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Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. In this study, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 °C. At 120 °C and 2% NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. These results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (Mw) by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a "microbial sink" improves the extent of enzymatic lignin depolymerization.

White-rot fungi (WRF) are the most efficient lignin-degrading organisms in nature. However, their capacity to use lignin-related aromatic compounds, such as 4-hydroxybenzoate, as carbon sources has only been described recently. Previously, the hydroxyquinol pathway was proposed for the bioconversion of these compounds in fungi, but gene- and structure-function relationships of the full enzymatic pathway remain uncharacterized in any single fungal species. Here, we characterize seven enzymes from two WRF, Trametes versicolor and Gelatoporia subvermispora, which constitute a four-enzyme cascade from 4-hydroxybenzoate to β-ketoadipate via the hydroxyquinol pathway. Furthermore, we solve the crystal structure of four of these enzymes and identify mechanistic differences with the closest bacterial and fungal structural homologs. Overall, this research expands our understanding of aromatic catabolism by WRF and establishes an alternative strategy for the conversion of lignin-related compounds to the valuable molecule β-ketoadipate, contributing to the development of biological processes for lignin valorization., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Determining accurate counts and size distributions for biological particles (bioparticles) is crucial in wide-ranging fields, but current methods to this end are susceptible to bias from polydispersity in size. This bias can be mitigated by incorporating a separation step prior to characterization. For this reason, asymmetrical flow field-flow fractionation (AF4) with on-line multiangle light scattering (MALS) has become an important platform for determining particle size. AF4-MALS has also been increasingly used to report particle concentration, particularly for complex biological particles, yet the impact of light scattering models and particle refractive indices (RI) have not been quantitatively evaluated. Here, we develop an analysis workflow using AF4-MALS to simultaneously separate and determine particles sizes and concentrations. The impacts of the MALS particle counting model used to process data and the chosen RI value(s) on particle counts are systematically assessed for polystyrene latex (PSL) particles and bacterial outer membrane vesicles (OMVs) in the 20-500 nm size range. Across spherical models, PSL and OMV particle counts varied up to 13 % or 200 %, respectively. For the coated-sphere model used in the analysis of OMV samples, the sphere RI value greatly impacts particle counts. As the sphere RI value approaches the RI of the suspending medium, the model becomes increasingly sensitive to the light scattering signal-to-noise ratio ultimately causing erroneous particle counts. Overall, this work establishes the importance of selecting appropriate MALS models and RI values for bioparticles to obtain accurate counts and provides an AF4-MALS method to separate, enumerate, and size polydisperse bioparticles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Feedstock variability represents a challenge in lignocellulosic biorefineries, as it can influence both lignocellulose deconstruction and microbial conversion processes for biofuels and biochemicals production. The impact of feedstock variability on microbial performance remains underexplored, and predictive tools for microbial behaviour are needed to mitigate risks in biorefinery scale-up. Here, twelve batches of corn stover were deconstructed via deacetylation, mechanical refining, and enzymatic hydrolysis to generate lignin-rich and sugar streams. These batches and their derived streams were characterised to identify their chemical components, and the streams were used as substrates for producing muconate and butyrate by engineered Pseudomonas putida and wildtype Clostridium tyrobutyricum, respectively. Bacterial performance (growth, product titers, yields, and productivities) differed among the batches, but no strong correlations were identified between feedstock composition and performance. To provide metabolic insights into the origin of these differences, we evaluated the effect of twenty-three isolated chemical components on these microbes, including three components in relevant bioprocess settings in bioreactors, and we found that growth-inhibitory concentrations were outside the ranges observed in the streams. Overall, this study generates a foundational dataset on P. putida and C. tyrobutyricum performance to enable future predictive models and underscores their resilience in effectively converting fluctuating lignocellulose-derived streams into bioproducts., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Pseudomonas putida KT2440 is a robust, aromatic catabolic bacterium that has been widely engineered to convert bio-based and waste-based feedstocks to target products. Towards industrial domestication of P. putida KT2440, rational genome reduction has been previously conducted, resulting in P. putida strain EM42, which exhibited characteristics that could be advantageous for production strains. Here, we compared P. putida KT2440- and EM42-derived strains for cis,cis-muconic acid production from an aromatic compound, p-coumarate, and in separate strains, from glucose. To our surprise, the EM42-derived strains did not outperform the KT2440-derived strains in muconate production from either substrate. In bioreactor cultivations, KT2440- and EM42-derived strains produced muconate from p-coumarate at titers of 45 g/L and 37 g/L, respectively, and from glucose at 20 g/L and 13 g/L, respectively. To provide additional insights about the differences in the parent strains, we analyzed growth profiles of KT2440 and EM42 on aromatic compounds as the sole carbon and energy sources. In general, the EM42 strain exhibited reduced growth rates but shorter growth lags than KT2440. We also observed that EM42-derived strains resulted in higher growth rates on glucose compared to KT2440-derived strains, but only at the lowest glucose concentrations tested. Transcriptomics revealed that genome reduction in EM42 had global effects on transcript levels and showed that the EM42-derived strains that produce muconate from glucose exhibit reduced modulation of gene expression in response to changes in glucose concentrations. Overall, our results highlight that additional studies are warranted to understand the effects of genome reduction on microbial metabolism and physiology, especially when intended for use in production strains., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Cross-linked elastomers are stretchable materials that typically are not recyclable or biodegradable. Medium-chain-length polyhydroxyalkanoates (mcl-PHAs) are soft and ductile, making these bio-based polymers good candidates for biodegradable elastomers. Elasticity is commonly imparted by a cross-linked network structure, and covalent adaptable networks have emerged as a solution to prepare recyclable thermosets via triggered rearrangement of dynamic covalent bonds. Here, we develop biodegradable and recyclable elastomers by chemically installing the covalent adaptable network within biologically produced mcl-PHAs. Specifically, an engineered strain of Pseudomonas putida was used to produce mcl-PHAs containing pendent terminal alkenes as chemical handles for postfunctionalization. Thiol-ene chemistry was used to incorporate boronic ester (BE) cross-links, resulting in PHA-based vitrimers. mcl-PHAs cross-linked with BE at low density (<6 mole %) affords a soft, elastomeric material that demonstrates thermal reprocessability, biodegradability, and denetworking at end of life. The mechanical properties show potential for applications including adhesives and soft, biodegradable robotics and electronics.

Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl- and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O -demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $2.01 per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($1.10 to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.

Muconic acid is a bioprivileged molecule that can be converted into direct replacement chemicals for incumbent petrochemicals and performance-advantaged bioproducts. In this study, Pseudomonas putida KT2440 is engineered to convert glucose and xylose, the primary carbohydrates in lignocellulosic hydrolysates, to muconic acid using a model-guided strategy to maximize the theoretical yield. Using adaptive laboratory evolution (ALE) and metabolic engineering in a strain engineered to express the D-xylose isomerase pathway, we demonstrate that mutations in the heterologous D-xylose:H + symporter (XylE), increased expression of a major facilitator superfamily transporter (PP&#95;2569), and overexpression of aroB encoding the native 3-dehydroquinate synthase, enable efficient muconic acid production from glucose and xylose simultaneously. Using the rationally engineered strain, we produce 33.7 g L -1 muconate at 0.18 g L -1 h -1 and a 46% molar yield (92% of the maximum theoretical yield). This engineering strategy is promising for the production of other shikimate pathway-derived compounds from lignocellulosic sugars., (© 2022. The Author(s).)

Plant-derived biomass is the most abundant biogenic carbon source on Earth. Despite this, only a small clade of organisms known as white-rot fungi (WRF) can efficiently break down both the polysaccharide and lignin components of plant cell walls. This unique ability imparts a key role for WRF in global carbon cycling and highlights their potential utilization in diverse biotechnological applications. To date, research on WRF has primarily focused on their extracellular 'digestive enzymes' whereas knowledge of their intracellular metabolism remains underexplored. Systems biology is a powerful approach to elucidate biological processes in numerous organisms, including WRF. Thus, here we review systems biology methods applied to WRF to date, highlight observations related to their intracellular metabolism, and conduct comparative extracellular proteomic analyses to establish further correlations between WRF species, enzymes, and cultivation conditions. Lastly, we discuss biotechnological opportunities of WRF as well as challenges and future research directions., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP + /NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products., (Copyright © 2022 International Metabolic Engineering Society. All rights reserved.)

