Thermotolerant microbial hosts offer advantages for industrial bioprocesses, yet direct oil-based bioconversion requires efficient coupling of extracellular lipid hydrolysis with intracellular carbon assimilation. In the thermotolerant bacterium Cupriavidus cauae PHS1, direct utilization of triacylglycerol-rich substrates is constrained by the lack of extracellular lipolytic activity and limited glycerol metabolism. Here, the underexplored non-model bacterium C. cauae PHS1 was engineered as a thermotolerant chassis for direct conversion of palm oil (PO) and waste frying oil (WFO) into polyhydroxybutyrate (PHB) at 42 °C. Heterologous expression of a thermostable secretory lipase from C. necator H16 conferred extracellular oil-hydrolysis capability, and adaptive laboratory evolution was applied to improve glycerol metabolism. This combined strategy enabled direct utilization of triacylglycerol-derived carbon and enhanced PHB accumulation from both a model oil feedstock and a complex waste oil feedstock. Under non-optimized culture conditions, the engineered strain produced PHB from 10 g/L PO, reaching 5.11 g/L cell dry weight (CDW) with 64.18% PHB content, and from 10 g/L WFO, achieving 4.03 g/L CDW with 52.61% PHB content. These results support the potential of C. cauae PHS1 as a promising thermotolerant chassis for sustainable PHB biomanufacturing from complex waste oil feedstocks.

Clostridium acetobutylicum ATCC 824 (pCD07239), designated CD07239 in this study, was previously constructed by introducing the Clostridioides difficile CD630_0723–CD630_0729 gene cluster into the parental ATCC 824 strain. The CD07239 strain initiates butanol production during the early growth phase. To investigate the transcriptional basis of this phenotype, we performed RNA-seq-based transcriptome analysis using phenotype-defined RNA samples collected during batch fermentation. At 3 h, CD07239 had already produced approximately 0.1 g/L butanol, whereas butanol was not detected in ATCC 824. At 9 h, both strains produced butanol, but CD07239 accumulated a substantially higher level than ATCC 824. Principal component analysis revealed that the transcriptomes were separated according to fermentation stage and strain background. In ATCC 824, major solventogenic genes, including adhE1, ctfA, ctfB, and adc, were strongly induced at 9 h compared with 3 h. In contrast, early butanol production in CD07239 at 3 h was not accompanied by premature induction of these genes. Instead, adhE2 showed markedly high expression in CD07239, with log₂ fold changes of 6.4 at 3 h and 6.6 at 9 h compared with ATCC 824. Additional adhE2 expression in ATCC 824 did not reproduce early butanol production, suggesting that adhE2 alone is insufficient to induce this phenotype. These results indicate that early butanol production in CD07239 is associated with an adhE2-dominant transcriptional state distinct from the conventional solventogenic transition of ATCC 824.

Grapevine trunk diseases threaten sustainable viticulture, while wine lees remain an underexploited biocontrol-relevant by-product within the circular economy framework. An integrated bioprocess was developed to upgrade lees into high-molecular-weight biomolecule mixtures and low-molecular-weight peptide-enriched fractions through a defined sequence of unit operations: fermentation design by using Saccharomyces monocultures and sequential inoculations with non-Saccharomyces strains, ultrasound-assisted autolysis, enzymatic hydrolysis, and 3 kDa ultrafiltration. The antifungal activity of the products was tested in vitro and in vivo against Phaeomoniella chlamydospora and Phaeoacremonium minimum, the two main pathogens that cause Petri disease in grapevine. The results demonstrated that high-molecular-weight autolysates showed no inhibition. The post-hydrolysis fractions reduced mycelial growth of Phaeoacremonium minimum by 45%–75% and Phaeomoniella chlamydospora by 40%–57%, and decreased conidiation by up to 92% and 61%, respectively. They also reduced symptoms (wood discoloration) by 47%–52% and 35%–73% and endophytic fungal growth by 63%–84% and 80%–91% respectively. Those results indicate an association between hydrolysis and the appearance of antifungal activity. Untargeted metabolomics employing liquid chromatography–mass spectrometry (UHPLC–ESI–TIMS–QTOF) highlighted strain- and fermentation-dependent secondary metabolite signatures, with 448 features differentiating across fermentation conditions. Activity-based correlation fingerprinting identified 49 putative biomarkers negatively associated with pathogen growth. Overall, fermentation design functions as a controllable upstream quality parameter, offering a scalable circular economy pathway for the production of antifungal bioproducts derived from winery waste.

