Biotechnology for Biofuels最新文献

{"title":"Alternative Splicing of BnABF4L Mediates Response to Abiotic Stresses in Rapeseed (Brassica napus L.)","authors":"Ruijia Zhu,&nbsp;Chu Yue,&nbsp;Shifan Wu,&nbsp;Mingting Wu,&nbsp;Ziyue Xu,&nbsp;Xiaoqun Liu,&nbsp;Rui Wang,&nbsp;Maolin Wang","doi":"10.1186/s13068-025-02645-2","DOIUrl":"10.1186/s13068-025-02645-2","url":null,"abstract":"<div><p>ABRE BINDING FACTOR 4 (<i>ABF4</i>) is a pivotal regulatory gene in the abscisic acid (ABA) signaling pathway, and changes in its expression levels can modulate the plant's stress resistance. To further explore the specific regulatory mechanisms of alternative splicing (AS) in the ABA signaling pathway and to identify new breakthroughs for breeding high stress-resistant varieties of <i>Brassica napus</i>, we identified 17 homologous genes of <i>ABF4</i> in the genome. Utilizing bioinformatics techniques, we analyzed their motifs, conserved domains, and <i>cis</i>-acting elements of their promoters. Through transcriptome data from the stress-tolerant dwarf strain <i>ndf2</i> and its parental line <i>3529</i>, we uncovered a significantly differentially expressed <i>ABF4</i> gene, which we named <i>BnABF4L</i>. Subsequently, we analyzed the AS events of <i>BnABF4L</i> under normal growth conditions and different abiotic stresses, as well as the impact of different transcript variants' 5’ untranslated region (5'UTR) on gene translation. <i>BnABF4L</i> undergoes alternative 3' splice site (A3SS) selection to produce three transcripts (V1-V3) with divergent 5'UTRs. While V1 translation is suppressed by upstream ORFs (uORFs), V2/V3 exhibit enhanced translational efficiency. Under stress, <i>ndf2</i> shifts splicing toward V3, circumventing uORF-mediated repression to upregulate stress-adapted isoforms. We validated the inhibitory effect of upstream open reading frames (uORFs) on protein-coding open reading frame (pORFs) and, based on the collective experimental results, proposed the flexible regulatory mechanism of AS events of <i>BnABF4L</i> in response to stress. Our findings provide new insights for future studies on stress resistance in rapeseed as well as for research on the regulation of alternative splicing mechanisms in the ABA signaling pathway.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02645-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143919113","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":"Identification of key transcription factors, including DAL80 and CRZ1, involved in heat and ethanol tolerance in Saccharomyces cerevisiae","authors":"Rong-Rong Chen,&nbsp;Li Wang,&nbsp;Xue-Xue Ji,&nbsp;Cai-Yun Xie,&nbsp;Yue-Qin Tang","doi":"10.1186/s13068-025-02653-2","DOIUrl":"10.1186/s13068-025-02653-2","url":null,"abstract":"<div><h3>Background</h3><p>High temperature and ethanol are two critical stress factors that significantly challenge bioethanol production using <i>Saccharomyces cerevisiae</i>. In this study, the tolerance mechanisms of the multi-tolerant <i>S. cerevisiae</i> strain E-158 to heat stress and combined heat-ethanol stress were investigated using comparative transcriptomics.</p><h3>Results</h3><p>Under heat stress at 44 °C, glucose transport and reactive oxygen species (ROS) scavenging were significantly upregulated, while gluconeogenesis, acetate formation, and dNDP formation showed significant downregulation. Under combined heat (43 °C) and ethanol (3% v/v) stress, glucose transport, glycolysis, acetate formation, peroxisome activity, ROS scavenging, and ribosome synthesis were significantly upregulated, while glycerol formation, cellular respiration and dNDP formation exhibited significant downregulation. Fourteen transcription factors (TFs), considered to play a key role in both stress conditions, were individually overexpressed and deleted in <i>S. cerevisiae</i> strain KF-7 in this study. Among these TFs, Gis1p, Crz1p, Tos8p, Yap1p, Dal80p, Uga3p, Mig1p, and Opi1p were found to contribute to enhanced heat tolerance in <i>S. cerevisiae</i>. Compared with KF-7, strains overexpressing <i>DAL80</i> and <i>CRZ1</i> demonstrated markedly improved fermentation performance under stress conditions. Under heat stress at 44 °C, glucose consumption increased by 10% and 12%, respectively, for strains KF7DAL80 and KF7CRZ1, while ethanol production increased by 12% and 15%, respectively, compared to KF-7. Under combined stress conditions of 43 °C and 3% (v/v) ethanol, glucose consumption increased by 67% and 44%, ethanol production by 116% and 77%, and ethanol yield by 29% and 22%, respectively, for KF7DAL80 and KF7CRZ1 compared to KF-7. KF7CRZ1 performs comparably to E-158, while KF7DAL80 outperforms E-158.