Lignin is an energy-dense, heterogeneous polymer comprised of phenylpropanoid monomers used by plants for structure, water transport, and defense, and it is the second most abundant biopolymer on Earth after cellulose. In production of fuels and chemicals from biomass, lignin is typically underused as a feedstock and burned for process heat because its inherent heterogeneity and recalcitrance make it difficult to selectively valorize. In nature, however, some organisms have evolved metabolic pathways that enable the utilization of lignin-derived aromatic molecules as carbon sources. Aromatic catabolism typically occurs via upper pathways that act as a "biological funnel" to convert heterogeneous substrates to central intermediates, such as protocatechuate or catechol. These intermediates undergo ring cleavage and are further converted via the β-ketoadipate pathway to central carbon metabolism. Here, we use a natural aromatic-catabolizing organism, Pseudomonas putida KT2440, to demonstrate that these aromatic metabolic pathways can be used to convert both aromatic model compounds and heterogeneous, lignin-enriched streams derived from pilot-scale biomass pretreatment into medium chain-length polyhydroxyalkanoates (mcl-PHAs). mcl-PHAs were then isolated from the cells and demonstrated to be similar in physicochemical properties to conventional carbohydratederived mcl-PHAs, which have applications as bioplastics. In a further demonstration of their utility, mcl-PHAs were catalytically converted to both chemical precursors and fuel-range hydrocarbons. Overall, this work demonstrates that the use of aromatic catabolic pathways enables an approach to valorize lignin by overcoming its inherent heterogeneity to produce fuels, chemicals, and materials. biofuels | lignocellulose | biorefinery | aromatic degradation

Lignin is an alkyl-aromatic polymer present in plant cell walls for defense, structure, and water transport. Despite exhibiting a high-energy content, lignin is typically slated for combustion in modern biorefineries due to its inherent heterogeneity and recalcitrance, whereas cellulose and hemicellulose are converted to renewable fuels and chemicals. However, it is critical for the viability of third-generation biorefineries to valorize lignin alongside polysaccharides. To that end, we employ metabolic engineering, separations, and catalysis to convert lignin-derived species into cis,cis-muconic acid, for subsequent hydrogenation to adipic acid, the latter being the most widely produced dicarboxylic acid. First, Pseudomonas putida KT2440 was metabolically engineered to funnel lignin-derived aromatics to cis,cis-muconate, which is an atom-efficient biochemical transformation. This engineered strain was employed in fed-batch biological cultivation to demonstrate a cis,cis-muconate titer of 13.5 g/L in 78.5 h from a model lignin-derived compound. cis,cis-Muconic acid was recovered in high purity (>97%) and yield (74%) by activated carbon treatment and crystallization (5°C, pH 2). Pd/C was identified as a highly active catalyst for cis,cis-muconic acid hydrogenation to adipic acid with high conversion (>97%) and selectivity (>97%). Under surface reaction controlling conditions (24°C, 24 bar, ethanol solvent), purified cis,cis-muconic acid exhibits a turnover frequency of 23-30 sec -1 over Pd/C, with an apparent activation energy of 70 kJ/mol. Lastly, cis,cis-muconate was produced with engineered P. putida grown on a biomass-derived, lignin-enriched stream, demonstrating an integrated strategy towards lignin valorization to an important commodity chemical.

Summary The mechanisms whereby chromatin structure and cell cycle progression are restored after DNA repair are largely unknown. We show that chromatin is reassembled following double-strand break (DSB) repair and that this requires the histone chaperone Asf1. Absence of Asf1 causes persistent activation of the DNA damage checkpoint after DSB repair as a consequence of defective checkpoint recovery, leading to cell death. The contribution of Asf1 towards chromatin assembly after DSB repair is due to its role in promoting acetylation of free histone H3 on lysine 56 (K56) by the histone acetyl transferase Rtt109, because mimicking acetylation of K56 bypasses the requirement for Asf1 for chromatin reassembly and checkpoint recovery after repair, while mutations that prevent K56 acetylation block chromatin reassembly after repair. These results indicate that restoration of the chromatin following DSB repair is driven by acetylated H3 K56 and that this is a signal for the completion of repair.

