The range of applications for industrial hemp has consistently increased in various sectors over the years. For example, hemp hurd can be used as a resource to produce biodegradable packaging ...materials when incorporated into a fungal mycelium composite, a process that has been commercialized. Although these packaging materials can be composted after usage, they may present an opportunity for valorization in a biorefinery setting. Here, we demonstrate the potential of using this type of discarded packaging composite as a feedstock for biofuel production. A one-pot ionic liquid-based biomass deconstruction and conversion process was implemented, and the results from the packaging material were compared with those obtained from untreated hemp hurd. At a 120 °C reaction temperature, 7.5% ionic liquid loading, and 2 h reaction time, the packaging materials showed a higher lignocellulosic sugar yield and sugar concentrations than hemp hurd. Hydrolysates prepared from packaging materials also promoted production of higher titers (1400 mg/L) of the jet-fuel precursor bisabolene when used to cultivate an engineered strain of the yeast
. Box-Behnken experiments revealed that pretreatment parameters affected the hemp hurd and packaging materials differently, evidencing different degrees of recalcitrance. This study demonstrated that a hemp hurd-based packaging material can be valorized a second time once it reaches the end of its primary use by supplying it as a feedstock to produce biofuels.
Endophytic fungi are ubiquitous plant endosymbionts that establish complex and poorly understood relationships with their host organisms. Many endophytic fungi are known to produce a wide spectrum of ...volatile organic compounds (VOCs) with potential energy applications, which have been described as "mycodiesel". Many of these mycodiesel hydrocarbons are terpenes, a chemically diverse class of compounds produced by many plants, fungi, and bacteria. Due to their high energy densities, terpenes, such as pinene and bisabolene, are actively being investigated as potential "drop-in" biofuels for replacing diesel and aviation fuel. In this study, we rapidly discovered and characterized 26 terpene synthases (TPSs) derived from four endophytic fungi known to produce mycodiesel hydrocarbons. The TPS genes were expressed in an E. coli strain harboring a heterologous mevalonate pathway designed to enhance terpene production, and their product profiles were determined using Solid Phase Micro-Extraction (SPME) and GC-MS. Out of the 26 TPS's profiled, 12 TPS's were functional, with the majority of them exhibiting both monoterpene and sesquiterpene synthase activity.
Aspergillus niger and other filamentous fungi are widely used in industry, but efficient genetic engineering of these hosts remains nascent. For example, while molecular genetic tools have been ...developed, including CRISPR/Cas9, facile genome engineering of A. niger remains challenging. To address these challenges, we have developed a simple Cas9-based gene targeting method that provides selectable, iterative, and ultimately marker-free generation of genomic deletions and insertions. This method leverages locus-specific "pop-out" recombination to suppress off-target integrations. We demonstrated the effectiveness of this method by targeting the phenotypic marker albA and validated it by targeting the glaA and mstC loci. After two selection steps, we observed 100% gene editing efficiency across all three loci. This method greatly reduces the effort required to engineer the A. niger genome and overcomes low Cas9 transformations efficiency by eliminating the need for extensive screening. This method represents a significant addition to the A. niger genome engineering toolbox and could be adapted for use in other organisms. It is expected that this method will impact several areas of industrial biotechnology, such as the development of new strains for the secretion of heterologous enzymes and the discovery and optimization of metabolic pathways.
Aspergillus section Nigri comprises filamentous fungi relevant to biomedicine, bioenergy, health, and biotechnology. To learn more about what genetically sets these species apart, as well as about ...potential applications in biotechnology and biomedicine, we sequenced 23 genomes de novo, forming a full genome compendium for the section (26 species), as well as 6 Aspergillus niger isolates. This allowed us to quantify both inter- and intraspecies genomic variation. We further predicted 17,903 carbohydrate-active enzymes and 2,717 secondary metabolite gene clusters, which we condensed into 455 distinct families corresponding to compound classes, 49% of which are only found in single species. We performed metabolomics and genetic engineering to correlate genotypes to phenotypes, as demonstrated for the metabolite aurasperone, and by heterologous transfer of citrate production to Aspergillus nidulans. Experimental and computational analyses showed that both secondary metabolism and regulation are key factors that are significant in the delineation of Aspergillus species.