</p><h3>Conclusions</h3><p>This study provides valuable theoretical insights and identifies critical TF targets, contributing to the development of robust <i>S. cerevisiae</i> strains for improved bioethanol production.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02653-2","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143900678","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":"Hi-TARGET: a fast, efficient and versatile CRISPR type I-B genome editing tool for the thermophilic acetogen Thermoanaerobacter kivui","authors":"Angeliki Sitara,&nbsp;Rémi Hocq,&nbsp;Alexander Jiwei Lu,&nbsp;Stefan Pflügl","doi":"10.1186/s13068-025-02647-0","DOIUrl":"10.1186/s13068-025-02647-0","url":null,"abstract":"<div><h3>Background</h3><p>Due to its ability to grow fast on CO₂, CO and H₂ at high temperatures and with high energy efficiency, the thermophilic acetogen <i>Thermoanaerobacter</i> <i>kivui</i> could become an attractive host for industrial biotechnology. In a circular carbon economy, diversification and upgrading of C1 platform feedstocks into value-added products (e. g., ethanol, acetone and isopropanol) could become crucial. To that end, genetic and bioprocess engineering tools are required to facilitate the development of bioproduction scenarios. Currently, the genome editing tools available for <i>T. kivui</i> present some limitations in speed and efficiency, thus restricting the development of a powerful strain chassis for industrial applications.</p><h3>Results</h3><p>In this study, we developed the versatile genome editing tool Hi-TARGET, based on the endogenous CRISPR Type I-B system of <i>T. kivui</i>. Hi-TARGET demonstrated 100% efficiency for gene knock-out (from both purified plasmid and cloning mixture) and knock-in, and 49% efficiency for creating point mutations. Furthermore, we optimized the transformation and plating protocol and increased transformation efficiency by 245-fold to 1.96 × 10⁴ ± 8.7 × 10³ CFU μg⁻¹. Subsequently, Hi-TARGET was used to demonstrate gene knock-outs (<i>pyrE</i>, <i>rexA</i>, <i>hrcA</i>), a knock-in (<i>ldh</i>::pFAST), a single nucleotide mutation corresponding to PolC^C629Y, and knock-down of the fluorescent protein pFAST. Analysis of the ∆<i>rexA</i> deletion mutant created with Hi-TARGET revealed that the transcriptional repressor <i>rexA</i> is likely involved in the regulation of the expression of lactate dehydrogenase (<i>ldh</i>). Following genome engineering, an optimized curing procedure for edited strains was devised. In total, the time required from DNA to a clean, edited strain is 12 days, rendering Hi-TARGET a fast, robust and complete method for engineering <i>T. kivui</i>.</p><h3>Conclusions</h3><p>The CRISPR-based genome editing tool Hi-TARGET developed for <i>T. kivui</i> can be used for scarless deletion, insertion, point mutation and gene knock-down, thus fast-tracking the generation of industrially-relevant strains for the production of carbon-negative chemicals and fuels as well as facilitating studies of acetogen metabolism and physiology.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02647-0","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143892745","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":"Enhancement of microalgal CO2 fixation in photobioreactors by means of spiral flow vortices","authors":"Santosh Kumar,&nbsp;Ameer Ali Kubar,&nbsp;Xinjuan Hu,&nbsp;Feifei Zhu,&nbsp;Shahid Mehmood,&nbsp;Michael Schagerl,&nbsp;Yajie Zhang,&nbsp;Muhammad Abdur Rehman Shah,&nbsp;Bin Zou,&nbsp;Obaid Ur Rehman,&nbsp;Shuhao Huo","doi":"10.1186/s13068-025-02650-5","DOIUrl":"10.1186/s13068-025-02650-5","url":null,"abstract":"<div><p>Microalgae have received a lot of interest as a sustainable solution for carbon dioxide fixation due to their great efficiency in capturing CO₂ and converting it into valuable biomass, making them a promising tool for mitigating climate change and expanding carbon capture technology. This study examines the efficacy of fixed shaped portable conical helix baffles (PCHB) in enhancing gas–liquid mixing to promote microalgal growth in column photobioreactors (PBRs). Flat (90° angle from cone surface), round, and inclined (60° angle from cone surface) baffles were compared for performance. Modeling the gas flow indicated that round PCHB produced more spiral vortices and achieved better mixing performance than flat and inclined designs. Increasing the baffle size from 3 to 7 cm resulted in a 21% higher mass transfer coefficient. The simulation was verified by experiments. Notably, the implementation of a PCHB with a round helix-shaped structure (5 cm) led to a 33% (2.102 ± 0.08 g/L) and 17% (2.419 ± 0.07 g/L) dry mass increase of <i>Limnospira fusiformis</i> when compared to flat and incline-shaped baffles, respectively. Our study revealed that using a round-shaped PCHB resulted to higher spiral movement, which in turn increases CO₂ utilization and cell proliferation. Our approach demonstrates high potential to further optimize industrial PBRs, thereby facilitating CO₂ sequestration during microalgal cultivation to combat global warming.