Biotechnology for Biofuels最新文献

{"title":"A characterized promoter suite enables metabolic rewiring of the acetogen Acetobacterium woodii toward ethanol production","authors":"Renaud Eynard,&nbsp;Sephora Justine,&nbsp;Rémi Hocq,&nbsp;Stefan Pflügl","doi":"10.1186/s13068-026-02790-2","DOIUrl":"10.1186/s13068-026-02790-2","url":null,"abstract":"<div><p>Acetogenic bacteria are promising platforms for converting gaseous and liquid one-carbon (C1) substrates such as carbon monoxide, carbon dioxide (CO₂), formate and methanol into fuels and chemicals, but their application is limited by sparse genetic tools and poor control over metabolic fluxes. Here, we developed a modular plasmid assembly platform and a suite of characterized native, heterologous and methanol-inducible promoters for the model acetogen <i>Acetobacterium woodii</i>, enabling rapid and tunable control of gene expression together with CRISPR-based genome editing. Using an anaerobe-compatible fluorescent reporter (pFAST), promoter strength was quantified, from medium to high expression levels. Subsequently, a minimal promoter replacement at the native <i>adhE</i> (bifunctional aldehyde-alcohol dehydrogenase) locus along with deletion of the neighboring LysR-type regulator was done employing the newly characterized promoters, demonstrating that a single promoter replacement in the genome was sufficient to shift <i>A. woodii</i> from a primarily acetogenic toward a partly ethanologenic phenotype. The engineered strain was able to produce ethanol not only from fructose but also from the C1 substrates methanol + CO₂ and formate + syngas. Together with the promoter replacement strategy, these findings underline that ethanol formation in <i>A. woodii</i> is constrained both by energy conservation and by endogenous transcriptional control. Relieving energetic bottlenecks through the choice of energetically favorable C1 substrates and decoupling <i>adhE</i> expression from its native promoter suffices to enable ethanol production from methanol + CO₂ and formate + syngas, directly linking the Wood–Ljungdahl pathway to ethanol formation. These results illustrate how small, targeted genome edits can significantly reshape carbon flux in acetogens and positions <i>A. woodii</i> as a viable chassis for C1-based bioproduction.</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":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-07-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02790-2.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148440082","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}

{"title":"Comprehensive assessment of fungal peroxygenases for the selective synthesis of 25-hydroxyvitamins D2 and D3","authors":"Alejandro González-Benjumea,&nbsp;Andrés Olmedo,&nbsp;Katharina Ebner,&nbsp;Anton Glieder,&nbsp;Ana Gutiérrez","doi":"10.1186/s13068-026-02792-0","DOIUrl":"10.1186/s13068-026-02792-0","url":null,"abstract":"<div><h3>Background</h3><p>The metabolite 25-hydroxyvitamin D is significantly better for clinical use in several diseases and food supplementations than vitamin D (calciferol) itself. The present study reports an efficient biotechnological method for the synthesis of 25-hydroxyvitamin D₂ and 25-hydroxyvitamin D₃, in one step, starting from the readily available and comparatively inexpensive vitamin D₂ (from fungi and yeast) and vitamin D₃ (from animals).</p><h3>Results</h3><p>A widespread investigation of a series of up to 71 unspecific peroxygenases (UPOs), including UPO samples from 41 fungal species (together with several isozymes and variants) expressed in <i>Komagataella phaffii</i> (formerly <i>Pichia pastoris</i>), has been performed for the regioselective C25-hydroxylation of vitamin D (D₂ and D₃). First experiments revealed that 42 UPOs were able to transform vitamin D₂ into its 25-hydroxy derivative within a wide range of conversion rates (from 5% to &gt; 99%), with strict regioselectivity in most cases, determined using GC–MS. The 16 best UPOs in terms of conversion and regioselectivity were tested for vitamin D₃ hydroxylation. With vitamin D₃, other hydroxylated derivatives were produced by UPOs in addition to the 25-hydroxy derivative, with the only exception of the UPO from <i>Hypoxylon</i> sp. (<i>Hsp</i>UPO). Finally, the synthesis of both 25-hydroxyvitamin D₂ and D₃ was scaled up (up to 20 mM of substrate loading) with <i>Hsp</i>UPO, using different cosubstrates, such as H₂O₂ or O₂/reductants, with ascorbic acid, gallic acid, or pyrogallol as reductants. High conversions (68–97% for vitamin D₂ and 63–75% for vitamin D₃) and product yields (14–19 mM for vitamin D₂ and 13–15 mM for D₃) were attained with all cosubstrates, with pyrogallol/O₂ and H₂O₂ showing the best results with both vitamins.</p><h3>Conclusions</h3><p>This approach represents a green and sustainable alternative to the chemical synthesis of these hydroxylated compounds that is tedious, occurs with little regioselectivity, and includes several steps and strong oxidizing agents. Moreover, <i>K. phaffii</i> a widely used yeast platform for recombinant protein production ‒ including high-yield secretory expression of industrial enzymes ‒ has been firmly established as a favorable host for recombinant UPO production in recent years.</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":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13640091/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148427355","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}

