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Background To expand on the range of products which can be obtained from lignocellulosic biomass, the lignin component should be utilized as feedstock for value-added chemicals such as substituted aromatics, instead of being incinerated for heat and energy. Enzymes could provide an effective means for lignin depolymerization into products of interest. In this study, soil bacteria were isolated by enrichment on Kraft lignin and evaluated for their ligninolytic potential as a source of novel enzymes for waste lignin valorization. Results Based on 16S rRNA gene sequencing and phenotypic characterization, the organisms were identified as Pandoraea norimbergensis LD001, Pseudomonas sp LD002 and Bacillus sp LD003. The ligninolytic capability of each of these isolates was assessed by growth on high-molecular weight and low-molecular weight lignin fractions, utilization of lignin-associated aromatic monomers and degradation of ligninolytic indicator dyes. Pandoraea norimbergensis LD001 and Pseudomonas sp. LD002 exhibited best growth on lignin fractions, but limited dye-decolourizing capacity. Bacillus sp. LD003, however, showed least efficient growth on lignin fractions but extensive dye-decolourizing capacity, with a particular preference for the recalcitrant phenothiazine dye class (Azure B, Methylene Blue and Toluidene Blue O). Conclusions Bacillus sp. LD003 was selected as a promising source of novel types of ligninolytic enzymes. Our observations suggested that lignin mineralization and depolymerization are separate events which place additional challenges on the screening of ligninolytic microorganisms for specific ligninolytic enzymes.

Plastics pollution is causing an environmental crisis, prompting the development of new approaches for recycling, and upcycling. Here, we review challenges and opportunities in chemical and biological catalysis for plastics deconstruction, recycling, and upcycling. We stress the need for rigorous characterization and use of widely available substrates, such that catalyst performance can be compared across studies. Where appropriate, we draw parallels between catalysis on biomass and plastics, as both substrates are low-value, solid, recalcitrant polymers. Innovations in catalyst design and reaction engineering are needed to overcome kinetic and thermodynamic limitations of plastics deconstruction. Either chemical and biological catalysts will need to act interfacially, where catalysts function at a solid surface, or polymers will need to be solubilized or processed to smaller intermediates to facilitate improved catalyst–substrate interaction. Overall, developing catalyst-driven technologies for plastics deconstruction and upcycling is critical to incentivize improved plastics reclamation and reduce the severe global burden of plastic waste. Plastics are invaluable materials for modern society, although they result in the generation of large amounts of litter at the end of their life cycle. This Review explores the challenges and opportunities associated with the catalytic transformation of waste plastics, looking at both chemical and biological approaches to transforming such spent materials into a resource.

Background Phenylpropanoids such as p -coumaric acid represent important precursors for the synthesis of a broad range of plant secondary metabolites including stilbenoids, flavonoids, and lignans, which are of pharmacological interest due to their health-promoting properties. Although extraction from plant material or chemical synthesis is possible, microbial synthesis of p -coumaric acid from glucose has the advantage of being less expensive and more resource efficient. In this study, Corynebacterium glutamicum was engineered for the production of the plant polyphenol precursor p -coumaric acid from glucose. Results Heterologous expression of the tyrosine ammonia-lyase encoding gene from Flavobacterium johnsoniae enabled the conversion of endogenously provided tyrosine to p -coumaric acid. Product consumption was avoided by abolishing essential reactions of the phenylpropanoid degradation pathway. Accumulation of anthranilate as a major byproduct was eliminated by reducing the activity of anthranilate synthase through targeted mutagenesis to avoid tryptophan auxotrophy. Subsequently, the carbon flux into the shikimate pathway was increased, phenylalanine biosynthesis was reduced, and phosphoenolpyruvate availability was improved to boost p -coumaric acid accumulation. A maximum titer of 661 mg/L p -coumaric acid (4 mM) in defined mineral medium was reached. Finally, the production strain was utilized in co-cultivations with a C.   glutamicum strain previously engineered for the conversion of p -coumaric acid into the polyphenol resveratrol. These co-cultivations enabled the synthesis of 31.2 mg/L (0.14 mM) resveratrol from glucose without any p -coumaric acid supplementation. Conclusions The utilization of a heterologous tyrosine ammonia-lyase in combination with optimization of the shikimate pathway enabled the efficient production of p -coumaric acid with C. glutamicum . Reducing the carbon flux into the phenylalanine and tryptophan branches was the key to success along with the introduction of feedback-resistant enzyme variants.