Poly(ethylene terephthalate) (PET) is the most abundantly consumed synthetic polyester and accordingly a major source of plastic waste. The development of chemocatalytic approaches for PET depolymerization to monomers offers new options for open-loop upcycling of PET, which can leverage biological transformations to higher-value products. To that end, here we perform four sequential metabolic engineering efforts in Pseudomonas putida KT2440 to enable the conversion of PET glycolysis products via: (i) ethylene glycol utilization by constitutive expression of native genes, (ii) terephthalate (TPA) catabolism by expression of tphA2 II A3 II B II A1 II from Comamonas and tpaK from Rhodococcus jostii, (iii) bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis to TPA by expression of PETase and MHETase from Ideonella sakaiensis, and (iv) BHET conversion to a performance-advantaged bioproduct, β-ketoadipic acid (βKA) by deletion of pcaIJ. Using this strain, we demonstrate production of 15.1 g/L βKA from BHET at 76% molar yield in bioreactors and conversion of catalytically depolymerized PET to βKA. Overall, this work highlights the potential of tandem catalytic deconstruction and biological conversion as a means to upcycle waste PET., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Pleurotus eryngii is a grassland-inhabiting fungus of biotechnological interest due to its ability to colonize non-woody lignocellulosic material. Genomic, transcriptomic, exoproteomic, and metabolomic analyses were combined to explain the enzymatic aspects underlaying wheat-straw transformation. Up-regulated and constitutive glycoside-hydrolases, polysaccharide-lyases, and carbohydrate-esterases active on polysaccharides, laccases active on lignin, and a surprisingly high amount of constitutive/inducible aryl-alcohol oxidases (AAOs) constituted the suite of extracellular enzymes at early fungal growth. Higher enzyme diversity and abundance characterized the longer-term growth, with an array of oxidoreductases involved in depolymerization of both cellulose and lignin, which were often up-regulated since initial growth. These oxidative enzymes included lytic polysaccharide monooxygenases (LPMOs) acting on crystalline polysaccharides, cellobiose dehydrogenase involved in LPMO activation, and ligninolytic peroxidases (mainly manganese-oxidizing peroxidases), together with highly abundant H 2 O 2 -producing AAOs. Interestingly, some of the most relevant enzymes acting on polysaccharides were appended to a cellulose-binding module. This is potentially related to the non-woody habitat of P. eryngii (in contrast to the wood habitat of many basidiomycetes). Additionally, insights into the intracellular catabolism of aromatic compounds, which is a neglected area of study in lignin degradation by basidiomycetes, were also provided. The multiomic approach reveals that although non-woody decay does not result in dramatic modifications, as revealed by detailed 2D-NMR and other analyses, it implies activation of the complete set of hydrolytic and oxidative enzymes characterizing lignocellulose-decaying basidiomycetes.

Expanding the portfolio of products that can be made from lignin will be critical to enabling a viable bio-based economy. Here, we engineer Pseudomonas putida for high-yield production of the tricarboxylic acid cycle-derived building block chemical, itaconic acid, from model aromatic compounds and aromatics derived from lignin. We develop a nitrogen starvation-detecting biosensor for dynamic two-stage bioproduction in which itaconic acid is produced during a non-growth associated production phase. Through the use of two distinct itaconic acid production pathways, the tuning of TCA cycle gene expression, deletion of competing pathways, and dynamic regulation, we achieve an overall maximum yield of 56% (mol/mol) and titer of 1.3 g/L from p-coumarate, and 1.4 g/L titer from monomeric aromatic compounds produced from alkali-treated lignin. This work illustrates a proof-of-principle that using dynamic metabolic control to reroute carbon after it enters central metabolism enables production of valuable chemicals from lignin at high yields by relieving the burden of constitutively expressing toxic heterologous pathways.

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 13 C-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., Competing Interests: The authors declare no competing interest.

