Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism.

SignificanceMore than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.

Integration of pH Control into Chi.Bio Reactors and Demonstration with Small-Scale Enzymatic Poly(ethylene terephthalate) Hydrolysis.

Small-scale bioreactors that are affordable and accessible would be of major benefit to the research community. In previous work, an open-source, automated bioreactor system was designed to operate up to the 30 mL scale with online optical monitoring, stirring, and temperature control, and this system, dubbed Chi.Bio, is now commercially available at a cost that is typically 1-2 orders of magnitude less than commercial bioreactors. In this work, we further expand the capabilities of the Chi.Bio system by enabling continuous pH monitoring and control through hardware and software modifications. For hardware modifications, we sourced low-cost, commercial pH circuits and made straightforward modifications to the Chi.Bio head plate to enable continuous pH monitoring. For software integration, we introduced closed-loop feedback control of the pH measured inside the Chi.Bio reactors and integrated a pH-control module into the existing Chi.Bio user interface. We demonstrated the utility of pH control through the small-scale depolymerization of the synthetic polyester, poly(ethylene terephthalate) (PET), using a benchmark cutinase enzyme, and compared this to 250 mL bioreactor hydrolysis reactions. The results in terms of PET conversion and rate, measured both by base addition and product release profiles, are statistically equivalent, with the Chi.Bio system allowing for a 20-fold reduction of purified enzyme required relative to the 250 mL bioreactor setup. Through inexpensive modifications, the ability to conduct pH control in Chi.Bio reactors widens the potential slate of biochemical reactions and biological cultivations for study in this system, and may also be adapted for use in other bioreactor platforms.

The catabolism of lignin-derived p-methoxylated aromatic compounds by Rhodococcus jostii RHA1.

Emergent strategies to valorize lignin, an abundant but underutilized aromatic biopolymer, include tandem processes that integrate chemical depolymerization and biological catalysis. To date, aromatic monomers from C-O bond cleavage of lignin have been converted to bioproducts, but the presence of recalcitrant C-C bonds in lignin limits the product yield. A promising chemocatalytic strategy that overcomes this limitation involves phenol methyl protection and autoxidation. Incorporating this into a tandem process requires microbial cell factories able to transform the p-methoxylated products in the resulting methylated lignin stream. In this study, we assessed the ability of Rhodococcus jostii RHA1 to catabolize the major aromatic products in a methylated lignin stream and elucidated the pathways responsible for this catabolism. RHA1 grew on a methylated pine lignin stream, catabolizing the major aromatic monomers: p-methoxybenzoate (p-MBA), veratrate, and veratraldehyde. Bioinformatic analyses suggested that a cytochrome P450, PbdA, and its cognate reductase, PbdB, are involved in p-MBA catabolism. Gene deletion studies established that both pbdA and pbdB are essential for growth on p-MBA and several derivatives. Furthermore, a deletion mutant of a candidate p-hydroxybenzoate (p-HBA) hydroxylase, ΔpobA, did not grow on p-HBA. Veratraldehyde and veratrate catabolism required both vanillin dehydrogenase (Vdh) and vanillate O-demethylase (VanAB), revealing previously unknown roles of these enzymes. Finally, a ΔpcaL strain grew on neither p-MBA nor veratrate, indicating they are catabolized through the ß-ketoadipate pathway. This study expands our understanding of the bacterial catabolism of aromatic compounds and facilitates the development of biocatalysts for lignin valorization.IMPORTANCELignin, an abundant aromatic polymer found in plant biomass, is a promising renewable replacement for fossil fuels as a feedstock for the chemical industry. Strategies for upgrading lignin include processes that couple the catalytic fractionation of biomass and biocatalytic transformation of the resulting aromatic compounds with a microbial cell factory. Engineering microbial cell factories for this biocatalysis requires characterization of bacterial pathways involved in catabolizing lignin-derived aromatic compounds. This study identifies new pathways for lignin-derived aromatic degradation in Rhodococcus, a genus of bacteria well suited for biocatalysis. Additionally, we describe previously unknown activities of characterized enzymes on lignin-derived compounds, expanding their utility. This work advances the development of strategies to replace fossil fuel-based feedstocks with sustainable alternatives.

Molecular simulation of lignin-related aromatic compound permeation through gram-negative bacterial outer membranes.

