Biotechnology for Biofuels最新文献

Biomass is greatly influenced by geographic location, soil composition, environment, and climate, making the efficient and accurate identification of growing areas highly significant. This study proposes a classification model for tobacco growing areas based on time series features from thermogravimetric analysis (TGA). This study combines Convolutional Neural Networks (CNN) with Long Short-Term Memory (LSTM) model to process the derivative thermogravimetric (DTG) data, aiming to uncover the inherent time series properties and the continuous and dynamic relationship between temperatures for classifying tobacco growing areas. By analyzing 375 tobacco samples from ten different provinces, CNN is employed to extract local features, while LSTM captures long-term dependencies in the DTG data. The dataset used in this study has a limited sample size, a wide variety of classes, and an imbalance in the number of samples across these classes. Despite these challenges, the model achieves 86.4% accuracy on the test set, significantly surpassing the performance of the traditional Support Vector Machine model, which only achieves 68.2% accuracy. Additionally, the model reveals key temperature ranges crucial for growing area classification associated with the pyrolysis temperature ranges of volatile components, hemicellulose, cellulose, lignin, and CaCO₃ in the tobacco. This model lays the groundwork for the future use of geographical labels to accurately represent tobacco’s style and quality, enabling more precise differentiation and improved quality control.

Background

Industrial lignocellulosic side streams are considered an attractive carbon source for the cultivation of biotechnologically important fungi, although the presence of toxic pretreatment by-products is a major challenge yet to be overcome. Aspergillus oryzae is a filamentous fungus with a large secretion capacity, high tolerance for toxins, and a wide substrate range, making it a promising candidate for side stream utilization. In the present study, the cellular mechanisms of tolerance against furfural, 5-hydroxymethylfurfural (HMF), levulinic acid, ferulic acid, and vanillin were studied at the transcriptome level.

Results

A. oryzae RIB40 was grown in the presence of different inhibitors commonly found in lignocellulosic side streams, and RNA sequencing was utilized to investigate the transcriptomic changes in response to the inhibitors. Analysis of the transcriptomic response in all conditions indicates that a large fraction of differentially expressed genes responded to the inhibitor-induced formation of reactive oxygen species (ROS). Apart from levulinic acid, all inhibitors showed strong initial suppression of metabolic pathways related to cell cycle, ribosome functions, protein folding, and sorting in the endoplasmic reticulum. Genes associated with cellular detoxification, namely, NAD(P)H-dependent oxidoreductases and efflux transporters, such as the ATP-Binding Cassette (ABC) transporters and major facilitator superfamily (MFS) transporters, showed strong upregulation upon exposure to the inhibitors.

Conclusions

The results obtained provide important insights into the stress response of A. oryzae to the xenobiotic compounds and their cellular detoxification. Aldehydic inhibitors, especially HMF, caused a strong and severe stress response in A. oryzae RIB40. Additionally, we identified several highly upregulated uncharacterized genes upon exposure to the inhibitors. These genes serve as promising targets for strain engineering to build robust industrial strains capable of utilizing lignocellulosic side streams as feedstock.

Advances in tissue printing and wound healing necessitate a continuous global supply of collagen. Microbial systems are highly desirable to meet these demands as recombinant collagenous proteins can be guaranteed as free from animal viruses. The filamentous cell factory Aspergillus niger has been instrumental for decades in the production of organic acids, enzymes and proteins, yet this fungus has not been explored for recombinant collagen production. In this study, we conducted extensive genetic engineering and fermentation optimization to provide proof of principle that A. niger can produce hydroxylated collagen. We used a modular cloning system to generate a suite of cassettes encoding numerous N-terminal secretion signals, native collagen genes and, additionally, various prolyl-4-hydroxylases (P4H) for protein hydroxylation. Collagen transcription was supported by both luciferase reporter and eGFP tagged approaches. Peptide sequencing from culture supernatant confirmed A. niger produced partially hydroxylated collagen. We then conducted a range of media optimizations and RNA sequencing to, respectively, increase collagen production and identify proteases which we hypothesized were detrimental to recombinant protein titers. Thus, we deleted an endopeptidase encoding gene, protA, which was likely responsible for degrading secreting collagen. Ultimately, we were able to generate an isolate capable of producing hydroxylated collagen at titers of 5 mgL⁻¹ in shake flask models of fermentation. This study thus proves A. niger is a promising heterologous system to address the demand for virus-free collagen.

Background

The red alga Palmaria palmata is a rich source of sugar compounds, particularly mixed-linkage xylans present in the cell walls of the algae. In contrast to their terrestrial lignocellulosic counterparts, these xylans are more easily accessible. They can be hydrolyzed enzymatically into valuable xylooligosaccharides (XOS), known for their prebiotic, antioxidant, and immunomodulatory properties.

