The solubilization and efficient upgrading of high loadings of polyethylene terephthalate (PET) are important challenges, and most solvents for PET are highly toxic. Herein, a low-cost (ca. $1.2 kg ) and biocompatible ionic liquid (IL), cholinium phosphate ([Ch] [PO ]), is demonstrated for the first time to play bifunctional roles in the solubilization and glycolytic degradation of PET. A high loading of PET (10 wt %) was readily dissolved in [Ch] [PO ] at relatively low temperatures (120 °C, 3 h) and under water-rich conditions. In-depth analysis of the solution revealed that high PET solubilization in [Ch] [PO ] could be ascribed to significant PET depolymerization. Acid precipitation yielded terephthalic acid as the dominant depolymerized monomer with a theoretical yield of approximately 95 %. Further exploration showed that in the presence of ethylene glycol (EG), the [Ch] [PO ]-catalyzed glycolysis of PET could efficiently occur with approximately 100 % conversion of PET and approximately 60.6 % yield of bis(2-hydroxyethyl)terephthalate under metal-free conditions. The IL could be reused at least three times without an apparent decrease in activity. NMR spectroscopy analysis revealed that strong hydrogen-bonding interactions between EG and the IL played an important role in the activation of EG and promotion of the glycolysis reaction. This study opens up avenues for exploring environmentally benign and efficient IL technology for solubilizing and recycling postconsumer polyester plastics.
Background: Lignocellulosic biomass is an important resource for renewable production of biofuels and bioproducts. Enzymes that deconstruct this biomass are critical for the viability of biomass-based biofuel production processes. Current commercial enzyme mixtures have limited thermotolerance. Thermophilic fungi may provide enzyme mixtures with greater thermal stability leading to more robust processes. Understanding the induction of biomassdeconstructing enzymes in thermophilic fungi will provide the foundation for strategies to construct hyper-production strains.Results: Induction of cellulases using xylan was demonstrated during cultivation of the thermophilic fungus Thermoascus aurantiacus. Simulated fed-batch conditions with xylose induced comparable levels of cellulases. These fed-batch conditions were adapted to produce enzymes in 2 and 19 L bioreactors using xylose and xylose-rich hydrolysate from dilute acid pretreatment of corn stover. Enzymes from T. aurantiacus that were produced in the xylose-fed bioreactor demonstrated comparable performance in the saccharification of deacetylated, dilute acid-pretreated corn stover when compared to a commercial enzyme mixture at 50 °C. The T. aurantiacus enzymes retained this activity at of 60 °C while the commercial enzyme mixture was largely inactivated. Conclusions:Xylose induces both cellulase and xylanase production in T. aurantiacus and was used to produce enzymes at up to the 19 L bioreactor scale. The demonstration of induction by xylose-rich hydrolysate and saccharification of deacetylated, dilute acid-pretreated corn stover suggests a scenario to couple biomass pretreatment with onsite enzyme production in a biorefinery. This work further demonstrates the potential for T. aurantiacus as a thermophilic platform for cellulase development.
Carbohydrate active enzymes (CAZymes) are vital for the lignocellulose-based biorefinery. The development of hypersecreting fungal protein production hosts is therefore a major aim for both academia and industry. However, despite advances in our understanding of their regulation, the number of promising candidate genes for targeted strain engineering remains limited. Here, we resequenced the genome of the classical hypersecreting Neurospora crassa mutant exo-1 and identified the causative point of mutation to reside in the F-box protein–encoding gene, NCU09899. The corresponding deletion strain displayed amylase and invertase activities exceeding those of the carbon catabolite derepressed strain Δcre-1, while glucose repression was still mostly functional in Δexo-1. Surprisingly, RNA sequencing revealed that while plant cell wall degradation genes are broadly misexpressed in Δexo-1, only a small fraction of CAZyme genes and sugar transporters are up-regulated, indicating that EXO-1 affects specific regulatory factors. Aiming to elucidate the underlying mechanism of enzyme hypersecretion, we found the high secretion of amylases and invertase in Δexo-1 to be completely dependent on the transcriptional regulator COL-26. Furthermore, misregulation of COL-26, CRE-1, and cellular carbon and nitrogen metabolism was confirmed by proteomics. Finally, we successfully transferred the hypersecretion trait of the exo-1 disruption by reverse engineering into the industrially deployed fungus Myceliophthora thermophila using CRISPR-Cas9. Our identification of an important F-box protein demonstrates the strength of classical mutants combined with next-generation sequencing to uncover unanticipated candidates for engineering. These data contribute to a more complete understanding of CAZyme regulation and will facilitate targeted engineering of hypersecretion in further organisms of interest.
Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.
Background Filamentous fungi are excellent lignocellulose degraders, which they achieve through producing carbohydrate active enzymes (CAZymes). CAZyme production is highly orchestrated and gene expression analysis has greatly expanded understanding of this important biotechnological process. The thermophilic fungus Thermoascus aurantiacus secretes highly active thermostable enzymes that enable saccharifications at higher temperatures; however, the genome-wide measurements of gene expression in response to CAZyme induction are not understood. Results A fed-batch system with plant biomass-derived sugars d-xylose, l-arabinose and cellobiose established that these sugars induce CAZyme expression in T. aurantiacus. The C5 sugars induced both cellulases and hemicellulases, while cellobiose specifically induced cellulases. A minimal medium formulation was developed to enable gene expression studies of T. aurantiacus with these inducers. It was found that d-xylose and l-arabinose strongly induced a wide variety of CAZymes, auxiliary activity (AA) enzymes and carbohydrate esterases (CEs), while cellobiose facilitated lower expression of mostly cellulase genes. Furthermore, putative orthologues of different unfolded protein response genes were up-regulated during the C5 sugar feeding together with genes in the C5 sugar assimilation pathways. Conclusion This work has identified two additional CAZyme inducers for T. aurantiacus, l-arabinose and cellobiose, along with d-xylose. A combination of biochemical assays and RNA-seq measurements established that C5 sugars induce a suite of cellulases and hemicellulases, providing paths to produce broad spectrum thermotolerant enzymatic mixtures.
35Background 36 Fungal enzymes are vital for industrial biotechnology, including the conversion of plant 37 biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using 38 enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance 39 compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus 40 aurantiacus produces large amounts of highly thermostable plant cell wall degrading enzymes. 41 However, no genetic tools have yet been developed for this fungus, which prevents strain 42 engineering efforts. The goal of this study was to develop strain engineering tools such as a 43 transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to 44 improve enzyme production. 45 46 Results 47Here we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. 48 aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly 49 developed transformation protocol was optimized and used to integrate an expression cassette of 50 the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. 51Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different 52 gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, 53 respectively. Lastly, a sexual crossing protocol was established using a hygromycin B-and a 5-54 fluororotic acid-resistant parent strain. Crossing and isolation of progeny on selective media was 55 completed in a week.56 57 4 Conclusion 58The genetic tools developed for T. aurantiacus can now be used individually or in 59 combination to further improve thermostable enzyme production by this fungus. 60 61 64 65 5 Background 66 Due to the potentially deleterious impacts of climate change, which is mainly caused by the 67 use of fossil resources, great efforts have been made to explore the applicability of lignocellulosic 68 plant biomass as sustainable alternative to fossil fuels. Lignocellulosic biomass is the most 69 abundant organic material on earth, consisting primarily of the sugar polymers cellulose and 70 hemicellulose and the aromatic polymer lignin [1,2]. These sugar polymers can be deconstructed 71 by enzymes (cellulases and hemicellulases) into simple sugars that can be further converted into 72 biofuels and other bio-based products using metabolically engineered bacterial and fungal hosts, 73 which will reduce our dependence on finite fossil resources [3]. The cost-efficient deconstruction 74 of lignocellulose is currently the largest obstacle preventing biofuels from becoming competitive 75 with fossil fuels. 76 Filamentous fungi are very effective lignocellulose degraders, possessing an arsenal of 77 secreted enzymes that digest cellulose and hemicellulose [4]. These organisms have evolved an 78 elaborate sensing system to detect the components of lignocellulosic biomass and fine-tune 79 expression of cellulase and hemicellulase gene...
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