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1. Adaptive Enrichment of a Thermophilic Bacterial Isolate for Enhanced Enzymatic Activity

   https://doi.org/10.3390/microorganisms8060871  

   Govil, Tanvi; Saxena, Priya; Samanta, Dipayan; Singh, Sindhu Suresh; Kumar, Sudhir; Salem, David R.; Sani, Rajesh K. (June 2020, Microorganisms)

   null (Ed.)

   The mimicking of evolution on a laboratory timescale to enhance biocatalyst specificity, substrate utilization activity, and/or product formation, is an effective and well-established approach that does not involve genetic engineering or regulatory details of the microorganism. The present work employed an evolutionary adaptive approach to improve the lignocellulose deconstruction capabilities of the strain by inducing the expression of laccase, a multicopper oxidase, in Geobacillus sp. strain WSUCF1. This bacterium is highly efficient in depolymerizing unprocessed lignocellulose, needing no preprocessing/pretreatment of the biomasses. However, it natively produces low levels of laccase. After 15 rounds of serially adapting this thermophilic strain in the presence of unprocessed corn stover as the selective pressure, we recorded a 20-fold increase in catalytic laccase activity, at 9.23 ± 0.6 U/mL, in an adapted yet stable strain of Geobacillus sp. WSUCF1, compared with the initial laccase production (0.46 ± 0.04 U/mL) obtained with the unadapted strain grown on unprocessed corn stover before optimization. Chemical composition analysis demonstrated that lignin removal by the adapted strain was 22 wt.% compared with 6 wt.% removal by the unadapted strain. These results signify a favorable prospect for fast, cost competitive bulk production of this thermostable enzyme. Also, this work has practical importance, as this fast adaptation of the Geobacillus sp. strain WSUCF1 suggests the possibility of growing industrial quantities of Geobacillus sp. strain WSUCF1 cells as biocatalysts on reasonably inexpensive carbon sources for commercial use. This work is the first application of the adaptive laboratory evolution approach for developing the desired phenotype of enhanced ligninolytic capability in any microbial strain. 

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

   https://doi.org/10.1073/pnas.2212246120  

   Kuatsjah, Eugene; Zahn, Michael; Chen, Xiangyang; Kato, Ryo; Hinchen, Daniel J.; Konev, Mikhail O.; Katahira, Rui; Orr, Christian; Wagner, Armin; Zou, Yike; et al (January 2023, Proceedings of the National Academy of Sciences)

   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 oferythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived β-1 dimer, was reported inNovosphingobium 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 convertserythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of theN. aromaticivoransLdpA and another homolog identified inSphingobiumsp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C₁reaction product, and we further demonstrated that both enantiomers oferythro-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. 

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3. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

   https://doi.org/10.1021/acssuschemeng.3c06266  

   DeChellis, Antonio; Nemmaru, Bhargava; Sammond, Deanne; Douglass, Jenna; Patil, Nivedita; Reste, Olivia; Chundawat, Shishir_P S (March 2024, ACS Sustainable Chemistry & Engineering)

   Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates. 

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4. Experimental–theoretical study of laccase as a detoxifier of aflatoxins

   https://doi.org/10.1038/s41598-023-27519-1  

   Zaccaria, Marco; Dawson, William; Russel Kish, Darius; Reverberi, Massimo; Bonaccorsi di Patti, Maria Carmela; Domin, Marek; Cristiglio, Viviana; Chan, Bun; Dellafiora, Luca; Gabel, Frank; et al (January 2023, Scientific Reports)

   Abstract We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB₁and AFG₂) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB₁is more challenging to degrade, to the point of complete degradation stalling; (2) AFG₂is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π–π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization. 

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5. One-pot production of oxygenated monomers and selectively oxidized lignin from biomass based on plasma electrolysis

   https://doi.org/10.1039/d1gc03315h  

   A, Lusi; Radhakrishnan, Harish; Hu, Hui; Bai, Xianglan (November 2021, Green Chemistry)

   Herein, we report a novel method to obtain oxygenated chemicals and high-quality lignin from biomass in one-pot using a single step process. Plasma electrolysis of red oak was conducted by applying high-voltage alternating current electricity in γ-valerolactone using sulfuric acid as the electrolyte. Red oak was completely solubilized to produce levoglucosenone and furfural as the two major monomers with the respective yields of up to 44.9 mol% and 98.0 mol%. During the conversion, an oxidized lignin was also simultaneously produced in high purity. The valorization potential of the plasma electrolysis-derived lignin evaluated using the pyrolysis method showed that depolymerization of this lignin could produce significantly higher yields of phenolic monomers than the natural lignin or the lignin isolated during conventional solvolysis. Our investigation showed that benzylic carbon of the natural lignin was selectively modified during plasma electrolysis to limit the formation of interunit C–C bonds, significantly improving the subsequent lignin valorization to aromatic monomers. Overall, this study demonstrated a simple green approach to improve chemical production without using costly catalysts or tedious biomass fractionation. This study also presented a novel and highly efficient way to modify lignin for enhanced valorization. 

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