Valorization of all major lignocellulose components, including lignin, cellulose, and hemicellulose is critical for an economically viable bioeconomy. In most biochemical conversion approaches, the standard process separately upgrades sugar hydrolysates and lignin. Here, we present a new process concept based on an engineered microbe that could enable simultaneous upgrading of all lignocellulose streams, which has the ultimate potential to reduce capital cost and enable new metabolic engineering strategies. Pseudomonas putida is a robust microorganism capable of natively catabolizing aromatics, organic acids, and D-glucose. We engineered this strain to utilize D-xylose by tuning expression of a heterologous D-xylose transporter, catabolic genes xylAB, and pentose phosphate pathway (PPP) genes tal-tkt. We further engineered L-arabinose utilization via the PPP or an oxidative pathway. This resulted in a growth rate on xylose and arabinose of 0.32 h -1 and 0.38 h -1 , respectively. Using the oxidative L-arabinose pathway with the PPP xylose pathway enabled D-glucose, D-xylose, and L-arabinose co-utilization in minimal medium using model compounds as well as real corn stover hydrolysate, with a maximum hydrolysate sugar consumption rate of 3.3 g/L/h. After modifying catabolite repression, our engineered P. putida simultaneously co-utilized five representative compounds from cellulose (D-glucose), hemicellulose (D-xylose, L-arabinose, and acetic acid), and lignin-related compounds (p-coumarate), demonstrating the feasibility of simultaneously upgrading total lignocellulosic biomass to value-added chemicals., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Burkholderia, a bacterial genus comprising more than 120 species, is typically reported to inhabit soil and water environments. These Gram-negative bacteria harbor a variety of aromatic catabolic pathways and are thus potential organisms for bioremediation of sites contaminated with aromatic pollutants. However, there are still substantial gaps in our knowledge of these catabolic processes that must be filled before these pathways and organisms can be harnessed for biotechnological applications. This review presents recent discoveries on the catabolism of monoaromatic and polycyclic aromatic hydrocarbons, as well as of heterocyclic compounds, by a diversity of Burkholderia strains. We also present a perspective on the beneficial features of Burkholderia spp. and future directions for their potential utilization in the bioremediation and bioconversion of aromatic compounds., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Pseudomonas putida KT2440 is a promising bacterial chassis for the conversion of lignin-derived aromatic compound mixtures to biofuels and bioproducts. Despite the inherent robustness of this strain, further improvements to aromatic catabolism and toxicity tolerance of P. putida will be required to achieve industrial relevance. Here, tolerance adaptive laboratory evolution (TALE) was employed with increasing concentrations of the hydroxycinnamic acids p -coumaric acid ( p CA) and ferulic acid (FA) individually and in combination ( p CA ​+ ​FA). The TALE experiments led to evolved P. putida strains with increased tolerance to the targeted acids as compared to wild type. Specifically, a 37 ​h decrease in lag phase in 20 ​g/L p CA and a 2.4-fold increase in growth rate in 30 ​g/L FA was observed. Whole genome sequencing of intermediate and endpoint evolved P. putida populations revealed several expected and non-intuitive genetic targets underlying these aromatic catabolic and toxicity tolerance enhancements. PP&#95;3350 and ttgB were among the most frequently mutated genes, and the beneficial contributions of these mutations were verified via gene knockouts. Deletion of PP&#95;3350, encoding a hypothetical protein, recapitulated improved toxicity tolerance to high concentrations of p CA, but not an improved growth rate in high concentrations of FA. Deletion of ttgB, part of the TtgABC efflux pump, severely inhibited growth in p CA ​+ ​FA TALE-derived strains but did not affect growth in p CA ​+ ​FA in a wild type background, suggesting epistatic interactions. Genes involved in flagellar movement and transcriptional regulation were often mutated in the TALE experiments on multiple substrates, reinforcing ideas of a minimal and deregulated cell as optimal for domesticated growth. Overall, this work demonstrates increased tolerance towards and growth rate at the expense of hydroxycinnamic acids and presents new targets for improving P. putida for microbial lignin valorization., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2020 The Author(s).)

Pseudomonas putida KT2440 has received increasing attention as an important biocatalyst for the conversion of diverse carbon sources to multiple products, including the olefinic diacid, cis,cis-muconic acid (muconate). P. putida has been previously engineered to produce muconate from glucose; however, periplasmic oxidation of glucose causes substantial 2-ketogluconate accumulation, reducing product yield and selectivity. Deletion of the glucose dehydrogenase gene (gcd) prevents 2-ketogluconate accumulation, but dramatically slows growth and muconate production. In this work, we employed adaptive laboratory evolution to improve muconate production in strains incapable of producing 2-ketogluconate. Growth-based selection improved growth, but reduced muconate titer. A new muconate-responsive biosensor was therefore developed to enable muconate-based screening using fluorescence activated cell sorting. Sorted clones demonstrated both improved growth and muconate production. Mutations identified by whole genome resequencing of these isolates indicated that glucose metabolism may be dysregulated in strains lacking gcd. Using this information, we used targeted engineering to recapitulate improvements achieved by evolution. Deletion of the transcriptional repressor gene hexR improved strain growth and increased the muconate production rate, and the impact of this deletion was investigated using transcriptomics. The genes gntZ and gacS were also disrupted in several evolved clones, and deletion of these genes further improved strain growth and muconate production. Together, these targets provide a suite of modifications that improve glucose conversion to muconate by P. putida in the context of gcd deletion. Prior to this work, our engineered strain lacking gcd generated 7.0 g/L muconate at a productivity of 0.07 g/L/h and a 38% yield (mol/mol) in a fed-batch bioreactor. Here, the resulting strain with the deletion of hexR, gntZ, and gacS achieved 22.0 g/L at 0.21 g/L/h and a 35.6% yield (mol/mol) from glucose in similar conditions. These strategies enabled enhanced muconic acid production and may also improve production of other target molecules from glucose in P. putida., (Copyright © 2020 International Metabolic Engineering Society. All rights reserved.)