Results

This study introduces a simplified, one-step enzymatic process utilizing the endo-1,4-β-xylanase FO15_GH10 that directly hydrolyzes P. palmata biomass to produce XOS, eliminating the need for prior xylan extraction and improving efficiency. The exact structure of the resulting XOS was determined using NMR and MS/MS techniques. In addition, the xylosidase FO17_GH43 can be added to break down all residual 1,4-linked XOS. As a result, only 1,3- and mixed-linkage XOS (degree of polymerization (DP) 2–4) remains under simultaneous increase of the xylose obtained. Using FO15_GH10 alone, it was possible to produce approximately 17.6 (± 0.16) % (176 mg) XOS from 1 g of powdered biomass while combining both enzymes resulted in 22.6 (± 0.51) % (226 mg) XOS. Further optimization upon upscaling offers the possibility of achieving even greater improvements.

Conclusion

In summary, our one-step enzymatic approach offers an efficient and sustainable method for producing XOS directly from P. palmata biomass. This streamlined process overcomes the need for resource-consuming extraction processes. The further characterization of the obtained XOS and the potential to gain solely 1,3- and mixed-linkage XOS is paving the way for future studies on their functional properties.

3-Methyl-1-butanol (3MB) is a promising renewable solvent, drop-in fuel, and precursor for various industrial products, including flavors, fragrances, and surfactants. Due to the myriad of intertwined biosynthetic pathways that share metabolic precursors, conventional metabolic engineering strategies to overproduce 3MB in yeast have typically resulted in yields that are far too low for economic viability. However, because 3MB is naturally produced by yeast, 100 million liter of 3MB are already produced annually as a byproduct of bioethanol fermentations. Despite its significant commercial value, this 3MB fraction is currently discarded due to its low relative concentration within the fusel alcohol mixture. Here, we present a novel strategy to produce 3MB along with the conventional bioethanol fermentation, leveraging the existing bioethanol industry by valorizing the discarded fusel alcohol byproduct stream. We first identified a robust industrially relevant chassis strain and explored different strategies to alleviate the valine and leucine feedback inhibition within the 3MB pathway, showing that mutating the leucine-inhibition site of Leu4p increased 3MB yield by 2.9-fold. Finally, we tested in silico-predicted gene deletion targets to reduce the byproduct acetate. Our final engineered strain achieved a 4.4-fold increase in 3MB yield compared to the wild type (1.5 mg/g sugars), average productivity of 5 mg/Lh, and a 3MB proportion increase from 42 to 71% within the fusel alcohol mix, while ethanol production remained comparable to the Ethanol Red® industrial reference. Our study thus opens a new route for co-producing 3MB and ethanol from sugarcane molasses in Saccharomyces cerevisiae, laying the groundwork toward an economically viable and sustainable approach for 3MB production alongside existing bioethanol production.

Graphical Abstract

Background

Cyanobacteria are promising platforms for metabolic engineering to convert carbon dioxide into valuable fuels and chemicals, addressing both energy demands and global climate change. Among various fuels and chemicals, isobutanol (IB) and 3-methyl-1-butanol (3M1B) have gained increasing attention due to their superior physical properties, such as high energy density, low water solubility, and low hygroscopicity. Heterologously expressing α-ketoisovalerate decarboxylase (Kivd^S286T) in the unicellular cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis) enables microbial production of IB and 3M1B through the 2-keto acid pathway, with Kivd^S286T identified as a key bottleneck limiting production efficiency.

Results

To address this limitation, a high-throughput screening method based on the consumption of the substrate 2-ketoisovalerate was successfully established. This screen was coupled with random mutagenesis, via error-prone PCR, of Kivd^S286T. Out of the 1600 variants, 1B12, featuring dual substitutions K419E and T186S, exhibited a 55% increase in IB production and a 50% increase in 3M1B production in Synechocystis on the fourth day of cultivation. The crystal structure of Kivd^S286T was determined as a tetramer with a resolution of 2.8 Å to provide a framework for analyzing the structural basis for the enhanced butanol production conferred by the K419E and T186S substitutions.

Conclusions

A novel Kivd variant, 1B12, was successfully generated via directed evolution, with enhanced catalytic activity for microbial IB and 3M1B biosynthesis. To our knowledge, this study represents the first successful application of directed evolution on the rate-limiting enzyme of a specific metabolic pathway to enhance biochemical production in cyanobacteria.

Background

Anaerobic digestion (AD) or acidogenic fermentation (AF) of biomass can generate either biogas fuel or C₂ ‒ C₈ volatile fatty acids (VFAs) as feedstocks for synthesis of other petrochemical products. Typical AD feedstocks require large amounts of land that could otherwise be used for food production. Unlike these traditional bioenergy crops, plants using the crassulacean acid metabolism pathway (CAM), such as cacti and succulents, may be cultivated on degraded or semi-arid land that cannot support conventional agriculture. This could allow significant biorefinery feedstock to be sourced with minimal impact on existing agriculture or biodiversity. Several economically important CAM crops (e.g. pineapple, agave, prickly pear) are cultivated globally, with waste biomass that could be valorised as a biorefinery feedstock.