The toxic fermentation inhibitors in lignocellulosic hydrolysates pose significant problems for the production of second-generation biofuels and biochemicals. Among these inhibitors, 5-(hydroxymethyl)furfural (HMF) and furfural are specifically notorious. In this study, we describe the complete molecular identification and characterization of the pathway by which Cupriavidus basilensis HMF14 metabolizes HMF and furfural. The identification of this pathway enabled the construction of an HMF and furfural-metabolizing Pseudomonas putida. The genetic information obtained furthermore enabled us to predict the HMF and furfural degrading capabilities of sequenced bacterial species that had not previously been connected to furanic aldehyde metabolism. These results pave the way for in situ detoxification of lignocellulosic hydrolysates, which is a major step toward improved efficiency of utilization of lignocellulosic feedstock.

Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of multiple value‐added compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates. Owing to its role as a metabolic hub, malonyl‐CoA availability is limited by competition in several essential metabolic pathways. To address this limitation, we modified a genome‐reduced Pseudomonas putida strain to increase acetyl‐CoA carboxylation while limiting malonyl‐CoA utilization. Genes involved in sugar catabolism and its regulation, the tricarboxylic acid (TCA) cycle, and fatty acid biosynthesis were knocked‐out in specific combinations towards increasing the malonyl‐CoA pool. An enzyme‐coupled biosensor, based on the rppA gene, was employed to monitor malonyl‐CoA levels in vivo. RppA is a type III polyketide synthase that converts malonyl‐CoA into flaviolin, a red‐colored polyketide. We isolated strains displaying enhanced malonyl‐CoA availability via a colorimetric screening method based on the RppA‐dependent red pigmentation; direct flaviolin quantification identified four engineered strains had a significant increase in malonyl‐CoA levels. We further modified these strains by adding a non‐canonical pathway that uses malonyl‐CoA as precursor for poly(3‐hydroxybutyrate) biosynthesis. These manipulations led to increased polymer accumulation in the fully engineered strains, validating our general strategy to boost the output of malonyl‐CoA–dependent pathways in P. putida. Malonyl‐coenzyme A (CoA) is a key precursor for the biosynthesis of high‐value compounds by microbial cell factories, including polyketides, carboxylic acids, biofuels, and polyhydroxyalkanoates (PHAs). Here, we engineered Pseudomonas putida, a versatile bacterial platform, for enhanced malonyl‐CoA availability, validating the engineered strains for enhanced PHA accumulation from sugars.

Microbial metabolism of furanic compounds, especially furfural and 5-hydroxymethylfurfural (HMF), is rapidly gaining interest in the scientific community. This interest can largely be attributed to the occurrence of toxic furanic aldehydes in lignocellulosic hydrolysates. However, these compounds are also widespread in nature and in human processed foods, and are produced in industry. Although several microorganisms are known to degrade furanic compounds, the variety of species is limited mostly to Gram-negative aerobic bacteria, with a few notable exceptions. Furanic aldehydes are highly toxic to microorganisms, which have evolved a wide variety of defense mechanisms, such as the oxidation and/or reduction to the furanic alcohol and acid forms. These oxidation/reduction reactions constitute the initial steps of the biological pathways for furfural and HMF degradation. Furfural degradation proceeds via 2-furoic acid, which is metabolized to the primary intermediate 2-oxoglutarate. HMF is converted, via 2,5-furandicarboxylic acid, into 2-furoic acid. The enzymes in these HMF/furfural degradation pathways are encoded by eight hmf genes, organized in two distinct clusters in Cupriavidus basilensis HMF14. The organization of the five genes of the furfural degradation cluster is highly conserved among microorganisms capable of degrading furfural, while the three genes constituting the initial HMF degradation route are organized in a highly diverse manner. The genetic and biochemical characterization of the microbial metabolism of furanic compounds holds great promises for industrial applications such as the biodetoxifcation of lignocellulosic hydrolysates and the production of value-added compounds such as 2,5-furandicarboxylic acid.[PUBLICATION ABSTRACT]