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., Competing Interests: The authors declare no competing interest.

The economic viability of the biorefinery concept is limited by the valorization of lignin. One possible method of lignin valorization is biological upgrading with aromatic-catabolic microbes. In conjunction, lignin monomers can be produced by fast pyrolysis and fractionation. However, biological upgrading of these lignin monomers is limited by low water solubility. Here, we address the problem of low water solubility with an emulsifier blend containing approximately 70 wt% Tween ® 20 and 30 wt% Span ® 80. Pseudomonas putida KT2440 grew to an optical density (OD 600 ) of 1.0 ± 0.2 when supplied with 1.6 wt% emulsified phenolic monomer-rich product produced by fast pyrolysis of red oak using an emulsifier dose of 0.076 ± 0.002 g emulsifier blend per g of phenolic monomer-rich product. This approach partially mitigated the toxicity of the model phenolic monomer p-coumarate to the microbe, but not benzoate or vanillin. This study provides a proof of concept that processing of biomass-derived phenolics to increase aqueous availability can enhance microbial utilization.

Actinobacillus succinogenes , a Gram-negative facultative anaerobe, exhibits the native capacity to convert pentose and hexose sugars to succinic acid (SA) with high yield as a tricarboxylic acid (TCA) cycle intermediate. In addition, A. succinogenes is capnophilic, incorporating CO 2 into SA, making this organism an ideal candidate host for conversion of lignocellulosic sugars and CO 2 to an emerging commodity bioproduct sourced from renewable feedstocks. In this work, we report the development of facile metabolic engineering capabilities in A. succinogenes , enabling examination of SA flux determinants via knockout of the primary competing pathways-namely, acetate and formate production-and overexpression of the key enzymes in the reductive branch of the TCA cycle leading to SA. Batch fermentation experiments with the wild-type and engineered strains using pentose-rich sugar streams demonstrate that the overexpression of the SA biosynthetic machinery (in particular, the enzyme malate dehydrogenase) enhances flux to SA. Additionally, removal of competitive carbon pathways leads to higher-purity SA but also triggers the generation of by-products not previously described from this organism (e.g., lactic acid). The resultant engineered strains also lend insight into energetic and redox balance and elucidate mechanisms governing organic acid biosynthesis in this important natural SA-producing microbe. IMPORTANCE Succinic acid production from lignocellulosic residues is a potential route for enhancing the economic feasibility of modern biorefineries. Here, we employ facile genetic tools to systematically manipulate competing acid production pathways and overexpress the succinic acid-producing machinery in Actinobacillus succinogenes Furthermore, the resulting strains are evaluated via fermentation on relevant pentose-rich sugar streams representative of those from corn stover. Overall, this work demonstrates genetic modifications that can lead to succinic acid production improvements and identifies key flux determinants and new bottlenecks and energetic needs when removing by-product pathways in A. succinogenes metabolism., (Copyright © 2017 American Society for Microbiology.)

Background: The production of value-added chemicals alongside biofuels from lignocellulosic hydrolysates is critical for developing economically viable biorefineries. Here, the production of propionic acid (PA), a potential building block for C3-based chemicals, from corn stover hydrolysate is investigated using the native PA-producing bacterium Propionibacterium acidipropionici ., Results: A wide range of culture conditions and process parameters were examined and experimentally optimized to maximize titer, rate, and yield of PA. The effect of gas sparging during fermentation was first examined, and N 2 was found to exhibit improved performance over CO 2 . Subsequently, the effects of different hydrolysate concentrations, nitrogen sources, and neutralization agents were investigated. One of the best combinations found during batch experiments used yeast extract (YE) as the primary nitrogen source and NH 4 OH for pH control. This combination enabled PA titers of 30.8 g/L with a productivity of 0.40 g/L h from 76.8 g/L biomass sugars, while successfully minimizing lactic acid production. Due to the economic significance of downstream separations, increasing titers using fed-batch fermentation was examined by changing both feeding media and strategy. Continuous feeding of hydrolysate was found to be superior to pulsed feeding and combined with high YE concentrations increased PA titers to 62.7 g/L and improved the simultaneous utilization of different biomass sugars. Additionally, applying high YE supplementation maintains the lactic acid concentration below 4 g/L for the duration of the fermentation. Finally, with the aim of increasing productivity, high cell density fed-batch fermentations were conducted. PA titers increased to 64.7 g/L with a productivity of 2.35 g/L h for the batch stage and 0.77 g/L h for the overall process., Conclusion: These results highlight the importance of media and fermentation strategy to improve PA production. Overall, this work demonstrates the feasibility of producing PA from corn stover hydrolysate.