Results

Here, we investigate the fermentation kinetics of this novel feedstock class (CAM plants) against traditional bioenergy crops with two contrasting inocula: AD sludge and rumen fluid. Fermentations were performed under the influence of a methanogenesis inhibitor (bromoethane sulfonate) to isolate the acidogenic fermentation processes. CAM and non-CAM substrates in this study demonstrated distinct degradation kinetics (yields and degradation rates). We demonstrate that regardless of the inoculum type, CAM crops show higher hydrolysis rates for VFA production. Moreover, yields of VFAs from three CAM crops (0.41 ± 0.01 – 0.48 ± 0.02 g/g_vs) were higher than for the three non-CAM crops (0.21 ± 0.01 – 0.38 ± 0.01 g/g_vs) when AD sludge was used as the inoculum. This superior performance appeared to correlate with a higher abundance of soluble material and lower structural carbohydrate content in CAM biomass.

Conclusions

At industrial scale, the observed kinetic advantages of VFA production from CAM-plant feedstocks could translate into process enhancements that would greatly improve the cost-competitiveness of anaerobic biorefinery. Assuming comparable biomass productivities of CAM and non-CAM crops, this high yield could allow higher VFA production per unit of cultivated land, improving the environmental credentials of CAM biorefinery.

Graphical abstract

Unspecific peroxygenases (UPOs) are versatile enzymes capable of oxidizing a broad range of substrates, using hydrogen peroxide as the sole co-substrate. In this study, UPOs were evaluated for their potential in the selective oxyfunctionalization of the phenolic lignin monomer 4-propylguaiacol (4-PG) to generate versatile scaffolds for the synthesis of high-value compounds. In addition to the desired peroxygenase reaction, the phenolic group of 4-PG is susceptible to undesirable one-electron oxidation (peroxidase activity). Assessment of the activity of 19 UPOs from phylogenetically diverse clades toward 4-PG revealed that several UPOs could serve as potential biocatalysts for the functionalization of 4-PG, with some enzymes showing both promising conversion yields (>50%) and regioselectivity for the peroxygenase reaction. Pronounced differences in peroxygenase:peroxidase activity ratios and regioselectivity were observed. Comparative analysis—supported by experimental activity profiles and structural data—suggest that a more constrained active-site topology contributes to the peroxygenase activity. UPOs from a clade within the Ascomycota phylum with high peroxygenase activity possess a unique aliphatic pocket in their catalytic centers. Our study provides valuable insights into the structure–function relationships underpinning enhanced peroxygenase activity of UPOs and provides a functional mapping of a broad UPO-sequence space for 4-PG, highlighting these enzymes as promising catalysts for the selective oxyfunctionalization of a phenolic lignin monomer.

Background

A deployable, continuous enzymatic hydrolysis (CEH) process can address cost and commercialization risks associated with second-generation (Gen2) biorefinery sugar/lignin/ethanol production while contributing to energy supply and security. Developments in commercial enzymatic hydrolysis formulations targeting Gen2 pretreated biomass such as deacetylated mechanically refined (DMR) biomass necessitate a reassessment of the existing hybrid simultaneous saccharification and fermentation (SSF) approach. Notably, the practice of "finishing hydrolysis" in SSF has become problematic with the introduction of oxidative enzymes, such as lytic polysaccharide monooxygenases (LPMOs), into commercial cellulase formulations as these require specific redox conditions and cofactor. Moreover, continuous SSF has not been demonstrated at commercial scale, limiting deployment and the associated economic benefits to farmers, producers, and support industries.

Results

Continuous enzymatic hydrolysis (CEH) was demonstrated at bench scale to enable optimal saccharification performance of deacetylated mechanically refined (DMR) pretreated biomass. Diafiltration was demonstrated to retain pretreated biomass solids and enzymes for continuous reaction while removing solubilized product sugars in situ. A significant breakthrough afforded by the CEH process is its ability to achieve equivalent endpoint conversions with approximately 50% lower enzyme loading. Yields of glucose and xylose were increased ~ 15% and ~ 4%, respectively, over batch hydrolysis. Unlike SSF using yeast or Zymomonas, CEH allows precise optimization of pH, temperature, oxygen tension, LPMO mediator concentration, and removal of end-product inhibitors.

Conclusions

Advanced CEH holds promise as a transformational, process-intensified, and cost-effective method for producing soluble clarified biomass sugars and insoluble lignin-rich streams. Enhancing saccharification performance, optimizing operating parameters, and employing membrane filtration will help overcome existing challenges and enable the efficient production of valuable biomaterials from lignocellulosic biomass.

Plant specialised metabolism generates a vast array of compounds with significant potential across agriculture, medicine, cosmetics, and the food industry. A key challenge lies in optimising their production in the plant, as these compounds are often present in trace amounts in a complex metabolic cocktail. Given their high economic value, extensive efforts have been made to elucidate their biosynthetic pathways and pinpoint key regulatory and enzymatic targets. This knowledge has been applied for metabolic engineering to enhance the carbon flux towards metabolites of interest, thereby broadening the utility of plants as a source of high-value compounds. This review examines different metabolic engineering strategies employed today using the phenylpropanoid pathway as a case study and highlights the potential of integrating plant and microbial research to drive cross-disciplinary innovation.