Lignin, an abundant aromatic heteropolymer in secondary plant cell walls, is the single largest source of renewable aromatics in the biosphere. Leveraging this resource for renewable bioproducts through targeted microbial action depends on lignin fragment uptake by microbial hosts and subsequent enzymatic action to obtain the desired product. Recent computational work has emphasized that bacterial inner membranes are permeable to many aromatic compounds expected from lignin depolymerization processes. In this study, we expand on these findings through simulations for 42 lignin-related compounds across a gram-negative bacterial outer membrane model. Unbiased simulation trajectories indicate that spontaneous crossing for the full outer membrane is relatively rare at molecular simulation timescales, primarily due to preferential membrane partitioning and slow diffusion within the lipopolysaccharide layer within the outer membrane. Membrane partitioning and permeability coefficients were determined through replica exchange umbrella sampling simulations to overcome sampling limitations. We find that the glycosylated lipopolysaccharides found in the outer membrane increase the permeation barrier to many lignin-related compounds, particularly the most hydrophobic compounds. However, the effect is relatively modest; at industrially relevant concentrations, uncharged lignin-related compounds will readily diffuse across the outer membrane without the need for specific porins. Together, our results provide insight into the permeability of the bacterial outer membrane for assessing lignin fragment uptake and the future production of renewable bioproducts.

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.

Stereoinversion via Alcohol Dehydrogenases Enables Complete Catabolism of ß-1-Type Lignin-Derived Aromatic Isomers.

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened ß-1-type dimer. Recently, LdpA-mediated catabolism of erythro-DGPD was reported in SYK-6, but the catabolic pathway for threo-DGPD was as yet unknown. Here, we elucidated the catabolism of threo-DGPD, which proceeds through conversion to erythro-DGPD. When threo-DGPD was incubated with SYK-6, the Cα hydroxy groups of threo-DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of ß-O-4-type dimers. Analysis of seven candidate genes revealed that NAD+-dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro-DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA-neighboring SDR genes. The gene products of SLG_12690 (ldpC) and SLG_12640 (ldpB) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four ß-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo-DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with ß-1 linkages. Therefore, elucidating the catabolic system of ß-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo-DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro-DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native ß-1-type dimer catabolic system.

Molecular mechanism of the chitinolytic peroxygenase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper-oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper-oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO-Cu(II) to LPMO-Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO-Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

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.

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 and engineering of a two-enzyme system for plastics depolymerization.

Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics.

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases.

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG). Despite the critical importance of GH7 enzymes, there remain gaps in our understanding of how GH7 sequence and structure relate to function. Here, we employed machine learning to gain data-driven insights into relationships between sequence, structure, and function across the GH7 family. Machine-learning models, trained only on the number of residues in the active-site loops as features, were able to discriminate GH7 CBHs and EGs with up to 99% accuracy, demonstrating that the lengths of loops A4, B2, B3, and B4 strongly correlate with functional subtype across the GH7 family. Classification rules were derived such that specific residues at 42 different sequence positions each predicted the functional subtype with accuracies surpassing 87%. A random forest model trained on residues at 19 positions in the catalytic domain predicted the presence of a CBM with 89.5% accuracy. Our machine learning results recapitulate, as top-performing features, a substantial number of the sequence positions determined by previous experimental studies to play vital roles in GH7 activity. We surmise that the yet-to-be-explored sequence positions among the top-performing features also contribute to GH7 functional variation and may be exploited to understand and manipulate function.

Structural and functional analysis of lignostilbene dioxygenases from Sphingobium sp. SYK-6.

Lignostilbene-α,ß-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from ß-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼106 M-1 s-1) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity.

Redesigned Hybrid Nylons with Optical Clarity and Chemical Recyclability.

Aliphatic polyamides, or nylons, are typically highly crystalline and thermally robust polymers used in high-performance applications. Nylon 6, a high-ceiling-temperature (HCT) polyamide from ε-caprolactam, lacks expedient chemical recyclability, while low-ceiling temperature (LCT) nylon 4 from pyrrolidone exhibits complete chemical recyclability, but it is thermally unstable and not melt-processable. Here, we introduce a hybrid nylon, nylon 4/6, based on a bicyclic lactam composed of both HCT ε-caprolactam and LCT pyrrolidone motifs in a hybridized offspring structure. Hybrid nylon 4/6 overcomes trade-offs in (de)polymerizability and performance properties of the parent nylons, exhibiting both excellent polymerization and facile depolymerization characteristics. This stereoregular polyamide forms nanocrystalline domains, allowing optical clarity and high thermal stability, however, without displaying a melting transition before decomposition. Of a series of statistical copolymers comprising nylon 4/6 and nylon 4, a 50/50 copolymer achieves the greatest synergy in both reactivity and polymer properties of each homopolymer, offering an amorphous nylon with favorable properties, including optical clarity, a high glass transition temperature, melt processability, and full chemical recyclability.

Passive membrane transport of lignin-related compounds.