{"title":"Discovery of a multifunctional chitinase–cellulase from Thermococcus chitonophagus with expanded polysaccharide specificity","authors":"Alex Windels,&nbsp;Shari Dhaene,&nbsp;Tom Desmet","doi":"10.1186/s13068-026-02793-z","DOIUrl":"10.1186/s13068-026-02793-z","url":null,"abstract":"<div><h3>Background</h3><p>The discovery of novel biocatalysts for the sustainable valorization of complex biomass feedstocks remains a significant challenge. Domain-centric exploration of characterized CAZyme families offers a promising but underexplored strategy for identifying enzymes with unusual architectures and potentially expanded substrate specificities.</p><h3>Results</h3><p>Systematic analysis of archaeal glycoside hydrolase family 18 (GH18) chitinases using the CANDy domain annotation pipeline led to the identification of <i>Tc</i>Chi from <i>Thermococcus chitonophagus</i>, a multidomain enzyme combining a GH12 and a GH18 catalytic domain alongside two carbohydrate-binding modules. Given that <i>T. chitonophagus</i> also encodes dedicated standalone cellulases and chitinases, we hypothesized that this multidomain assembly may have evolved a broader functional range than either composing domain alone. Biochemical assays of truncated constructs confirmed this hypothesis: the GH18 domain hydrolyzed chitin, chitosan, and β-1,3-glucan, marking the first report of β-1,3-glucanase activity (EC 3.2.1.58) in a GH18 chitinase, while the GH12 domain exhibited strong cellulase activity alongside unexpected chitosanase activity (EC 3.2.1.132), extending the known functional range of this family. Both domains demonstrated high thermostability consistent with the hyperthermophilic origin of <i>T. chitonophagus</i>.</p><h3>Conclusions</h3><p><i>Tc</i>Chi is a thermostable, multifunctional biocatalyst capable of degrading chitin, chitosan, cellulose, and β-1,3-glucan from a single protein scaffold, making it a promising candidate for consolidated biomass deconstruction and waste valorization. These findings also demonstrate that domain-centric analysis of CAZyme families is an effective strategy for uncovering hidden functional diversity in well-characterized enzyme families.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13629093/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148414249","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}

{"title":"From contamination to valorisation: a case study assessing culture contamination effect on the biofuel potential and nanomechanical properties of Parachlorella kessleri","authors":"Alba Zurita,&nbsp;Simona Sebastiano,&nbsp;Catherine Dupré,&nbsp;Jordan Prieto,&nbsp;Esther Torrens,&nbsp;Cécile Formosa-Dague,&nbsp;Christophe Bengoa","doi":"10.1186/s13068-026-02789-9","DOIUrl":"10.1186/s13068-026-02789-9","url":null,"abstract":"<div><p>Culture contamination is a critical problem in microalgae cultivation, often leading to the loss of affected batches and significant financial losses. Therefore, repurposing this contaminated biomass as a fuel source is one of the most viable valorisation outlooks, as the presence of contaminants in the energy sector is not as relevant as in cosmetic or food applications. This work evaluated the viability of this approach using a culture of <i>Parachlorella kessleri</i> contaminated with initially unknown micro-organisms. Microscopy analyses identified <i>Tetradesmus</i> sp. and bacilli as contaminants. The biochemical composition of this contaminated sample was compared to regular and nitrogen-starved cultures, showing no statistically significant differences from the regular batch. This resemblance also extended to the biodiesel profile, highlighting the suitability of valorising contaminated microalgae in biofuel applications. However, the evaluation of nanomechanical properties revealed that <i>P. kessleri</i> cells from the contaminated batch had Young’s modulus remarkably higher than values reported in the literature, indicating that the cell disruption step could be more energy intensive. Hence, contaminated biomass remains biochemically suitable for biofuel valorisation, but potentially requires more energy-intensive processing.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13621546/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148400456","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}