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02650-5","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888734","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":"Synergistic cell-free enzyme cocktails for enhanced fiber matrix development: improving dewatering, strength, and decarbonization in the paper industry","authors":"Nelson Barrios,&nbsp;María Gonzalez,&nbsp;Richard Venditti,&nbsp;Lokendra Pal","doi":"10.1186/s13068-025-02646-1","DOIUrl":"10.1186/s13068-025-02646-1","url":null,"abstract":"<div><h3>Background</h3><p>The pulp and paper industry is under increasing pressure to adopt sustainable solutions that address its substantial energy consumption and environmental impact. One of the most energy-intensive operations is the thermal drying, which presents significant opportunities for efficiency improvements. This study evaluates a cell-free mild enzyme pretreatment, utilizing a cocktail of cellulases and xylanases, combined with cationic starch, to enhance dewatering efficiency and improve paper strength utilizing bleached hardwood pulp fibers. Life cycle and economic analysis were also conducted to quantify the environmental impact and economic benefits, with a particular focus on direct greenhouse gas emissions. Enhanced water removal during pressing can significantly reduce energy consumption during thermal drying, facilitating the decarbonization of the paper industry.</p><h3>Results</h3><p>The cell-free enzyme pretreatment, applied with mild refining and cationic starch, achieved significant improvements in dewatering efficiency and paper strength. The treatment led to an 11% point increase in solids and a 25% improvement in tensile strength. Morphological analyses revealed no changes in fiber length and width; however, reductions in kink and curl indexes indicated enhanced fiber flexibility and bonding potential. Furthermore, the enzyme–starch combination decreased water retention value by 27%, including substantial reductions in bound and hard-to-remove water content. Environmental assessments estimated a 12% reduction in global warming potential (GWP), with the technology yielding net savings of $11.29 per air-dried ton of paper through reduced natural gas consumption.</p><h3>Conclusions</h3><p>This study demonstrates the technical feasibility and economic viability of incorporating enzyme and cationic starch treatments into papermaking. The treatment improves paper quality while reducing energy consumption, costs, and carbon emissions. These findings support the broader adoption of enzyme-based innovations for sustainable manufacturing, aligning with decarbonization goals and industry demands for greater efficiency. The results highlight a promising avenue for achieving significant environmental and economic benefits in the pulp and paper sector.</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":"2025-04-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02646-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143888733","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":"Solid-state NMR at natural isotopic abundance for bioenergy applications","authors":"Bennett Addison,&nbsp;Malitha C. Dickwella Widange,&nbsp;Yunqiao Pu,&nbsp;Arthur J. Ragauskas,&nbsp;Anne E. Harman-Ware","doi":"10.1186/s13068-025-02648-z","DOIUrl":"10.1186/s13068-025-02648-z","url":null,"abstract":"<div><p>Lignocellulosic biomass offers a vast and renewable resource for biofuel production and carbon management solutions. The effective conversion of lignocellulosic biomass into economically competitive biofuels and bioproducts demands a comprehensive understanding of its complex structure and composition, often requiring a range of analytical tools to achieve meaningful insights. However, for the analysis of rigid solids, many traditional methods necessitate dissolution or chemical/physical modification of the sample, which limit our ability to capture an intact view of its structural components. This highlights the need for non-destructive approaches, such as solid-state nuclear magnetic resonance (ssNMR), which preserves the sample’s natural state while providing deep, molecular-level insights. While advanced multi-dimensional ssNMR on ¹³C-enriched materials has recently proven exceptionally valuable for elucidating the complex macrostructure of biomass, isotopic enrichment is expensive, laborious and is clearly infeasible at large scales. In