Lignin is nature's largest source of phenolic compounds. Its recalcitrance to enzymatic conversion is still a limiting step to increase the value of lignin. Although bacteria are able to degrade ...lignin in nature, most studies have focused on lignin degradation by fungi. To understand which bacteria are able to use lignin as the sole carbon source, natural selection over time was used to obtain enriched microbial consortia over a 12-week period. The source of microorganisms to establish these microbial consortia were commercial and backyard compost soils. Cultivation occurred at two different temperatures, 30°C and 37°C, in defined culture media containing either Kraft lignin or alkaline-extracted lignin as carbon source. iTag DNA sequencing of bacterial 16S rDNA gene was performed for each of the consortia at six timepoints (passages). The initial bacterial richness and diversity of backyard compost soil consortia was greater than that of commercial soil consortia, and both parameters decreased after the enrichment protocol, corroborating that selection was occurring. Bacterial consortia composition tended to stabilize from the fourth passage on. After the enrichment protocol, Firmicutes phylum bacteria were predominant when lignin extracted by alkaline method was used as a carbon source, whereas Proteobacteria were predominant when Kraft lignin was used. Bray-Curtis dissimilarity calculations at genus level, visualized using NMDS plots, showed that the type of lignin used as a carbon source contributed more to differentiate the bacterial consortia than the variable temperature. The main known bacterial genera selected to use lignin as a carbon source were Altererythrobacter, Aminobacter, Bacillus, Burkholderia, Lysinibacillus, Microvirga, Mycobacterium, Ochrobactrum, Paenibacillus, Pseudomonas, Pseudoxanthomonas, Rhizobiales and Sphingobium. These selected bacterial genera can be of particular interest for studying lignin degradation and utilization, as well as for lignin-related biotechnology applications.
R. toruloides is an oleaginous yeast, with diverse metabolic capacities and high tolerance for inhibitory compounds abundant in plant biomass hydrolysates. While R. toruloides grows on several ...pentose sugars and alcohols, further engineering of the native pathway is required for efficient conversion of biomass-derived sugars to higher value bioproducts. A previous high-throughput study inferred that R. toruloides possesses a non-canonical L-arabinose and D-xylose metabolism proceeding through D-arabitol and D-ribulose. In this study, we present a combination of genetic and metabolite data that refine and extend that model. Chiral separations definitively illustrate that D-arabitol is the enantiomer that accumulates under pentose metabolism. Deletion of putative D-arabitol-2-dehydrogenase (RTO4_9990) results in > 75% conversion of D-xylose to D-arabitol, and is growth-complemented on pentoses by heterologous xylulose kinase expression. Deletion of putative D-ribulose kinase (RTO4_14368) arrests all growth on any pentose tested. Analysis of several pentose dehydrogenase mutants elucidates a complex pathway with multiple enzymes mediating multiple different reactions in differing combinations, from which we also inferred a putative L-ribulose utilization pathway. Our results suggest that we have identified enzymes responsible for the majority of pathway flux, with additional unknown enzymes providing accessory activity at multiple steps. Further biochemical characterization of the enzymes described here will enable a more complete and quantitative understanding of R. toruloides pentose metabolism. These findings add to a growing understanding of the diversity and complexity of microbial pentose metabolism.
The basidiomycete yeast
(also known as
) accumulates high concentrations of lipids and carotenoids from diverse carbon sources. It has great potential as a model for the cellular biology of lipid ...droplets and for sustainable chemical production. We developed a method for high-throughput genetics (RB-TDNAseq), using sequence-barcoded
T-DNA insertions. We identified 1,337 putative essential genes with low T-DNA insertion rates. We functionally profiled genes required for fatty acid catabolism and lipid accumulation, validating results with 35 targeted deletion strains. We identified a high-confidence set of 150 genes affecting lipid accumulation, including genes with predicted function in signaling cascades, gene expression, protein modification and vesicular trafficking, autophagy, amino acid synthesis and tRNA modification, and genes of unknown function. These results greatly advance our understanding of lipid metabolism in this oleaginous species and demonstrate a general approach for barcoded mutagenesis that should enable functional genomics in diverse fungi.
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•Sequential bioprocess demonstrated effective conversion of cellulose to bisabolene.•Maximum bisabolene titre of 7.8 g/L achieved, the highest reported microbial titre.•Organic acids ...are an effective carbon source for R. toruloides.•Bisabolene titres improved 7.4-fold through pH controlled organic acid feeding.•Fermentation of cellulose by R. flavefaciens yielded up to 5.1 g/L organic acids.
In this study, organic acids were demonstrated as a promising carbon source for bisabolene production by the non-conventional yeast, Rhodosporidium toruloides, at microscale with a maximum titre of 1055 ± 7 mg/L. A 125-fold scale-up of the optimal process, enhanced bisabolene titres 2.5-fold to 2606 mg/L. Implementation of a pH controlled organic acid feeding strategy at this scale lead to a further threefold improvement in bisabolene titre to 7758 mg/L, the highest reported microbial titre. Finally, a proof-of-concept sequential bioreactor approach was investigated. Firstly, the cellulolytic bacterium Ruminococcus flavefaciens was employed to ferment cellulose, yielding 4.2 g/L of organic acids. R. toruloides was subsequently cultivated in the resulting supernatant, producing 318 ± 22 mg/L of bisabolene. This highlights the feasibility of a sequential bioprocess for the bioconversion of cellulose, into biojet fuel candidates. Future work will focus on enhancing organic acid yields and the use of real lignocellulosic feedstocks to further enhance bisabolene production.
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