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Directed evolution of hydrocarbon-producing enzymes 产烃酶的定向进化
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-12 DOI: 10.1186/s13068-025-02689-4
Jochem R. Nielsen, Joseph Kennerley, Wei E. Huang
{"title":"Directed evolution of hydrocarbon-producing enzymes","authors":"Jochem R. Nielsen,&nbsp;Joseph Kennerley,&nbsp;Wei E. Huang","doi":"10.1186/s13068-025-02689-4","DOIUrl":"10.1186/s13068-025-02689-4","url":null,"abstract":"<div><p>Enzymes capable of catalysing the production of hydrocarbons hold promise for sustainable fuel synthesis. However, the native activities of these enzymes are often insufficient for their exploitation in industrial bioprocesses. Enzyme engineering approaches including directed evolution (DE) can be used to improve the properties of enzymes to meet desirable standards for their industrial application. In this review, we summarise DE methods for engineering hydrocarbon-producing enzymes, including both screening- and selection procedures. The efficacy of DE depends on several factors, including sensitive and accurate detection of enzyme activity, the throughput of screening or selection steps, and the scale of diversity generation. Although DE is a well-established approach, its application in engineering hydrocarbon-producing enzymes has not been widely demonstrated. This can be attributed to the physiochemical properties of the target molecules, such as aliphatic hydrocarbons, which can be insoluble, gaseous, and chemically inert. Detection of these molecules in vivo presents several unique challenges, as does dynamically coupling their abundance to cell fitness. We conclude with a discussion on future directions and potential advancements in this field.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02689-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144832253","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
A model for tobacco growing area classification based on time series features of thermogravimetric analysis 基于热重分析时间序列特征的烟草种植区分类模型
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-11 DOI: 10.1186/s13068-025-02682-x
Jiaxu Xia, Yunong Tian, Xianwei Hao, Yuhan Peng, Guanqun Luo, Zhihua Gan
{"title":"A model for tobacco growing area classification based on time series features of thermogravimetric analysis","authors":"Jiaxu Xia,&nbsp;Yunong Tian,&nbsp;Xianwei Hao,&nbsp;Yuhan Peng,&nbsp;Guanqun Luo,&nbsp;Zhihua Gan","doi":"10.1186/s13068-025-02682-x","DOIUrl":"10.1186/s13068-025-02682-x","url":null,"abstract":"<div><p>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 CaCO<sub>3</sub> 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.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02682-x","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144810856","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Transcriptome analysis of Aspergillus oryzae RIB40 under chemical stress reveals mechanisms of adaptation to fungistatic compounds of lignocellulosic side streams 化学胁迫下米曲霉RIB40转录组分析揭示了其对木质纤维素侧流抑菌化合物的适应机制。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-08 DOI: 10.1186/s13068-025-02688-5
Miika-Erik Korpioja, Emmi Sveholm, Adiphol Dilokpimol, Tanja Paasela, Andriy Kovalchuk
{"title":"Transcriptome analysis of Aspergillus oryzae RIB40 under chemical stress reveals mechanisms of adaptation to fungistatic compounds of lignocellulosic side streams","authors":"Miika-Erik Korpioja,&nbsp;Emmi Sveholm,&nbsp;Adiphol Dilokpimol,&nbsp;Tanja Paasela,&nbsp;Andriy Kovalchuk","doi":"10.1186/s13068-025-02688-5","DOIUrl":"10.1186/s13068-025-02688-5","url":null,"abstract":"<div><h3>Background</h3><p>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. <i>Aspergillus oryzae</i> 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.</p><h3>Results</h3><p><i>A. oryzae</i> 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.</p><h3>Conclusions</h3><p>The results obtained provide important insights into the stress response of <i>A. oryzae</i> to the xenobiotic compounds and their cellular detoxification. Aldehydic inhibitors, especially HMF, caused a strong and severe stress response in <i>A. oryzae</i> 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.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12335040/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144805384","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
The multipurpose cell factory Aspergillus niger can be engineered to produce hydroxylated collagen 多用途细胞工厂黑曲霉可以工程生产羟基化胶原。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-08 DOI: 10.1186/s13068-025-02681-y