this review, we explore the role of solid-state NMR methods at natural isotopic abundance as essential tools for the non-destructive, in-depth characterization of lignocellulosic biomass and bioenergy materials in their native and unaltered state. After a brief introduction to the basic principles of solid-state NMR, we first describe the acquisition and interpretation of routine 1D ¹³C ssNMR spectra of lignocellulose and other related biopolymers and products. We then delve into more advanced ssNMR approaches, including key spectral editing techniques, probing polymer dynamics, and various 2D methods applicable at natural abundance. Understanding of domain miscibility as observed from proton-based spin diffusion effects is a theme throughout. Our aim is to highlight key examples where ssNMR provides valuable insights into the composition, structure, dynamics, and morphology of rigid biomaterials relevant to the bioenergy economy, revealing both the native structures and fundamental transformations that occur across conversion and decomposition pathways. We hope that this review encourages a broader adoption of ssNMR methods in bioenergy research, where it can serve as a pivotal analytical tool for achieving sustainable biomass utilization and advancing a carbon-efficient bioeconomy.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-04-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02648-z","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143883554","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":"Disruption-induced changes in syntrophic propionate and acetate oxidation: flocculation, cell proximity, and microbial activity","authors":"Nils Weng,&nbsp;Hossein Nadali Najafabadi,&nbsp;Maria Westerholm","doi":"10.1186/s13068-025-02644-3","DOIUrl":"10.1186/s13068-025-02644-3","url":null,"abstract":"<div><h3>Background</h3><p>Syntrophic propionate- and acetate-oxidising bacteria (SPOB and SAOB) play a crucial role in biogas production, particularly under high ammonia conditions that are common in anaerobic degradation of protein-rich waste streams. These bacteria rely on close interactions with hydrogenotrophic methanogens to facilitate interspecies electron transfer and maintain thermodynamic feasibility. However, the impact of mixing-induced disruption of these essential syntrophic interactions in biogas systems remains largely unexplored. This study investigates how magnetic stirring and orbital shaking influence degradation dynamics, microbial community composition, and gene expression in syntrophic enrichment communities under high-ammonia conditions.</p><h3>Results</h3><p>Stirring significantly delayed the initiation of propionate degradation in one culture and completely inhibited it in the other two parallel cultures, whereas acetate degradation was less affected. Computational fluid dynamics modelling revealed that stirring generated higher shear rates (~ 20 s⁻¹) and uniform cell distribution, while shaking led to lower shear rates and cell accumulation at the bottom of the culture bottle. Visual observations confirmed that stirring inhibited floc formation, while shaking promoted larger flocs compared to the static control condition, which formed smaller flocs and a sheet-like biofilm. Microbial community analysis identified substrate type and degradation progress as primary drivers of community structure, with motion displaying minimal influence. However, metatranscriptomic analysis revealed that motion-induced gene downregulation was associated with motility, surface sensing, and biofilm formation in SAOB and another bacterial species expressing genes for the glycine synthase reductase pathway. Stirring also suppressed oxalate–formate antiporter expression in SPOB, suggesting its dependence on spatial proximity for this energy-conserving mechanism. The strongest gene expression changes of stirring were observed in methanogens, indicating a coupling of the first and last steps of hydrogenotrophic methanogenesis, likely an adaptive strategy for efficient energy conservation. Other downregulated genes included ferrous iron transporters and electron transfer-associated enzymes.</p><h3>Conclusions</h3><p>This study highlights that stirring critically disrupts the initial syntrophic connection between SPOB and methanogens, whereas SAOB communities exhibit greater tolerance to shear stress and disruptive conditions that inhibits aggregate formation. These findings emphasize the importance of carefully managing mixing regimes, especially when attempting to reactivate ammonia-tolerant syntrophic propionate degraders in biogas systems experiencing rapid propionate accumulation under high-ammonia conditions.