Laccases from white-rot fungi catalyze lignin depolymerization, a critical first step to upgrading lignin to valuable biodiesel fuels and chemicals. In this study, a wildtype laccase from the basidiomycete Fomitiporia mediterranea (Fom_lac) and a variant engineered to have a carbohydrate-binding module (Fom_CBM) were studied for their ability to catalyze cleavage of β-O-4′ ether and C–C bonds in phenolic and non-phenolic lignin dimers using a nanostructure-initiator mass spectrometry-based assay. Fom_lac and Fom_CBM catalyze β-O-4′ ether and C–C bond breaking, with higher activity under acidic conditions (pH < 6). The potential of Fom_lac and Fom_CBM to enhance saccharification yields from untreated and ionic liquid pretreated pine was also investigated. Adding Fom_CBM to mixtures of cellulases and hemicellulases improved sugar yields by 140% on untreated pine and 32% on cholinium lysinate pretreated pine when compared to the inclusion of Fom_lac to the same mixtures. Adding either Fom_lac or Fom_CBM to mixtures of cellulases and hemicellulases effectively accelerates enzymatic hydrolysis, demonstrating its potential applications for lignocellulose valorization. We postulate that additional increases in sugar yields for the Fom_CBM enzyme mixtures were due to Fom_CBM being brought more proximal to lignin through binding to either cellulose or lignin itself.
The plant polymer lignin is the most abundant renewable source of aromatics on the planet and conversion of it to valuable fuels and chemicals is critical to the economic viability of a lignocellulosic biofuels industry and to meeting the DOE’s 2022 goal of $\$2.50$/gallon mean biofuel selling price. Presently, there is no efficient way of converting lignin into valuable commodities. Current biological approaches require mixtures of expensive ligninolytic enzymes and engineered microbes. This project was aimed at circumventing these problems by discovering commensal relationships among fungi and bacteria involved in biological lignin utilization and using this knowledge to engineer microbial communities capable of converting lignin into renewable fuels and chemicals. Essentially, we aimed to learn from, mimic and improve on nature. We discovered fungi that synergistically work together to degrade lignin, engineered fungal systems to increase expression of the required enzymes and engineered organisms to produce products such as biodegradable plastics precursors.
The generating value from lignin through depolymerization and biological conversion to valuable fuels, chemicals, or intermediates has great promise but is limited by several factors including lack of cost-effective depolymerization methods, toxicity within the breakdown products, and low bioconversion of the breakdown products. High yield depolymerization of natural lignins requires cleaving carbon-carbon bonds in addition to ether bonds. To address that need, we report that a chelator-mediated Fenton reaction can efficiently cleave C-C bonds in sulfonated polymers at or near room temperature, and that unwanted repolymerization can be minimized through optimizing reaction conditions. This method was used to depolymerize lignosulfonate from Mw = 28,000 g/mol to Mw = 800 g/mol. The breakdown products were characterized by SEC, FTIR and NMR and evaluated for bioavailability. The breakdown products are rich in acid, aldehyde, and alcohol functionalities but are largely devoid of aromatics and aliphatic dienes. A panel of nine organisms were tested for the ability to grow on the breakdown products. Growth at a low level was observed for several monocultures on the depolymerized LS in absence of glucose. Much stronger growth was observed in the presence of 0.2% glucose and for one organism we demonstrate doubling of melanin production in the presence of depolymerized LS. The results suggest that this chelator-mediated Fenton method is a promising new approach for biological conversion of lignin into higher value chemicals or intermediates.
Ing, Nicole; Deng, Kai; Chen, Yan; Aulitto, Martina; Gin, Jennifer W.; Pham, Thanh L.; Petzold, Christopher J.; Singer, Steve W.; Bowen, Benjamin; Sale, Kenneth L.; Simmons, Blake A.; Singh, Anup K.; Adams, Paul D.; Northen, Trent R.
Lignocellulosic biomass is composed of three major biopolymers: cellulose, hemicellulose and lignin. Analytical tools capable of quickly detecting both glycan and lignin deconstruction are needed to support the development and characterization of efficient enzymes/enzyme cocktails. Previously we have described nanostructure-initiator mass spectrometry-based assays for the analysis of glycosyl hydrolase and most recently an assay for lignin modifying enzymes. Here we integrate these two assays into a single multiplexed assay against both classes of enzymes and use it to characterize crude commercial enzyme mixtures. Application of our multiplexed platform based on nanostructure-initiator mass spectrometry enabled us to characterize crude mixtures of laccase enzymes from fungi Agaricus bisporus (Ab) and Myceliopthora thermophila (Mt) revealing activity on both carbohydrate and aromatic substrates. Using time-series analysis we determined that crude laccase from Ab has the higher GH activity and that laccase from Mt has the higher activity against our lignin model compound. Inhibitor studies showed a significant reduction in Mt GH activity under low oxygen conditions and increased activities in the presence of vanillin (common GH inhibitor). Ultimately, this assay can help to discover mixtures of enzymes that could be incorporated into biomass pretreatments to deconstruct diverse components of lignocellulosic biomass.
Laccases are oxidative enzymes containing 4 conserved copper heteroatoms. Laccases catalyze cleavage of bonds in lignin using radical chemistry, yet their exact specificity for bonds (such as the β-O-4 or C-C) in lignin remains unknown and may vary with the diversity of laccases across fungi, plants and bacteria. Bond specificity may perhaps even vary for the same enzyme across different reaction conditions. Determining these differences has been difficult due to the fact that heterologous expression of soluble, active laccases has proven difficult. Here we describe the successful heterologous expression of functional laccases in two strains of Saccharomyces cerevisiae, including one we genetically modified with CRISPR. We phylogenically map the enzymes that we successfully expressed, compared to those that did not express. We also describe differences protein sequence differences and pH and temperature profiles and their ability to functionally express, leading to a potential future screening platform for directed evolution of laccases and other ligninolytic enzymes such as peroxidases.