{"title":"Sustainable aviation fuels: a review of atomization, combustion and emission characteristics","authors":"A. M. Abdelmagid,&nbsp;D. O. Glushkov,&nbsp;A. S. Sviridenko,&nbsp;V. A. Pershukov","doi":"10.1186/s13068-026-02777-z","DOIUrl":"10.1186/s13068-026-02777-z","url":null,"abstract":"<div><p>Sustainable aviation fuels (SAFs) are considered a projected alternative to widely used petroleum-based fuels for reducing the environmental impact of aviation, including the potential mitigation of greenhouse gas emissions and the reduction of particulate matter formation. While numerous studies have addressed feedstocks and production technologies of SAFs, comparatively limited attention has been given to experimental investigations associated with the second stage of the ASTM D4054 fuel certification process, which includes fuel atomization, ignition, combustion characteristics, and emission behavior under realistic engine operating conditions. This review provides a comprehensive analysis of recent experimental studies devoted to these processes. Particular attention is given to the influence of physicochemical fuel properties, including volatility, viscosity, cetane number, and chemical composition, on atomization quality, ignition delay, flame stability, and combustion efficiency. The relationship between fuel molecular structure and soot formation tendencies is also examined. The analysis shows that the lower density, viscosity, and aromatic content typical of many SAF pathways generally improve atomization and evaporation processes, leading to more homogeneous fuel–air mixtures and reduced particulate matter emissions compared with conventional jet fuels. Overall, the review highlights the importance of integrating atomization and combustion studies into SAF development and certification, providing insights that can support the optimization of fuel formulations and contribute to the safe and efficient large-scale deployment of sustainable aviation fuels.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02777-z.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148378506","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}

{"title":"Engineering thiamine pyrophosphate metabolism improves consolidated bioprocessing for cellulosic ethanol with inorganic nitrogen in Myceliophthora thermophila","authors":"Jiaxing Chen,&nbsp;Jia Liu,&nbsp;Tao Sun,&nbsp;Rui Pang,&nbsp;Jinyi Yu,&nbsp;Defei Liu,&nbsp;Jingen Li,&nbsp;Chaoguang Tian","doi":"10.1186/s13068-026-02786-y","DOIUrl":"10.1186/s13068-026-02786-y","url":null,"abstract":"<div><h3>Background</h3><p>Consolidated bioprocessing (CBP) by cellulolytic fungi offers a direct route for cellulosic ethanol production, but the process still relies heavily on costly complex nitrogen sources. Replacing complex nitrogen with inorganic nitrogen is a potential strategy to reduce the cost of the medium, yet it often leads to impaired fermentation performance, and the metabolic basis for this limitation remains unclear.</p><h3>Results</h3><p>Replacement of yeast extract with inorganic nitrogen was evaluated in the engineered thermophilic fungus <i>Myceliophthora thermophila</i> YL913, a CBP strain capable of producing ethanol directly from cellulose. Ammonium sulfate supported higher ethanol production than nitrate, but still resulted in a lower titer than yeast extract. Under ammonium sulfate, transcriptomic and metabolomic analyses revealed altered amino acid metabolism together with reduced carbohydrate metabolic and cellulose catabolic functions, while supplementation with selected amino acids provided only limited improvement. Supplementation with 5 mg/L thiamine pyrophosphate (TPP) increased ethanol production under ammonium sulfate from 14.3 g/L to 23 g/L, reaching a level comparable to that obtained with yeast extract. Moreover, α-ketoglutarate was identified as a responsive metabolic node associated with the altered fermentation state. Stepwise strengthening of the endogenous TPP biosynthetic pathway progressively improved ammonium-based ethanol fermentation, and additional overexpression of pyruvate decarboxylase further enhanced ethanol production. In a 5-L fermenter, the final engineered strain produced 49.5 g/L ethanol under ammonium sulfate without exogenous TPP, close to the 51.2 g/L obtained with the TPP-supplemented parental strain.</p><h3>Conclusions</h3><p>This study establishes ammonium sulfate as a feasible low-cost alternative to yeast extract for CBP-based cellulosic ethanol production by the engineered <i>M. thermophila</i> strain. These findings provide a practical strategy for developing low-cost fungal CBP processes for cellulosic ethanol production supported by inorganic nitrogen.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02786-y.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148364486","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}