</p><h3>Graphical abstract</h3>\u0000<div><figure><div><div><picture><source><img></source></picture></div></div></figure></div><","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-04-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02644-3","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143850896","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":"Two routes for tyrosol production by metabolic engineering of Corynebacterium glutamicum","authors":"Nora Junker,&nbsp;Sara-Sophie Poethe,&nbsp;Volker F. Wendisch","doi":"10.1186/s13068-025-02641-6","DOIUrl":"10.1186/s13068-025-02641-6","url":null,"abstract":"<div><h3>Background</h3><p>The phenolic compound tyrosol is widely used in the pharmaceutical industry, owing to its beneficial effects on human health and its use as a precursor for key pharmaceuticals, including β₁-receptor blockers. Tyrosol can be found in olive oil, but despite its natural biosynthesis in plants, low extraction efficiencies render microbial production a more viable alternative.</p><h3>Results</h3><p>Here, we engineered the <span>l</span>-tyrosine overproducing <i>Corynebacterium glutamicum</i> strain AROM3 for the de novo production of tyrosol. Two routes were established and compared: one via 4-OH-phenylpyruvate as intermediate and the other via tyramine. We initially expected the first route to require heterologous expression of a prephenate dehydrogenase gene, given that <i>C. glutamicum</i> lacks this enzymatic function. However, heterologous expression of <i>ARO10</i> from <i>Saccharomyces cerevisiae</i> (<i>ARO10</i>_<i>Sc</i>), which encodes a phenylpyruvate decarboxylase, was sufficient to establish tyrosol production in strain AROM3. We identified that 4-OH-phenylpyruvate is synthesized from<span> l</span>-tyrosine by native aminotransferases, which is subsequently decarboxylated by Aro10_<i>Sc</i><i>,</i> and reduced to tyrosol by native alcohol dehydrogenases, leading to a titer of 9.4 ± 1.1 mM (1.30 ± 0.15 g/L). We identified the furfural dehydrogenase FudC as major enzyme involved in this pathway, as its gene deletion reduced tyrosol production by 75%. Given the instability of 4-OH-phenylpyruvate, the synthesis of tyrosol via the stable intermediate tyramine was pursued via the second route. Decarboxylation of<span> l</span>-tyrosine followed by oxidative deamination was accomplished by overexpression of the <span>l</span>-tyrosine decarboxylase gene <i>tdc</i> from <i>Levilactobacillus brevis</i> (<i>tdc</i>_<i>Lb</i>) and the tyramine oxidase gene <i>tyo</i> from <i>Kocuria rhizophila</i> (<i>tyo</i>_<i>Kr</i>). Using this route, tyrosol production was increased by 44% compared to the route via 4-OH-phenylpyruvate. With a division of labor approach by co-cultivating <span>l</span>-tyrosine producing strains that either express <i>tdc</i>_<i>Lb</i> or <i>tyo</i>_<i>Kr</i>, the highest titer of 14.1 ± 0.3 mM (1.95 ± 0.04 g/L) was achieved.</p><h3>Conclusions</h3><p>This study demonstrates the potential of endotoxin-free <i>C. glutamicum</i> as production host for the <span>l-</span>tyrosine-derived product tyrosol. Due to its <span>l</span>-arogenate pathway for <span>l</span>-tyrosine synthesis, the unstable 4-OH-phenylpyruvate could be excluded as intermediate in the Tdc–Tyo pathway, outcompeting the most often utilized production route via phenylpyruvate decarboxylases.</p></div>","PeriodicalId":494,"journal":{"name":"Biotechnology for Biofuels","volume":"18 1","pages":""},"PeriodicalIF":6.1,"publicationDate":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02641-6","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784211","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":"Development of a β-glucosidase improved for glucose retroinhibition for cellulosic ethanol production: an integrated bioinformatics and genetic engineering approach","authors":"Raíza dos Santos Azevedo,&nbsp;Hugo Santana,&nbsp;Vinícius Rosa Seus,&nbsp;Alex Dias Camargo,&nbsp;Adriano Velasque Werhli,&nbsp;Karina dos Santos Machado,&nbsp;Letícia Jungmann Cançado,&nbsp;Betania Ferraz Quirino,&nbsp;Luis Fernando Marins","doi":"10.1186/s13068-025-02643-4","DOIUrl":"10.1186/s13068-025-02643-4","url":null,"abstract":"<div><h3>Background</h3><p>The global energy crisis, driven by economic growth and the increasing demand for energy, highlights the urgency of searching for alternative energy sources to mitigate environmental pollution and climate change. β-Glucosidases act in the final step of the enzymatic hydrolysis of cellulose, cleaving the β-1,4-glycosidic bonds in cellobiose to produce second-generation ethanol. However, these enzymes are easily inhibited by glucose, their final product, which limits the production of this biofuel. Genetic engineering combined with bioinformatics tools can improve key enzymatic characteristics, such as catalytic activity and glucose tolerance, in a more precise, faster, and cost-effective manner compared to traditional methods. In this work, a variant of a β-glucosidase from the GH1 family, isolated from the microbial community of Amazonian soil (Brazil), with enhanced catalytic activity and improved for glucose retroinhibition, was developed.