We are working to generate fundamental understanding of enzymatic depolymerization of lignin and using this understanding to engineer mixtures of enzymes that catalyze the reactions necessary to efficiently depolymerize lignin into defined fragments. Over the years the enzymes involved in these processes have been difficult to study, because 1) the enzymes thought to be most important, fungal laccases and peroxidases, are very difficult to express in soluble, active form; 2) the full complement of required enzymes and whether or not they act synergistically is not known; 3) analysis of bond cleavage events is difficult due to the lack of analytical tools for measuring bond cleavage events in either polymeric lignin or model lignin-like compounds.
Ray, Allison E.; Williams, C.L.; Hoover, Amber N.; Li, Chenlin; Sale, Kenneth L.; Emerson, Rachel M.; Klinger, Jordan; Oksen, Ethan; Narani, Akash; Sun, Ning; Yan, Jipeng; Tanjore, Deepti; Yunes, Manal; Bose, Elizabeth; Leal, Juan H.; Bowen, Julie L.; Wolfrum, Edward J.; Resch, Michael G.; Semelsberger, Troy A.; Donohoe, Bryon S.
Feedstock variability that originates from biomass production and field conditions propagates through the value chain, posing a significant challenge to the emerging biorefinery industry. Variability in feedstock properties impacts feeding, handling, equipment operations, and conversion performance. Feedstock quality attributes, and their variations, are often overlooked in assessing feedstock value and utilization for conversion to fuels, chemicals, and products. This study developed and employed a multiscale analytical characterization approach coupled with data analytic methods to better understand the sources and distribution of feedstock quality variability through evaluation of 24 corn stover bales collected in 4 counties of Iowa. In total, 216 core samples were generated by sampling nine positions on each bale using a reliable bale coring process. The samples were characterized for a broad suite of physicochemical properties ranging across field and bale, macro, micro, and molecular scales. Results demonstrated that feedstock quality attributes can vary at all spatial scales and that multiple sources of variability must be considered in order to establish and manage biomass quality for conversion processes.
Ju, Zhaoyang; Xiao, Weihua; Yao, Xiaoqian; Tan, Xin; Simmons, Blake A.; Sale, Kenneth L.; Sun, Ning
Keggin-type polyoxometalate derived ionic liquids (POM-ILs) have recently been presented as effective solvent systems for biomass delignification. To investigate the mechanism of lignin dissolution in POM-ILs, the system involving POM-IL ([C4C1Im]3[PW12O40]) and guaiacyl glycerol-β-guaiacyl ether (GGE), which contains a β-O-4 bond (the most dominant bond moiety in lignin), was studied using quantum mechanical calculations and molecular dynamics simulations. These studies show that more stable POM-IL structures are formed when [C4C1Im]+ is anchored in the connecting four terminal oxygen region of the [PW12O40]3- surface. The cations in POM-ILs appear to stabilize the geometry by offering strong and positively charged sites, and the POM anion is a good H-bond acceptor. Calculations of POM-IL interacting with GGE show the POM anion interacts strongly with GGE through many H-bonds and π-π interactions which are the main interactions between the POM-IL anion and GGE and are strong enough to force GGE into highly bent conformations. These simulations provide fundamental models of the dissolution mechanism of lignin by POM-IL, which is promoted by strong interactions of the POM-IL anion with lignin.
The goal of this project was to develop compounds that kill biothreat bacteria by inhibiting the activity of bacterial acetyl CoA carboxylase (ACC) by blocking dimerization of one component of that enzyme complex, biotin carboxylase (BC). The ultimate goal is to combine a BC dimerization inhibitor with an existing active site inhibitor for carboxytransferase, another component of ACC, to form a new dual inhibitor therapy for this essential enzyme to avoid onset of resistance. Developing medical countermeasures to defeat antibiotic resistance in biothreat agents is of interest for national security for protecting both warfighters and the public.
Open algal ponds are likely to succumb to unpredictable, devastating crashes by one or several deleterious species. Developing methodology to mitigate or prevent pond crashes will increase algal biomass production, drive down costs for algae farmers, and reduce the risk involved with algae cultivation, making it more favorable for investment by entrepreneurs and biotechnology companies. Here, we show that specific algal-bacterial co-cultures grown with the green alga Microchloropsis salina prevented grazing by the marine rotifer, Brachionus plicatilis. We obtained seven algal-bacterial co-cultures from crashed rotifer cultures, maintained them in co-culture with Microchloropsis salina, and used a microalgal survival assay to determine that algae present in each co-culture were protected from rotifer grazing and culture crash. After months of routinely diluting and maintaining these seven algal-bacterial co-cultures, we repeated the assay and found the opposite result: none of the seven bacterial communities protected the microalgae from rotifer grazing. We performed 16S rRNA gene amplicon sequencing on the protective and nonprotective co-culture samples and identified substantial differences in the makeup of the bacterial communities. Protective bacterial communities consisted primarily of Alphaproteobacteria (Rhodobacteraceae) and Gammaproteobacteria (Marinobacter, Pseudomonas, Methylophaga) while nonprotective bacterial communities were less diverse and missing many putatively crucial members. We compared the seven protective communities with the seven nonprotective communities and we correlated specific bacterial amplicon sequence variants with algal protection. With these data, our future work will aim to define and develop an engineered-microbiome that can stabilize industrial Microchloropsis salina cultures by protecting against grazer-induced pond crashes.