation (HDO) of 4-propylguaicol (4PG), a lignin-derived model compound. Both Co/TiO2 catalysts promoted the HDO of 4PG following a similar pathway, but the Co/TiO2-R catalyst exhibited a higher activity in the early stages of the reaction due to the formation of abundant Ti3+ species, as detected by X-ray photoelectron spectroscopy (XPS) and hydrogen-temperature programed reduction (H2-TPR) analyses. On the other hand, the Co/TiO2-A catalyst possessed a higher acidity that enhanced propylcyclohexane production at prolonged reaction times. In terms of reusability, the Co/TiO2-A catalyst showed a higher stability (less Co leaching) and reusability compared to Co/TiO2-R, as confirmed by transmission electron microscopy (TEM) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses. The HDO of the real bio-oil derived from pyrolysis of Leucaena leucocephala revealed that the Co/TiO2-A catalyst could convert high oxygenated aromatics (methoxyphenols, dimethoxyphenols, and benzenediols) to phenols and enhanced the phenols content, hinting at its potential to produce green chemicals from bio-feedstock.

A new approach to zip-lignin: 3,4-dihydroxybenzoate is compatible with lignification.

Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. This study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of 'zip-lignin' derived from the incorporation of DHB into poplar lignin.

Developing Rhodococcus opacus and Sphingobium sp. coculture systems for valorization of lignin-derived dimers.

Bioconversion is being regarded as a promising way for lignin valorization because it enables funneling diverse lignin components into single compounds, overcoming the heterogeneity of lignin. Although numerous lignin-derived aromatic monomers have been funneled to target compounds in previous studies, the bioconversion of low-molecular-weight lignin (LMW-lignin) fragments, for example, lignin-derived dimers, has been rarely systematically studied, impeding further conversion of lignin. In this study, coculture systems were designed and developed to funnel multiple lignin-derived dimers to cis, cis-muconate and gallate by combining lignin-derived dimers cleavage bacterium Sphingobium sp. and monomers conversion bacterium Rhodococcus opacus. With the developed coculture systems, ß-O-4 type dimer guaiacylglycerol-ß-guaiacyl ether, 4-O-5 type dimer 4,4'-dihydroxydiphenyl ether, ß-5 type dimer balanophonin, ß-ß type dimer pinoresinol, ß-1 type dimer 1,2-bis(4-hydroxy-3-methoxyphehyl)-1,3-propanediol and 5-5 type dimer 5,5'-dehydrodivanillate were converted to cis, cis-muconate. Additionally, the developed coculture systems also showed potential in conversion of lignin-derived dimers to gallate. The application of alkali lignin for cis, cis-muconate production further demonstrated the effectiveness of the designed coculture systems. Overall, the developed coculture systems are beneficial to lignin biological valorization, and also provide references for the valorization of other bio-resources.

Engineered Pseudomonas putida for biosynthesis of catechol from lignin-derived model compounds and biomass hydrolysate.

Catechol is an industrially relevant chemical with myriad applications. Its production via chemical route suffers from several drawbacks the major being a non-green and nonselective route. Currently, bio-based products using biocatalyst are gaining attention due to the growing environmental and health hazards concerns over the use of petroleum-derived feedstock. Lignocellulosic biomass serves as a promising feedstock. Lignin valorization is the demand of the current scenario which is complicated task by its complexity, heterogeneity and diversity of lignin structures posing limitations toward lignin valorization via chemical routes. There are several microorganisms that possess the ability to metabolize lignin monomers via their central metabolic pathways and this paves the way to the synthesis of a number of products. Pseudomonas putida KT2440 is one such organism and was chosen for genetic manipulations for catechol biosynthesis using lignin-derived model compounds and biomass hydrolysate stream comprising of various lignin monomers. Catechol production was engineered by diverting various lignin monomers and addressing the identified metabolic bottlenecks particularly vanillic acid accumulation toward catechol biosynthesis. The engineered strain could convert the model lignin monomers as well as monomers in the biomass hydrolysates to catechol and vanillic acid in more than 60% and 90% molar yields, respectively.

