SignificanceSynthetic polymers are ubiquitous in the modern world but pose a global environmental problem. While plastics such as poly(ethylene terephthalate) (PET) are highly versatile, their resistance to natural degradation presents a serious, growing risk to fauna and flora, particularly in marine environments. Here, we have characterized the 3D structure of a newly discovered enzyme that can digest highly crystalline PET, the primary material used in the manufacture of single-use plastic beverage bottles, in some clothing, and in carpets. We engineer this enzyme for improved PET degradation capacity and further demonstrate that it can also degrade an important PET replacement, polyethylene-2,5-furandicarboxylate, providing new opportunities for biobased plastics recycling.

Plant cell walls are composed primarily of cellulose, hemicelluloses, lignins, and pectins. Of these components, lignins exhibit unique chemistry and physiological functions. Although lignins can be used as a product feedstock or as a fuel, lignins are also generally seen as a barrier to efficient enzymatic breakdown of biomass to sugars. Indeed, many pretreatment strategies focus on removing a significant fraction of lignin from biomass to better enable saccharification. In order to better understand the fate of biomass lignins that remain with the solids following dilute acid pretreatment, we undertook a structural investigation to track lignins on and in biomass cell walls. SEM and TEM imaging revealed a range of droplet morphologies that appear on and within cell walls of pretreated biomass; as well as the specific ultrastructural regions that accumulate the droplets. These droplets were shown to contain lignin by FTIR, NMR, antibody labeling, and cytochemical staining. We provide evidence supporting the idea that thermochemical pretreatments reaching temperatures above the range for lignin phase transition cause lignins to coalesce into larger molten bodies that migrate within and out of the cell wall, and can redeposit on the surface of plant cell walls. This decompartmentalization and relocalization of lignins is likely to be at least as important as lignin removal in the quest to improve the digestibility of biomass for sugars and fuels production.

Deconstruction of lignocellulosic plant cell walls to fermentable sugars by thermochemical and/or biological means is impeded by several poorly understood ultrastructural and chemical barriers. A promising thermochemical pretreatment called ammonia fiber expansion (AFEX) overcomes the native recalcitrance of cell walls through subtle morphological and physicochemical changes that enhance cellulase accessibility without extracting lignin and hemicelluloses into separate liquid streams. Multiscale visualization and characterization of Zea mays (i.e., corn stover) cell walls were carried out by laser scanning confocal fluorescence microscopy (LSCM), Raman spectroscopy, atomic force microscopy (AFM), electron microscopy (SEM, TEM), nuclear magnetic resonance (NMR), and electron spectroscopy for chemical analysis (ESCA) to elucidate the mechanism of AFEX pretreatment. AFEX first dissolves, then extracts and, as the ammonia evaporates, redeposits cell wall decomposition products (e.g., amides, arabinoxylan oligomers, lignin-based phenolics) on outer cell wall surfaces. As a result, nanoporous tunnel-like networks, as visualized by 3D-electron tomography, are formed within the cell walls. We propose that this highly porous structure greatly enhances enzyme accessibility to embedded cellulosic microfibrils. The shape, size (10 to 1000 nm), and spatial distribution of the pores depended on their location within the cell wall and the pretreatment conditions used. Exposed pore surface area per unit AFEX pretreated cell wall volume, estimated via TEMtomogram image analysis, ranged between 0.005 and 0.05 nm 2 per nm 3 . AFEX results in ultrastructural and physicochemical modifications within the cell wall that enhance enzymatic hydrolysis yield by 4-5 fold over that of untreated cell walls.

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

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.