{"title":"Towards sustainable production of biofuels via milking the cells of Synechococcus elongatus PCC 7942 engineered for free fatty acid excretion","authors":"Akihiro Kato,&nbsp;Nobuyuki Takatani,&nbsp;Shin-ichi Maeda,&nbsp;Makiko Aichi,&nbsp;Tatsuo Omata","doi":"10.1186/s13068-026-02782-2","DOIUrl":"10.1186/s13068-026-02782-2","url":null,"abstract":"<div><h3>Background</h3><p>Free fatty acids are considered a potential source of biodiesel because they are excreted by engineered cyanobacterial cells, eliminating the processing costs associated with cell recovery and extraction. Although the cyanobacterial mutants engineered for the production of free fatty acids do excrete significant amounts of the product, the yield per dry cell weight is low due to their rapid proliferation and the consequent substantial cell biomass, which makes it impossible to support sustainable biofuel production. Development of strategies to increase the production of free fatty acids per cell is urgently needed.</p><h3>Results</h3><p>To increase the <i>per-cell</i> production of free fatty acids, we attempted to increase the allocation of photosynthetically fixed carbon into fatty acid production by restricting cell proliferation. Growth limitation of free fatty acid-producing cells was achieved by cultivating dAS2T, a free fatty acid-producing mutant of <i>Synechococcus elongatus</i> PCC 7942 defective in active nitrate uptake, in media containing nitrate as the sole source of nitrogen. Although the nitrogen-limited cultivation increased the <i>per-cell</i> yield of free fatty acids, it significantly decreased the extracellular free fatty acid level, reducing the volumetric productivity. Overexpressing an endogenous free fatty acid-efflux pump in the dAS2T strain elevated extracellular free fatty acid level to those seen under nitrate-sufficient conditions, while maintaining slow cell growth. By decoupling the production of fatty acids from cell growth in this manner, excretion of 0.90 g of free fatty acids per g of dry cell weight was achieved in 240 h, with a free fatty acid productivity of 1.8 mg L⁻¹ h⁻¹. This represents the highest yield of free fatty acids obtained to date without significantly decreasing the volumetric productivity.</p><h3>Conclusion</h3><p>Growth limitation and enhancement of excretion of the product are essential to increase the <i>per-cell</i> yield of the free fatty acid production system using cyanobacteria. The high yield and productivity of extracellular free fatty acids achieved in this study suggest that the cyanobacteria-based production of free fatty acids provides a reliable approach for sustainable biofuel production through the “milking” strategy.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-06-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13576201/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148355030","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}

{"title":"Deciphering the genetic background of an industrial 2-ketogluconic acid-producing strain Pseudomonas plecoglossicida JUIM01 using whole-genome sequencing","authors":"Lulu Li,&nbsp;Lei Sun,&nbsp;Xinyi Zan,&nbsp;Fengjie Cui,&nbsp;Mei Zhao,&nbsp;Wenjing Sun","doi":"10.1186/s13068-026-02784-0","DOIUrl":"10.1186/s13068-026-02784-0","url":null,"abstract":"<div><p>2-Ketogluconic acid (2KGA) is an important precursor for the food antioxidant erythorbic acid, currently produced via microbial fermentation using <i>Pseudomonas</i> species. To facilitate the genetic improvement of production strains, the complete genome of an industrial 2KGA producer <i>P. plecoglossicida</i> JUIM01 was sequenced and analyzed. The genome consists of a 5.13-Mb circular chromosome with a GC content of 63.58%, encoding 4,517 predicted proteins. Comprehensive functional annotation identified a putative global regulatory network comprising 75 core regulators, which were classified into six functionally cooperative modules, potentially governing the strain’s metabolism and environmental adaptability. We further delineated the genetic determinants hypothetically linked to efficient 2KGA synthesis, including glucose metabolism, fatty acid metabolism, and the oxidative phosphorylation system. These outputs could provide the genomic resource for elucidating high productivity and robustness, and rationally engineering the high-performance chassis cells toward robust 2KGA production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-06-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02784-0.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148335107","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}