Pathway discovery and engineering for cleavage of a ß-1 lignin-derived biaryl compound.

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial ß-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a ß-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization.

Lignin valorization using biological approach.

Due to the structural complexity and recalcitrance nature of lignin, its depolymerization into monomeric units becomes one of the biggest challenges in the bioconversion of lignin into value-added products. Depolymerization of lignin produces a blend of many compounds that are problematic for isolating components in a cost-effective way. Lignin valorization using a biological approach facilitates sustainable and commercially viable biorefineries. The use of microbes for the conversion of depolymerized lignin compounds into target products can be a solution to the heterogeneity issue. Several studies have been carried out to develop robust strains that can utilize all relevant lignin-derived compounds, but constructing these strains is difficult. As an alternative, designing multiple microbes to convert a mixture of various compounds into the desired product seems realistic. This review provides an overview of lignin bioconversion using various approaches such as metabolic engineering and synthetic biology. Ligninolytic strains have a broad enzymatic machine for depolymerization of lignin and its conversion into intermediates such as catechol or protocatechuate. These intermediates can be further converted to metabolite products such as polyhydroxyalkanoates and triacylglycerol. Synthetic biology offers encouraging methodologies to construct pathways for lignin conversion and to engineer ligninolytic microbes as prospective strains for lignin bioconversion.

Toward engineering E. coli with an autoregulatory system for lignin valorization.

Efficient lignin valorization could add more than 10-fold the value gained from burning it for energy and is critical for economic viability of future biorefineries. However, lignin-derived aromatics from biomass pretreatment are known to be potent fermentation inhibitors in microbial production of fuels and other value-added chemicals. In addition, isopropyl-ß-d-1-thiogalactopyranoside and other inducers are routinely added into fermentation broth to induce the expression of pathway enzymes, which further adds to the overall process cost. An autoregulatory system that can diminish the aromatics' toxicity as well as be substrate-inducible can be the key for successful integration of lignin valorization into future lignocellulosic biorefineries. Toward that goal, in this study an autoregulatory system is demonstrated that alleviates the toxicity issue and eliminates the cost of an external inducer. Specifically, this system is composed of a catechol biosynthesis pathway coexpressed with an active aromatic transporter CouP under induction by a vanillin self-inducible promoter, ADH7, to effectively convert the lignin-derived aromatics into value-added chemicals using Escherichia coli as a host. The constructed autoregulatory system can efficiently transport vanillin across the cell membrane and convert it to catechol. Compared with the system without CouP expression, the expression of catechol biosynthesis pathway with transporter CouP significantly improved the catechol yields about 30% and 40% under promoter pTrc and ADH7, respectively. This study demonstrated an aromatic-induced autoregulatory system that enabled conversion of lignin-derived aromatics into catechol without the addition of any costly, external inducers, providing a promising and economically viable route for lignin valorization.

Effect of N2 flow rate on kinetic investigation of lignin pyrolysis.

Fast pyrolysis of lignin can obtain valuable products such as bio-oil, bio-chemical, syngas, and biochar. In this study, two types of lignin known as brown solid from the byproduct of cellulosic ethanol fermentation and commercial dealkaline lignin from the papermaking process were used for pyrolysis in a 3-L batch reactor at 300-450 °C. The product composition in the liquid and gas phases were analyzed by using gas chromatography-mass spectrometry/Flame-ionization detector/thermal conductivity detector (GC-MS/FID/TCD). Increasing the N2 flow rate to 150 mL/min was sufficient to increase the production of bio-oil/bio-organics up to 15% for brown solid pyrolysis. In contrast, the biochemical production during dealkaline lignin pyrolysis was not sensitive to the change of the N2 flow rate. The amount of biochar produced in the pyrolysis (~60%) slightly changed at various pyrolysis temperature and gas flow rate, which could be due to the relatively low pyrolysis temperature that was insufficient to decompose the lignin. The GC-MS analysis also revealed that C7-C8 compounds, which represented the phenolic compounds, were the most abundant in the liquid products. Kinetic models of the pyrolysis were established based on the thermogravimetric analysis.