Lignin is a primary component of lignocellulosic biomass that is an underutilized feedstock in the growing biofuels industry. Despite the fact that lignin depolymerization has long been studied, the intrinsic heterogeneity of lignin typically leads to heterogeneous streams of aromatic compounds, which in turn present significant technical challenges when attempting to produce lignin-derived chemicals where purity is often a concern. In Nature, microorganisms often encounter this same problem during biomass turnover wherein powerful oxidative enzymes produce heterogeneous slates of aromatics compounds. Some microbes have evolved metabolic pathways to convert these aromatic species via 'upper pathways' into central intermediates, which can then be funneled through 'lower pathways' into central carbon metabolism in a process we dubbed 'biological funneling'. This funneling approach offers a direct, biological solution to overcome heterogeneity problems in lignin valorization for the modern biorefinery. Coupled to targeted separations and downstream chemical catalysis, this concept offers the ability to produce a wide range of molecules from lignin. This perspective describes research opportunities and challenges ahead for this new field of research, which holds significant promise towards a biorefinery concept wherein polysaccharides and lignin are treated as equally valuable feedstocks. In particular, we discuss tailoring the lignin substrate for microbial utilization, host selection for biological funneling, ligninolytic enzyme-microbe synergy, metabolic engineering, expanding substrate specificity for biological funneling, and process integration, each of which presents key challenges. Ultimately, for biological solutions to lignin valorization to be viable, multiple questions in each of these areas will need to be addressed, making biological lignin valorization a multidisciplinary, co-design problem., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The production of chemicals alongside fuels will be essential to enhance the feasibility of lignocellulosic biorefineries. Succinic acid (SA), a naturally occurring C4-diacid, is a primary intermediate of the tricarboxylic acid cycle and a promising building block chemical that has received significant industrial attention. Basfia succiniciproducens is a relatively unexplored SA-producing bacterium with advantageous features such as broad substrate utilization, genetic tractability, and facultative anaerobic metabolism. Here B. succiniciproducens is evaluated in high xylose-content hydrolysates from corn stover and different synthetic media in batch fermentation. SA titers in hydrolysate at an initial sugar concentration of 60g/L reached up to 30g/L, with metabolic yields of 0.69g/g, and an overall productivity of 0.43g/L/h. These results demonstrate that B. succiniciproducens may be an attractive platform organism for bio-SA production from biomass hydrolysates., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The conversion of biomass-derived sugars and aromatic molecules to cis , cis -muconic acid (referred to hereafter as muconic acid or muconate) has been of recent interest owing to its facile conversion to adipic acid, an important commodity chemical. Metabolic routes to produce muconate from both sugars and many lignin-derived aromatic compounds require the use of a decarboxylase to convert protocatechuate (PCA, 3,4-dihydroxybenzoate) to catechol (1,2-dihydroxybenzene), two central aromatic intermediates in this pathway. Several studies have identified the PCA decarboxylase as a metabolic bottleneck, causing an accumulation of PCA that subsequently reduces muconate production. A recent study showed that activity of the PCA decarboxylase is enhanced by co-expression of two genetically associated proteins, one of which likely produces a flavin-derived cofactor utilized by the decarboxylase. Using entirely genome-integrated gene expression, we have engineered Pseudomonas putida KT2440-derived strains to produce muconate from either aromatic molecules or sugars and demonstrate in both cases that co-expression of these decarboxylase associated proteins reduces PCA accumulation and enhances muconate production relative to strains expressing the PCA decarboxylase alone. In bioreactor experiments, co-expression increased the specific productivity (mg/g cells/h) of muconate from the aromatic lignin monomer p -coumarate by 50% and resulted in a titer of >15 g/L. In strains engineered to produce muconate from glucose, co-expression more than tripled the titer, yield, productivity, and specific productivity, with the best strain producing 4.92±0.48 g/L muconate. This study demonstrates that overcoming the PCA decarboxylase bottleneck can increase muconate yields from biomass-derived sugars and aromatic molecules in industrially relevant strains and cultivation conditions.