Background Itaconic acid is a bio-derived platform chemical with uses ranging from polymer synthesis to biofuel production. The efficient conversion of cellulosic waste streams into itaconic acid could thus enable the sustainable production of a variety of substitutes for fossil oil based products. However, the realization of such a process is currently hindered by an expensive conversion of cellulose into fermentable sugars. Here, we present the stepwise development of a fully consolidated bioprocess (CBP), which is capable of directly converting recalcitrant cellulose into itaconic acid without the need for separate cellulose hydrolysis including the application of commercial cellulases. The process is based on a synthetic microbial consortium of the cellulase producer Trichoderma reesei and the itaconic acid producing yeast Ustilago maydis. A method for process monitoring was developed to estimate cellulose consumption, itaconic acid formation as well as the actual itaconic acid production yield online during co-cultivation. Results The efficiency of the process was compared to a simultaneous saccharification and fermentation setup (SSF). Because of the additional substrate consumption of T. reesei in the CBP, the itaconic acid yield was significantly lower in the CBP than in the SSF. In order to increase yield and productivity of itaconic acid in the CBP, the population dynamics was manipulated by varying the inoculation delay between T. reesei and U. maydis. Surprisingly, neither inoculation delay nor inoculation density significantly affected the population development or the CBP performance. Instead, the substrate availability was the most important parameter. U. maydis was only able to grow and to produce itaconic acid when the cellulose concentration and thus, the sugar supply rate, was high. Finally, the metabolic processes during fed-batch CBP were analyzed in depth by online respiration measurements. Thereby, substrate availability was again identified as key factor also controlling itaconic acid yield. In summary, an itaconic acid titer of 34 g/L with a total productivity of up to 0.07 g/L/h and a yield of 0.16 g/g could be reached during fed-batch cultivation. Conclusion This study demonstrates the feasibility of consortium-based CBP for itaconic acid production and also lays the fundamentals for the development and improvement of similar microbial consortia for cellulose-based organic acid production.

Ustilago maydis, a member of the Ustilaginaceae family, is a promising host for the production of several metabolites including itaconic acid. This dicarboxylate has great potential as a bio-based building block in the polymer industry, and is of special interest for pharmaceutical applications. Several itaconate overproducing Ustilago strains have been generated by metabolic and morphology engineering. This yielded stabilized unicellular morphology through fuz7 deletion, reduction of by-product formation through deletion of genes responsible for itaconate oxidation and (glyco)lipid production, and the overexpression of the regulator of the itaconate cluster ria1 and the mitochondrial tricarboxylate transporter encoded by mttA from Aspergillusterreus. In this study, itaconate production was further optimized by consolidating these different optimizations into one strain. The combined modifications resulted in itaconic acid production at theoretical maximal yield, which was achieved under biotechnologically relevant fed-batch fermentations with continuous feed.

Glucose is the most abundant monosaccharide and a principal substrate in biotechnological production processes. In Pseudomonas, this sugar is either imported directly into the cytosol or first oxidised to gluconate in the periplasm. While gluconate is taken up via a proton‐driven symporter, the import of glucose is mediated by an ABC‐type transporter, and hence both require energy. In this study, we heterologously expressed the energy‐independent glucose facilitator protein (Glf) from Zymomonas mobilis to replace the native energy‐demanding glucose transport systems, thereby increasing the metabolic energy efficiency. The implementation of passive glucose uptake in engineered production strains significantly increased product titres and yields of the two different aromatic products, cinnamic acid (+10%–15%) and resveratrol (+26%; 18.1 mg/g) in batch cultures. In this study, we heterologously expressed the energy‐independent glucose facilitator protein (Glf) from Zymomonas mobilis to replace the native energy‐demanding glucose transport systems, thereby increasing the metabolic energy efficiency. Implementation of passive glucose uptake in engineered production strains significantly increased product titres and yields of two different aromatic products.

Bio-upcycling of plastics is an emerging alternative process that focuses on extracting value from a wide range of plastic waste streams. Such streams are typically too contaminated to be effectively processed using traditional recycling technologies. Medium-chain-length (mcl) diols and dicarboxylates (DCA) are major products of chemically or enzymatically depolymerized plastics, such as polyesters or polyethers. In this study, we enabled the efficient metabolism of mcl-diols and -DCA in engineered Pseudomonas putida as a prerequisite for subsequent bio-upcycling. We identified the transcriptional regulator GcdR as target for enabling metabolism of uneven mcl-DCA such as pimelate, and uncovered amino acid substitutions that lead to an increased coupling between the heterologous β-oxidation of mcl-DCA and the native degradation of short-chain-length DCA. Adaptive laboratory evolution and subsequent reverse engineering unravelled two distinct pathways for mcl-diol metabolism in P. putida , namely via the hydroxy acid and subsequent native β-oxidation or via full oxidation to the dicarboxylic acid that is further metabolized by heterologous β-oxidation. Furthermore, we demonstrated the production of polyhydroxyalkanoates from mcl-diols and -DCA by a single strain combining all required metabolic features. Overall, this study provides a powerful platform strain for the bio-upcycling of complex plastic hydrolysates to polyhydroxyalkanoates and leads the path for future yield optimizations. Graphical Abstract