Isolation of a novel platform bacterium for lignin valorization and its application in glucose-free cis,cis-muconate production.

Microbial production of cis,cis-muconate (ccMA) from phenolic compounds obtained by chemical depolymerization of lignin is a promising approach to valorize lignin. Because microbial production requires a large amount of carbon and energy source, it is desirable to establish a ccMA-producing strain that utilizes lignin-derived phenols instead of general sources like glucose. We isolated Pseudomonas sp. strain NGC7 that grows well on various phenolic compounds derived from p-hydroxyphenyl, guaiacyl, and syringyl units of lignin. An NGC7 mutant of protocatechuate (PCA) 3,4-dioxygenase and ccMA cycloisomerase genes (NGC703) lost the ability to grow on vanillate and p-hydroxybenzoate but grew normally on syringate. Introduction of a plasmid carrying genes encoding PCA decarboxylase, flavin prenyltransferase, vanillate O-demethylase, and catechol 1,2-dioxygenase into NGC703 enabled production of 3.2 g/L ccMA from vanillate with a yield of 75% while growing on syringate. This strain also produced ccMA from birch lignin-derived phenols. All these results indicate the utility of NGC7 in glucose-free ccMA production.

Lignin-Only Polymeric Materials Based on Unmethylated Unfractionated Kraft and Ball-Milled Lignins Surpass Polyethylene and Polystyrene in Tensile Strength.

Functional polymeric materials composed solely of lignin preparations appeared only very recently. A gradual paradigm shift spanning 56 years has revealed how lignin-lignin blends can upgrade the performance of 100 wt% lignin-based plastics. The view, first espoused in 1960, that lignin macromolecules are crosslinked reduces the plausibility of creating functional polymeric materials that are composed only of lignin preparations. Lignin-based materials would be much weaker mechanically if interstices remain in significant numbers between adjoining macromolecular structures that consist of rigid crosslinked chains. In 1982, random-coil features in the hydrodynamic character of kraft lignin (KL) components were evident from ultracentrifugal sedimentation equilibrium studies of their SEC behavior. In 1997, it was recognized that the macromolecular species in plastics with 85 wt% levels of KL are associated complexes rather than individual components. Finally, in 2016, the first polymeric material composed entirely of ball-milled softwood lignin (BML) was found to support a tensile strength above polyethylene. Except in its molecular weight, the BML was similar in structure to the native biopolymer. It was composed of associated lignin complexes, each with aromatic rings arranged in two domains. The inner domain maintains structural integrity largely through noncovalent interactions between cofacially-offset aromatic rings; the peripheral domain contains a higher proportion of edge-on aromatic-ring arrangements. Interdigitation between peripheral domains in adjoining complexes creates material continuity during casting. By interacting at low concentrations with the peripheral domains, non-lignin blend components can improve the tensile strengths of BML-based plastics to values well beyond those seen in polystyrene. The KL-based plastics are weaker because the peripheral domains of adjoining complexes are less capable of interdigitation than those of BML. Blending with 5 wt% 1,8-dinitroanthraquinone results in a tensile strength above that of polyethylene. Analogous effects can be achieved with 10 wt% maple γ-valerolactone (GVL) lignin which, with a structure close to the native biopolymer, imparts some native character to the peripheral domains of the KL complexes. Comparable enhancements in the behavior of BML complexes upon blending with 10 wt% ball-milled corn-stover lignin (BMCSL) result in lignin-only polymeric materials with tensile strengths well beyond polystyrene.

Characterization and use of a bacterial lignin peroxidase with an improved manganese-oxidative activity.