</p><h3>Results</h3><p>Bioinformatics analyses suggested the substitution of tryptophan at position 404 with leucine. The produced variant (W404L) was expressed in <i>Escherichia coli</i> and showed activity 3.2 times higher in the presence of glucose than the non-mutated control. Moreover, the partially purified mutated variant of β-glucosidase exhibited a 26-fold increase in catalytic activity compared to the original form of the enzyme. The results confirmed that the mutation proposed by computational analyses had a significant impact on enzyme catalytic activity and glucose retroinhibition.</p><h3>Conclusions</h3><p>This new variant may become a promising alternative to reduce the costs of enzyme cocktails used in the hydrolysis of lignocellulosic biomass used as a raw material in the production of second-generation ethanol.</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":"2025-04-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02643-4","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143784210","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":"Impact of heterologous expression of Cannabis sativa tetraketide synthase on Phaeodactylum tricornutum metabolic profile","authors":"Nicolas Sene,&nbsp;Karen Cristine Gonçalves dos Santos,&nbsp;Natacha Merindol,&nbsp;Sarah-Eve Gélinas,&nbsp;Alexandre Custeau,&nbsp;Fatima Awwad,&nbsp;Elisa Fantino,&nbsp;Fatma Meddeb-Mouelhi,&nbsp;Hugo Germain,&nbsp;Isabel Desgagné-Penix","doi":"10.1186/s13068-025-02638-1","DOIUrl":"10.1186/s13068-025-02638-1","url":null,"abstract":"<div><h3>Background</h3><p>Pharmaceutical safety is an increasing global priority, particularly as the demand for therapeutic compounds rises alongside population growth. Phytocannabinoids, a class of bioactive polyketide molecules derived from plants, have garnered significant attention due to their interaction with the human endocannabinoid system, offering potential benefits for managing a range of symptoms and conditions. Traditional extraction from cannabis plants poses regulatory, environmental, and yield-related challenges. Consequently, microbial biosynthesis has emerged as a promising biotechnological alternative to produce cannabinoids in a controlled, scalable, and sustainable manner. Developing diatom-based biofactories represent a crucial step in advancing this biotechnology, enabling the efficient production of high-valued compounds such as cannabinoids.</p><h3>Results</h3><p>We engineered the diatom <i>Phaeodactylum tricornutum</i>, a unicellular photosynthetic model organism prized for its naturally high lipid content, to produce olivetolic acid (OA), a key metabolic precursor to most cannabinoids. The genes encoding tetraketide synthase and olivetolic acid cyclase from cannabis were cloned onto episomal vectors and introduced using bacterial conjugation in two separate <i>P. tricornutum</i> transconjugant lines to evaluate enzyme activity and OA production in vivo. Both genes were successfully expressed, and the corresponding enzymes accumulated within the transconjugant lines. However, despite testing the cell extracts individually and in combination, OA accumulation was not detected suggesting potential conversion or utilization of OA by endogenous metabolic pathways within the diatoms. To investigate this further, we analyzed the impact of <i>Cs</i>TKS expression on the diatom’s metabolome, revealing significant alterations that may indicate metabolic flux redirection or novel pathway interactions.</p><h3>Conclusions</h3><p>Our study demonstrates the successful expression of cannabinoid biosynthetic genes in <i>P. tricornutum</i> but highlights challenges in OA accumulation, likely due to endogenous metabolic interactions. These findings underscore the complexity of metabolic engineering in diatoms and suggest the need for further pathway optimization and metabolic flux analysis to achieve efficient cannabinoid biosynthesis. This research contributes to advancing sustainable biotechnological approaches for cannabinoid 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":"2025-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://biotechnologyforbiofuels.biomedcentral.com/counter/pdf/10.1186/s13068-025-02638-1","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778115","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}