{"title":"Strategies for GH functional annotation: toward harmonized and predictive CAZymology","authors":"D. S. Erdody,&nbsp;N. G. Griffin,&nbsp;A. E. Hughes,&nbsp;J. Munoz-Munoz,&nbsp;R. Berlemont","doi":"10.1186/s13068-026-02781-3","DOIUrl":"10.1186/s13068-026-02781-3","url":null,"abstract":"<div><h3>Background</h3><p>The functional annotation and biochemical characterization of glycoside hydrolases (GHs) for polysaccharides and oligosaccharides deconstruction are a major challenge. Here, we traced the transition from traditional “activity first” approaches, based on biochemical assays, to modern “sequence first” discovery methods enabled by modern genomics.</p><h3>Results</h3><p>We reported 169 glycosidase activities, the substrates, and the techniques used for the characterization of GHs. We highlight the challenges posed by the diversity of GH families including overlapping substrate specificities, heterogeneous assay conditions, and inconsistent reporting standards. To address these issues, we compiled a list of substrates for each GH family’s glycosidase activities as a practical guide to serve as an initial framework for characterizing newly identified GHs.</p><h3>Conclusions</h3><p>The adoption of standardized substrates and protocols, uniform reporting practices, and community-driven data sharing initiatives provides a practical roadmap toward harmonized GH functional annotation and predictive enzymology while acknowledging current limitations in data standardization and reporting heterogeneity.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02781-3.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148267230","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}

{"title":"SynBio-DSP: an integrated synthetic biology platform with fed-batch fermentation and continuous downstream processing for sustainable production of thermostable cellulases","authors":"Shah Faisal Mohammad,&nbsp;Fawad Ali,&nbsp;Mamirkulova Shynara","doi":"10.1186/s13068-026-02783-1","DOIUrl":"10.1186/s13068-026-02783-1","url":null,"abstract":"<div><h3>Background</h3><p>The economic viability of enzymatic lignocellulosic biomass conversion is hindered by high production costs and energy-intensive recovery of thermostable enzymes. Traditional optimization approaches treat strain engineering, fermentation, and downstream processing as separate domains, leading to suboptimal system performance. This study aimed to develop an integrated platform that synergistically combines synthetic biology, machine learning-guided bioprocess optimization, and continuous downstream processing for sustainable cellulase production.</p><h3>Results</h3><p>We engineered a <i>Bacillus subtilis</i> chassis with a Quorum-Lysis-Modulation (QLM) system for auto-induction of hyperthermostable cellulase CtCel9A and CRISPRi-mediated cell wall weakening. The QLM strain achieved a CtCel9A titer of 9.32 ± 0.29 IU/mL, a 3.6-fold increase over conventional methods. Fermentation was optimized using Gaussian Process Regression in high-throughput microbioreactors, yielding excellent predictive performance (R² = 0.94). A novel continuous Pulsed Electric Field-Assisted Synergistic Lysis (PEF-ASL) system was developed and optimized via Response Surface Methodology (Box-Behnken Design), achieving 96.8% enzyme recovery with specific energy consumption of 0.11 kWh/g, a 68% reduction compared to bead milling. The PEF system was operated at 100 Hz pulse frequency with 30 ± 2 µs pulse width using monopolar exponential decay waveform, and the specific energy input was calculated as 0.098 kWh/g, consistent with the measured value. Multi-objective optimization using NSGA-III balanced yield, energy consumption, water intensity, and production cost. After re-optimization with a full cost function including waste treatment, quality control, contingency, and facility overhead, Pareto front analysis identified a knee-point solution representing the optimal compromise among competing objectives. System-wide optimization reduced production costs by 52–58% and carbon footprint by 57% compared to conventional enzyme production, with techno-economic analysis for a 500 kg/year facility indicating a manufacturing cost of $132/kg, net present value of $4.7 million, and payback period of 3.8 years. The sfGFP fusion at the C-terminus reduced specific activity by 8.3% compared to unfused enzyme but enabled real-time fluorescence monitoring critical for auto-induction timing.</p><h3>Conclusions</h3><p>The SynBio-DSP platform establishes a new paradigm for sustainable biocatalyst manufacturing by co-optimizing cellular design and process configuration. The integrated approach demonstrates that simultaneous improvements in productivity, cost, and environmental impact are achievable for scalable, economically viable biofuel enzyme production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"19 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2026-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://link.springer.com/content/pdf/10.1186/s13068-026-02783-1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148254888","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}