Peroxidases are well-known biocatalysts produced by all organisms, especially microorganisms, and used in a number of biotechnological applications. The enzyme DypB from the lignin-degrading bacterium Rhodococcus jostii was recently shown to degrade solvent-obtained fractions of a Kraft lignin. In order to promote the practical use, the N246A variant of DypB, named Rh_DypB, was overexpressed in E. coli using a designed synthetic gene: by employing optimized conditions, the enzyme was fully produced as folded holoenzyme, thus avoiding the need for a further time-consuming and expensive reconstitution step. By a single chromatographic purification step, > 100 mg enzyme/L fermentation broth with a > 90% purity was produced. Rh_DypB shows a classical peroxidase activity which is significantly increased by adding Mn2+ ions: kinetic parameters for H2O2, Mn2+, ABTS, and 2,6-DMP were determined. The recombinant enzyme shows a good thermostability (melting temperature of 63-65 °C), is stable at pH 6-7, and maintains a large part of the starting activity following incubation for 24 h at 25-37 °C. Rh_DypB activity is not affected by 1 M NaCl, 10% DMSO, and 5% Tween-80, i.e., compounds used for dye decolorization or lignin-solubilization processes. The enzyme shows broad dye-decolorization activity, especially in the presence of Mn2+, oxidizes various aromatic monomers from lignin, and cleaves the guaiacylglycerol-ß-guaiacyl ether (GGE), i.e., the Cα-Cß bond of the dimeric lignin model molecule of ß-O-4 linkages. Under optimized conditions, 2 mM GGE was fully cleaved by recombinant Rh_DypB, generating guaiacol in only 10 min, at a rate of 12.5 µmol/min mg enzyme.

Aromatic catabolic pathway selection for optimal production of pyruvate and lactate from lignin.

Lignin represents an untapped feedstock for the production of fuels and chemicals, but its intrinsic heterogeneity makes lignin valorization a significant challenge. In nature, many aerobic organisms degrade lignin-derived aromatic molecules through conserved central intermediates including catechol and protocatechuate. Harnessing this microbial approach offers potential for lignin upgrading in modern biorefineries, but significant technical development is needed to achieve this end. Catechol and protocatechuate are subjected to aromatic ring cleavage by dioxygenase enzymes that, depending on the position, ortho or meta relative to adjacent hydroxyl groups, result in different products that are metabolized through parallel pathways for entry into the TCA cycle. These degradation pathways differ in the combination of succinate, acetyl-CoA, and pyruvate produced, the reducing equivalents regenerated, and the amount of carbon emitted as CO2-factors that will ultimately impact the yield of the targeted product. As shown here, the ring-cleavage pathways can be interchanged with one another, and such substitutions have a predictable and substantial impact on product yield. We demonstrate that replacement of the catechol ortho degradation pathway endogenous to Pseudomonas putida KT2440 with an exogenous meta-cleavage pathway from P. putida mt-2 increases yields of pyruvate produced from aromatic molecules in engineered strains. Even more dramatically, replacing the endogenous protocatechuate ortho pathway with a meta-cleavage pathway from Sphingobium sp. SYK-6 results in a nearly five-fold increase in pyruvate production. We further demonstrate the aerobic conversion of pyruvate to l-lactate with a yield of 41.1 ± 2.6% (wt/wt). Overall, this study illustrates how aromatic degradation pathways can be tuned to optimize the yield of a desired product in biological lignin upgrading.

Chemical intervention in bacterial lignin degradation pathways: Development of selective inhibitors for intradiol and extradiol catechol dioxygenases.

Bacterial lignin degradation could be used to generate aromatic chemicals from the renewable resource lignin, provided that the breakdown pathways can be manipulated. In this study, selective inhibitors of enzymatic steps in bacterial degradation pathways were developed and tested for their effects upon lignin degradation. Screening of a collection of hydroxamic acid metallo-oxygenase inhibitors against two catechol dioxygenase enzymes, protocatechuate 3,4-dioxygenase (3,4-PCD) and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB), resulted in the identification of selective inhibitors D13 for 3,4-PCD (IC50 15µM) and D3 for MhpB (IC50 110µM). Application of D13 to Rhodococcus jostii RHA1 in minimal media containing ferulic acid led to the appearance of metabolic precursor protocatechuic acid at low concentration. Application of 1mM disulfiram, an inhibitor of mammalian aldehyde dehydrogenase, to R. jostii RHA1, gave rise to 4-carboxymuconolactone on the ß-ketoadipate pathway, whereas in Pseudomonas fluorescens Pf-5 disulfiram treatment gave rise to a metabolite found to be glycine betaine aldehyde.