Background: Co-production of chemicals from lignocellulosic biomass alongside fuels holds promise for improving the economic outlook of integrated biorefineries. In current biochemical conversion processes that use thermochemical pretreatment and enzymatic hydrolysis, fractionation of hemicellulose-derived and cellulose-derived sugar streams is possible using hydrothermal or dilute acid pretreatment (DAP), which then offers a route to parallel trains for fuel and chemical production from xylose- and glucose-enriched streams. Succinic acid (SA) is a co-product of particular interest in biorefineries because it could potentially displace petroleum-derived chemicals and polymer precursors for myriad applications. However, SA production from biomass-derived hydrolysates has not yet been fully explored or developed., Results: Here, we employ Actinobacillus succinogenes 130Z to produce succinate in batch fermentations from various substrates including (1) pure sugars to quantify substrate inhibition, (2) from mock hydrolysates similar to those from DAP containing single putative inhibitors, and (3) using the hydrolysate derived from two pilot-scale pretreatments: first, a mild alkaline wash (deacetylation) followed by DAP, and secondly a single DAP step, both with corn stover. These latter streams are both rich in xylose and contain different levels of inhibitors such as acetate, sugar dehydration products (furfural, 5-hydroxymethylfurfural), and lignin-derived products (ferulate, p-coumarate). In batch fermentations, we quantify succinate and co-product (acetate and formate) titers as well as succinate yields and productivities. We demonstrate yields of 0.74 g succinate/g sugars and 42.8 g/L succinate from deacetylated DAP hydrolysate, achieving maximum productivities of up to 1.27 g/L-h. Moreover, A. succinogenes is shown to detoxify furfural via reduction to furfuryl alcohol, although an initial lag in succinate production is observed when furans are present. Acetate seems to be the main inhibitor for this bacterium present in biomass hydrolysates., Conclusion: Overall, these results demonstrate that biomass-derived, xylose-enriched hydrolysates result in similar yields and titers but lower productivities compared to clean sugar streams, which can likely be improved via fermentation process developments and metabolic engineering. Overall, this study comprehensively examines the behavior of A. succinogenes on xylose-enriched hydrolysates on an industrially relevant, lignocellulosic feedstock, which will pave the way for future work toward eventual SA production in an integrated biorefinery.

Background: Bio-manufacturing of high-value chemicals in parallel to renewable biofuels has the potential to dramatically improve the overall economic landscape of integrated lignocellulosic biorefineries. However, this will require the generation of carbohydrate streams from lignocellulose in a form suitable for efficient microbial conversion and downstream processing appropriate to the desired end use, making overall process development, along with selection of appropriate target molecules, crucial to the integrated biorefinery. Succinic acid (SA), a high-value target molecule, can be biologically produced from sugars and has the potential to serve as a platform chemical for various chemical and polymer applications. However, the feasibility of microbial SA production at industrially relevant productivities and yields from lignocellulosic biorefinery streams has not yet been reported., Results: Actinobacillus succinogenes 130Z was immobilised in a custom continuous fermentation setup to produce SA on the xylose-enriched fraction of a non-detoxified, xylose-rich corn stover hydrolysate stream produced from deacetylation and dilute acid pretreatment. Effective biofilm attachment, which serves as a natural cell retention strategy to increase cell densities, productivities and resistance to toxicity, was accomplished by means of a novel agitator fitting. A maximum SA titre, yield and productivity of 39.6 g L(-1), 0.78 g g(-1) and 1.77 g L(-1) h(-1) were achieved, respectively. Steady states were obtained at dilution rates of 0.02, 0.03, 0.04, and 0.05 h(-1) and the stirred biofilm reactor was stable over prolonged periods of operation with a combined fermentation time of 1550 h. Furthermore, it was found that a gradual increase in the dilution rate was required to facilitate adaptation of the culture to the hydrolysate, suggesting a strong evolutionary response to the toxic compounds in the hydrolysate. Moreover, the two primary suspected fermentation inhibitors, furfural and HMF, were metabolised during fermentation with the concentration of each remaining at zero across all steady states., Conclusions: The results demonstrate that immobilised A. succinogenes has the potential for effective conversion of an industrially relevant, biomass-derived feed stream to succinic acid. Furthermore, due to the attractive yields, productivities and titres achieved in this study, the process has the potential to serve as a means for value-added chemical manufacturing in the integrated biorefinery.