The packaging of the eukaryotic genome into chromatin is likely to be important for the maintenance of genomic integrity. Chromatin structures are assembled onto newly synthesized DNA by the action of chromatin assembly factors, including anti-silencing function 1 (ASF1). To investigate the role of chromatin structure in the maintenance of genomic integrity, we examined budding yeast lacking the histone chaperone Asf1p. We found that yeast lacking Asf1p accumulate in metaphase of the cell cycle due to activation of the DNA damage checkpoint. Furthermore, yeast lacking Asf1p are highly sensitive to mutations in DNA polymerase alpha and to DNA replicational stresses. Although yeast lacking Asf1p do complete DNA replication, they have greatly elevated rates of DNA damage occurring during DNA replication, as indicated by spontaneous Ddc2p-green fluorescent protein foci. The presence of elevated levels of spontaneous DNA damage in asf1 mutants is due to increased DNA damage, rather than the failure to repair double-strand DNA breaks, because asf1 mutants are fully functional for double-strand DNA repair. Our data indicate that the altered chromatin structure in asf1 mutants leads to elevated rates of spontaneous recombination, mutation, and DNA damage foci formation arising during DNA replication, which in turn activates cell cycle checkpoints that respond to DNA damage.

Biorefinery process development relies on techno-economic analysis (TEA) to identify primary cost drivers, prioritize research directions, and mitigate technical risk for scale-up through development of detailed process designs. Here, we conduct TEA of a model 2000 dry metric ton-per-day lignocellulosic biorefinery that employs a two-step pretreatment and enzymatic hydrolysis to produce biomass-derived sugars, followed by biological lipid production, lipid recovery, and catalytic hydrotreating to produce renewable diesel blendstock (RDB). On the basis of projected near-term technical feasibility of these steps, we predict that RDB could be produced at a minimum fuel selling price (MFSP) of USD $9.55/gasoline-gallon-equivalent (GGE), predicated on the need for improvements in the lipid productivity and yield beyond current benchmark performance. This cost is significant given the limitations in scale and high costs for aerobic cultivation of oleaginous microbes and subsequent lipid extraction/recovery. In light of this predicted cost, we developed an alternative pathway which demonstrates that RDB costs could be substantially reduced in the near term if upgradeable fractions of biomass, in this case hemicellulose-derived sugars, are diverted to coproducts of sufficient value and market size; here, we use succinic acid as an example coproduct. The coproduction model predicts an MFSP of USD $5.28/GGE when leaving conversion and yield parameters unchanged for the fuel production pathway, leading to a change in biorefinery RDB capacity from 24 to 15 MM GGE/year and 0.13 MM tons of succinic acid per year. Additional analysis demonstrates that beyond the near-term projections assumed in the models here, further reductions in the MFSP toward $2–3/GGE (which would be competitive with fossil-based hydrocarbon fuels) are possible with additional transformational improvements in the fuel and coproduct trains, especially in terms of carbon efficiency to both fuels and coproducts, recovery and purification of fuels and coproducts, and coproduct selection and price. Overall, this analysis documents potential economics for both a hydrocarbon fuel and bioproduct process pathway and highlights prioritized research directions beyond the current benchmark to enable hydrocarbon fuel production via an oleaginous microbial platform with simultaneous coproduct manufacturing from lignocellulosic biomass.

The disassembly of promoter nucleosomes appears to be a general property of highly transcribed eukaryotic genes. We have previously shown that the disassembly of chromatin from the promoters of the Saccharomyces cerevisiae PHO5 and PHO8 genes, mediated by the histone chaperone anti-silencing function 1 (Asf1), is essential for transcriptional activation upon phosphate depletion. This mechanism of transcriptional regulation is shared with the ADY2 and ADH2 genes upon glucose removal. Promoter chromatin disassembly by Asf1 is required for recruitment of TBP and RNA polymerase II, but not the Pho4 and Pho2 activators. Furthermore, accumulation of SWI/SNF and SAGA at the PHO5 promoter requires promoter chromatin disassembly. By contrast, the requirement for SWI/SNF and SAGA to facilitate Pho4 activator recruitment to the nucleosome-buried binding site in the PHO5 promoter occurs prior to chromatin disassembly and is distinct from the stable recruitment of SWI/SNF and SAGA that occurs after chromatin disassembly.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)-a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ 13C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora, funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts—a key step toward enabling a sustainable bioeconomy.