Lignin is an abundant aromatic polymer found in plant secondary cell walls. In recent years, lignin has attracted renewed interest as a feedstock for bio-based chemicals via catalytic and biological approaches and has emerged as a target for genetic engineering to improve lignocellulose digestibility by altering its composition. In lignin biosynthesis and microbial conversion, small phenolic lignin precursors or degradation products cross membrane bilayers through an unidentified translocation mechanism prior to incorporation into lignin polymers (synthesis) or catabolism (bioconversion), with both passive and transporter-assisted mechanisms postulated. To test the passive permeation potential of these phenolics, we performed molecular dynamics simulations for 69 monomeric and dimeric lignin-related phenolics with 3 model membranes to determine the membrane partitioning and permeability coefficients for each compound. The results support an accessible passive permeation mechanism for most compounds, including monolignols, dimeric phenolics, and the flavonoid, tricin. Computed lignin partition coefficients are consistent with concentration enrichment near lipid carbonyl groups, and permeability coefficients are sufficient to keep pace with cellular metabolism. Interactions between methoxy and hydroxy groups are found to reduce membrane partitioning and improve permeability. Only carboxylate-modified or glycosylated lignin phenolics are predicted to require transporters for membrane translocation. Overall, the results suggest that most lignin-related compounds can passively traverse plant and microbial membranes on timescales commensurate with required biological activities, with any potential transport regulation mechanism in lignin synthesis, catabolism, or bioconversion requiring compound functionalization.

The dissociation mechanism of processive cellulases.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Nanomechanics of cellulose deformation reveal molecular defects that facilitate natural deconstruction.

Technologies surrounding utilization of cellulosic materials have been integral to human society for millennia. In many materials, controlled introduction of defects provides a means to tailor properties, introduce reactivity, and modulate functionality for various applications. The importance of defects in defining the behavior of cellulose is becoming increasingly recognized. However, fully exploiting defects in cellulose to benefit biobased materials and conversion applications will require an improved understanding of the mechanisms of defect induction and corresponding molecular-level consequences. We have identified a fundamental relationship between the macromolecular structure and mechanical behavior of cellulose nanofibrils whereby molecular defects may be induced when the fibrils are subjected to bending stress exceeding a certain threshold. By nanomanipulation, imaging, and molecular modeling, we demonstrate that cellulose nanofibrils tend to form kink defects in response to bending stress, and that these macromolecular features are often accompanied by breakages in the glucan chains. Direct observation of deformed cellulose fibrils following partial enzymatic digestion reveals that processive cellulases exploit these defects as initiation sites for hydrolysis. Collectively, our findings provide a refined understanding of the interplay between the structure, mechanics, and reactivity of cellulose assemblies.

Enabling microbial syringol conversion through structure-guided protein engineering.

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Characterization of aromatic acid/proton symporters in Pseudomonas putida KT2440 toward efficient microbial conversion of lignin-related aromatics.

Pseudomonas putida KT2440 (hereafter KT2440) is a well-studied platform bacterium for the production of industrially valuable chemicals from heterogeneous mixtures of aromatic compounds obtained from lignin depolymerization. KT2440 can grow on lignin-related monomers, such as ferulate (FA), 4-coumarate (4CA), vanillate (VA), 4-hydroxybenzoate (4HBA), and protocatechuate (PCA). Genes associated with their catabolism are known, but knowledge about the uptake systems remains limited. In this work, we studied the KT2440 transporters of lignin-related monomers and their substrate selectivity. Based on the inhibition by protonophores, we focused on five genes encoding aromatic acid/H+ symporter family transporters categorized into major facilitator superfamily that uses the proton motive force. The mutants of PP_1376 (pcaK) and PP_3349 (hcnK) exhibited significantly reduced growth on PCA/4HBA and FA/4CA, respectively, while no change was observed on VA for any of the five gene mutants. At pH 9.0, the conversion of these compounds by hcnK mutant (FA/4CA) and vanK mutant (VA) was dramatically reduced, revealing that these transporters are crucial for the uptake of the anionic substrates at high pH. Uptake assays using 14C-labeled substrates in Escherichia coli and biosensor-based assays confirmed that PcaK, HcnK, and VanK have ability to take up PCA, FA/4CA, and VA/PCA, respectively. Additionally, analyses of the predicted protein structures suggest that the size and hydropathic properties of the substrate-binding sites of these transporters determine their substrate preferences. Overall, this study reveals that at physiological pH, PcaK and HcnK have a major role in the uptake of PCA/4HBA and FA/4CA, respectively, and VanK is a VA/PCA transporter. This information can contribute to the engineering of strains for the efficient conversion of lignin-related monomers to value-added chemicals.

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.