Tom Morris, Friederike Gerstl, Sascha Jung, Timothy C. Cairns, Vera Meyer
{"title":"The multipurpose cell factory Aspergillus niger can be engineered to produce hydroxylated collagen","authors":"Tom Morris,&nbsp;Friederike Gerstl,&nbsp;Sascha Jung,&nbsp;Timothy C. Cairns,&nbsp;Vera Meyer","doi":"10.1186/s13068-025-02681-y","DOIUrl":"10.1186/s13068-025-02681-y","url":null,"abstract":"<div><p>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 <i>Aspergillus niger</i> 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 <i>A. niger</i> 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 <i>A. niger</i> 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, <i>protA</i>, 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<sup>−1</sup> in shake flask models of fermentation. This study thus proves <i>A. niger</i> is a promising heterologous system to address the demand for virus-free collagen.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12333218/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144805383","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Producing mixed linked xylooligosaccharides from red algae biomass through single-step enzymatic hydrolysis 通过一步酶解从红藻生物质中生产混合连接低聚木糖。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-05 DOI: 10.1186/s13068-025-02686-7
Michelle Teune, Christoph Suster, Yannick Wolf, Nils Michels, Henrieke Mieth, Thorben Döhler, Daniel Bartosik, Joris Krull, Jan-Hendrik Hehemann, Thomas Schweder, Christian Stanetty, Uwe T. Bornscheuer
{"title":"Producing mixed linked xylooligosaccharides from red algae biomass through single-step enzymatic hydrolysis","authors":"Michelle Teune,&nbsp;Christoph Suster,&nbsp;Yannick Wolf,&nbsp;Nils Michels,&nbsp;Henrieke Mieth,&nbsp;Thorben Döhler,&nbsp;Daniel Bartosik,&nbsp;Joris Krull,&nbsp;Jan-Hendrik Hehemann,&nbsp;Thomas Schweder,&nbsp;Christian Stanetty,&nbsp;Uwe T. Bornscheuer","doi":"10.1186/s13068-025-02686-7","DOIUrl":"10.1186/s13068-025-02686-7","url":null,"abstract":"<div><h3>Background</h3><p>The red alga <i>Palmaria palmata</i> 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.</p><h3>Results</h3><p>This study introduces a simplified, one-step enzymatic process utilizing the <i>endo</i>-1,4-<i>β</i>-xylanase FO15_GH10 that directly hydrolyzes <i>P. palmata</i> 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.</p><h3>Conclusion</h3><p>In summary, our one-step enzymatic approach offers an efficient and sustainable method for producing XOS directly from <i>P. palmata</i> 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.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12323150/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144791018","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Metabolic engineering of Saccharomyces cerevisiae for co-production of ethanol and 3-methyl-1-butanol from sugarcane molasses 甘蔗糖蜜协同生产乙醇和3-甲基-1-丁醇的酿酒酵母代谢工程研究。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-08-01 DOI: 10.1186/s13068-025-02685-8
Sasha Yogiswara, Jonas Rombout, Giovanni Micharikopoulos, Sam De Craemer, Beatriz Herrera-Malaver, Lotte van Landschoot, Sofie Mannaerts, Marcelo do Amaral, Karin Voordeckers, Stijn Spaepen, Jan Steensels, Quinten Deparis, Bart Ghesquière, Kevin J. Verstrepen
{"title":"Metabolic engineering of Saccharomyces cerevisiae for co-production of ethanol and 3-methyl-1-butanol from sugarcane molasses","authors":"Sasha Yogiswara,&nbsp;Jonas Rombout,&nbsp;Giovanni Micharikopoulos,&nbsp;Sam De Craemer,&nbsp;Beatriz Herrera-Malaver,&nbsp;Lotte van Landschoot,&nbsp;Sofie Mannaerts,&nbsp;Marcelo do Amaral,&nbsp;Karin Voordeckers,&nbsp;Stijn Spaepen,&nbsp;Jan Steensels,&nbsp;Quinten Deparis,&nbsp;Bart Ghesquière,&nbsp;Kevin J. Verstrepen","doi":"10.1186/s13068-025-02685-8","DOIUrl":"10.1186/s13068-025-02685-8","url":null,"abstract":"<div><p>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 <i>Saccharomyces cerevisiae</i>, laying the groundwork toward an economically viable and sustainable approach for 3MB production alongside existing bioethanol production.</p><h3>Graphical Abstract</h3>\u0000<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12317478/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144765889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Directed evolution of α-ketoisovalerate decarboxylase for improved isobutanol and 3-methyl-1-butanol production in cyanobacteria α-酮异戊酸脱羧酶在蓝藻中促进异丁醇和3-甲基-1-丁醇生产的定向进化。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-07-31 DOI: 10.1186/s13068-025-02687-6