Background: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases., Results: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases., Conclusion: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

Irpex lacteus is a white rot basidiomycete proposed for a wide spectrum of biotechnological applications which presents an interesting, but still scarcely known, enzymatic oxidative system. Among these enzymes, the production, purification, and identification of a new dye-decolorizing peroxidase (DyP)-type enzyme, as well as its physico-chemical, spectroscopic, and catalytic properties, are described in the current work. According to its N-terminal sequence and peptide mass fingerprinting analyses, I. lacteus DyP showed high homology (>95%) with the hypothetical (not isolated or characterized) protein cpop21 from an unidentified species of the family Polyporaceae. The enzyme had a low optimal pH, was very stable to acid pH and temperature, and showed improved activity and stability at high H2O2 concentrations compared to other peroxidases. Other attractive features of I. lacteus DyP were its high catalytic efficiency oxidizing the recalcitrant anthraquinone and azo dyes assayed (kcat/Km of 1.6 × 10(6) s(-1) M(-1)) and its ability to oxidize nonphenolic aromatic compounds like veratryl alcohol. In addition, the effect of this DyP during the enzymatic hydrolysis of wheat straw was checked. The results suggest that I. lacteus DyP displayed a synergistic action with cellulases during the hydrolysis of wheat straw, increasing significantly the fermentable glucose recoveries from this substrate. These data show a promising biotechnological potential for this enzyme.

The modification and generation of new biomolecules intended to give higher molecular-mass species for biotechnological purposes, can be achieved by enzymatic cross-linking. The versatile peroxidase (VP) from Pleurotus eryngii is a high redox-potential enzyme with oxidative activity on a wide variety of substrates. In this study, VP was successfully used to catalyze the polymerization of low molecular mass compounds, such as lignans and peptides, as well as larger macromolecules, such as protein and complex polysaccharides. Different analytical, spectroscopic, and rheological techniques were used to determine structural changes and/or variations of the physicochemical properties of the reaction products. The lignans secoisolariciresinol and hydroxymatairesinol were condensed by VP forming up to 8 unit polymers in the presence of organic co-solvents and Mn(2+). Moreover, 11 unit of the peptides YIGSR and VYV were homogeneously cross-linked. The heterogeneous cross-linking of one unit of the peptide YIGSR and several lignan units was also achieved. VP could also induce gelation of feruloylated arabinoxylan and the polymerization of β-casein. These results demonstrate the efficacy of VP to catalyze homo- and hetero-condensation reactions, and reveal its potential exploitation for polymerizing different types of compounds., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Irpex lacteus is a white-rot fungus capable of increasing sugar recovery from wheat straw; however, in order to incorporate biopretreatment in bioethanol production, some process specifications need to be optimized. With this objective, I. lacteus was grown on different liquid culture media for use as inoculums. Additionally, the effect of wheat straw particle size, moisture content, organic and inorganic supplementations, and mild alkali washing during solid-state fermentation (SSF) on sugar yield were investigated. Wheat thin stillage was the best medium for producing inoculums. Supplementation of wheat straw with 0.3mM Mn(II) during SSF resulted in glucose yields of 68% as compared to yields of 62% and 33% for cultures grown without supplementation or on untreated raw material, respectively after 21 days. Lignin loss, wheat straw digestibility, peroxidase activity, and fungal biomass were also correlated with sugar yields in the search for biopretreatment efficiency indicators., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

O-linked heterogalactomannans with similar structural features have been purified from the fungal walls of the entomopathogenic fungi Lecanicillium muscarium, Beauveria bassiana, Beauveriabrongniartii, and Cordyceps sphingum. Their composition and structure have been determined using acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry (GC-MS) and Nuclear magnetic resonance spectroscopy (NMR). All structures have an α-(1→6)-mannose backbone, but one of the two strains of L. muscarium included in this study contained an acidic heterogalactomannan instead of the neutral polysaccharide isolated in the rest of the species analyzed. Sequence analysis of the internal transcribed spacer (ITS) region of this strain indicated that it belongs to the related genus Simplicillium, displaying low identity (83%) with the closest Lecanicillium species. This is a new demonstration of the structural diversity of fungal wall heteromannans and validates their interest as chemotaxonomic markers. The production of a pullulan-like extracellular polysaccharide in strain CBS 413.70C of L. muscarium is also reported., (Copyright © 2011 British Mycological Society. Published by Elsevier Ltd. All rights reserved.)

The potential of a fungal pretreatment combined with a mild alkali treatment to replace or complement current physico-chemical methods for ethanol production from wheat straw has been investigated. Changes in substrate composition, secretion of ligninolytic enzymes, enzymatic hydrolysis efficiency and ethanol yield after 7, 14 and 21 days of solid-state fermentation were evaluated. Most fungi degraded lignin with variable selectivity degrees, although only eight of them improved sugar recovery compared to untreated samples. Glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lacteus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of 62% in both cases. Conversions from glucose to ethanol reached around 90%, showing that no inhibitors were generated during this pretreatment. No close correlations were found between ligninolytic enzymes production and sugar yields., (Copyright © 2011 Elsevier Ltd. All rights reserved.)