Hao Xie, Afshan Begum, Laura H. Gunn, Peter Lindblad
{"title":"Directed evolution of α-ketoisovalerate decarboxylase for improved isobutanol and 3-methyl-1-butanol production in cyanobacteria","authors":"Hao Xie,&nbsp;Afshan Begum,&nbsp;Laura H. Gunn,&nbsp;Peter Lindblad","doi":"10.1186/s13068-025-02687-6","DOIUrl":"10.1186/s13068-025-02687-6","url":null,"abstract":"<div><h3>Background</h3><p>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 <i>α</i>-ketoisovalerate decarboxylase (Kivd<sup>S286T</sup>) in the unicellular cyanobacterium <i>Synechocystis</i> sp. PCC 6803 (<i>Synechocystis</i>) enables microbial production of IB and 3M1B through the 2-keto acid pathway, with Kivd<sup>S286T</sup> identified as a key bottleneck limiting production efficiency.</p><h3>Results</h3><p>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<sup>S286T</sup>. 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 <i>Synechocystis</i> on the fourth day of cultivation. The crystal structure of Kivd<sup>S286T</sup> 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.</p><h3>Conclusions</h3><p>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.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12312269/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144762673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Biorefinery for a circular carbon paradigm: process benefits to the use of dryland CAM crops for anaerobic volatile fatty acid production 循环碳范例的生物炼制:利用旱地CAM作物生产厌氧挥发性脂肪酸的过程有益。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-07-31 DOI: 10.1186/s13068-025-02636-3
Nicholas A. Tenci, Nichola Austen, Laura K. Martin, J. Andrew C. Smith, Ian P. Thompson
{"title":"Biorefinery for a circular carbon paradigm: process benefits to the use of dryland CAM crops for anaerobic volatile fatty acid production","authors":"Nicholas A. Tenci,&nbsp;Nichola Austen,&nbsp;Laura K. Martin,&nbsp;J. Andrew C. Smith,&nbsp;Ian P. Thompson","doi":"10.1186/s13068-025-02636-3","DOIUrl":"10.1186/s13068-025-02636-3","url":null,"abstract":"<div><h3>Background</h3><p>Anaerobic digestion (AD) or acidogenic fermentation (AF) of biomass can generate either biogas fuel or C<sub>2</sub> ‒ C<sub>8</sub> 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.</p><h3>Results</h3><p>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<sub>vs</sub>) were higher than for the three non-CAM crops (0.21 ± 0.01 – 0.38 ± 0.01 g/g<sub>vs</sub>) 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.</p><h3>Conclusions</h3><p>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.</p><h3>Graphical abstract</h3><div><figure><div><div><picture><source><img></source></picture></div></div></figure></div></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-07-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12315394/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144762672","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Structure–function relationships in unspecific peroxygenases revealed by a comparative study of their action on the phenolic lignin monomer 4-propylguaiacol 非特异性过氧酶对酚木质素单体4-丙基愈木酚作用的比较研究揭示了它们的结构-功能关系。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-07-28 DOI: 10.1186/s13068-025-02675-w
Marta Barros-Reguera, Esteban Lopez-Tavera, Gabriela C. Schröder, Greta Nardini, Kenneth A. Kristoffersen, Iván Ayuso-Fernández, Vincent G. H. Eijsink, Morten Sørlie
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引用次数: 0
Advancing continuous enzymatic hydrolysis for improved biomass saccharification 推进连续酶解改善生物质糖化。
IF 6.1 1区 工程技术
Biotechnology for Biofuels Pub Date : 2025-07-25 DOI: 10.1186/s13068-025-02680-z
Roman Brunecky, Yudong Li, Stephen R. Decker, Michael E. Himmel
{"title":"Advancing continuous enzymatic hydrolysis for improved biomass saccharification","authors":"Roman Brunecky,&nbsp;Yudong Li,&nbsp;Stephen R. Decker,&nbsp;Michael E. Himmel","doi":"10.1186/s13068-025-02680-z","DOIUrl":"10.1186/s13068-025-02680-z","url":null,"abstract":"<div><h3>Background</h3><p>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.</p><h3>Results</h3><p>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 <i>Zymomonas</i>, CEH allows precise optimization of pH, temperature, oxygen tension, LPMO mediator concentration, and removal of end-product inhibitors.</p><h3>Conclusions</h3><p>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.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12291225/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144719258","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
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