Lacto-N-neotetraose (LNnT), a pivotal oligosaccharide in human milk, plays a significant role in modulating the intestinal microbiota of infants, and exhibits potential applications in antiviral therapy. Corynebacterium glutamicum is widely used in industrial fermentation, but it is incapable of synthesizing LNnT under natural conditions. Previously, we constructed a recombinant C. glutamicum strain CL014 for LNnT production, but the yield was limited. In this study, the key enzyme β-1,4-galactosyltransferase LgtB was engineered to improve LNnT biosynthesis in CL014. Fourteen mutant strains were constructed by replacing the critical residue Arg²⁴ of LgtB to different amino acids and screened for the optimal LNnT production. The highest LNnT production was obtained in the mutant CL014-T. CL014-T could produce 1.40 g/L LNnT in shake flask cultivation, which is a 33.3% increase compared with the control strain CL014. Molecular docking simulations demonstrated that the mutation in CL014-T promotes the formation of additional hydrogen bonds in the flexible loop region, enhancing conformational flexibility and optimizing substrate interactions. Further mechanistic insights obtained through molecular dynamics simulations uncovered that the increased dynamic flexibility in two functional regions and the formation of more core hydrogen bonds collectively contribute to enhanced binding stability and improved product yield in the mutant. After 96 h feed-batch fermentation, CL014-T produced 2.29 g/L LNnT. This work demonstrates that C. glutamicum has the potential to be engineered as an efficient cell factory for human milk oligosaccharide production.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the irreversible α-decarboxylation of L-glutamate to produce γ-Aminobutyric acid (GABA). As the rate-limiting enzyme in GABA biosynthesis, GAD is central to research in neuroscience, plant stress physiology, and industrial biotechnology. This review systematically summarizes the structural characteristics, catalytic mechanisms, enzymatic properties, factors affecting the activity, and activity determination methods of GAD in microorganisms, plants, and mammals. Furthermore, from the perspectives of enzyme resource mining and protein engineering, this review presents an evaluation of strategies for enhancing GAD catalytic performance and stability, including site-directed mutagenesis, directed evolution, and semi-rational design. Finally, the applications of engineered GAD in GABA biosynthesis are comprehensively analyzed.

Paddy straw is one of the most abundant lignocellulosic agricultural residues generated worldwide, and its improper disposal through open-field burning contributes significantly to air pollution, greenhouse gas emissions, nutrient loss, and soil degradation. Sustainable management of paddy straw therefore requires efficient and eco-friendly biodegradation approaches. This review highlights the role of lignocellulolytic microorganisms and their enzyme systems in the biodegradation and valorization of paddy straw. The structural complexity of paddy straw, mainly due to cellulose, hemicellulose, and lignin, limits natural degradation and necessitates the synergistic action of specialized microbial communities and extracellular enzymes. The review discusses the contributions of bacteria, fungi, and actinomycetes in producing cellulases, xylanases, pectinases, laccases, manganese peroxidase, and lignin peroxidase involved in lignocellulose breakdown. It also summarizes microbial mechanisms of degradation and the influence of substrate characteristics, environmental conditions, and microbial interactions on enzymatic efficiency. Special emphasis is placed on microbial consortia, pretreatment strategies, and recent advances in consortium engineering for accelerated decomposition and biomass valorization. Overall, the review provides a comprehensive understanding of sustainable paddy straw management through integrated microbial and enzymatic biotechnological approaches.

While methanotroph-based bioconversion is a promising solution for mitigating methane emissions, its industrialization remains constrained. This limitation stems from substantial physicochemical disparities among real-world emission sources, highlighting a profound disconnect between laboratory-scale achievements and practical operational barriers. To bridge this divide, this review proposes a comprehensive, source-tailored decision-making framework. For diffuse emissions, the energy deficits caused by large volumetric overheads mandate an energy-minimized abatement strategy. Conversely, centralized point sources offer strong concentration driving forces that justify capital-intensive, high-efficiency biomanufacturing platforms to maximize productivity. Furthermore, guided by techno-economic analysis, we diagnose specific engineering bottlenecks, confirming that process improvements must strictly align with distinct market profiles, ranging from bulk single-cell protein and mid-value polyhydroxyalkanoates to high-value ectoine. Finally, we outline how prospective research frontiers can disrupt current technological paradigms. Strain-process multiscale modeling resolves the critical scale-up hurdle, and digital twin-based verification systems translate measurement uncertainties into tradable carbon assets. Moreover, co-producing site-integrated bio-fertilizers via nitrogen fixation can transform low-concentration abatement infrastructures into self-sustaining models. Although tailored approaches are currently necessary, central to future transitions is utilizing ultra-low-level methane enrichment to converge diffuse capture with centralized biomanufacturing. Advancing this frontier could eventually enable a truly integrated, universal methane biorefinery.

Glucoamylase is one of the most widely produced enzymes globally with diverse applications, such as in the fields of food, bioenergy, and papermaking. Microbially expressed glucoamylase serves as a potential additive in industrial alcohol fermentation to improve the utilization rate of starch. Komagataella phaffii is widely recognized for its capacity for high-density fermentation and efficient secretion of heterologous proteins, making it an ideal host for large-scale production under relatively simple fermentation conditions. However, the expression of glucoamylase in Komagataella phaffii currently faces challenges, including the potential toxicity of methanol as an inducer and overall low production yields. In this study, we first employed the improved transcription signal amplification device (iTSAD) in Komagataella phaffii to express the glucoamylase from Schizophyllum commune, achieving a titer of 3.31 g/L in a 5-L bioreactor along with a maximum glucoamylase activity of 5.2 U/mL. This study represents the first demonstration of high-efficiency methanol-free production of glucoamylase in Komagataella phaffii and provides strategies for the efficient expression and secretion of other industrial enzymes in Komagataella phaffii.

The untapped microbial diversity of the Amazon region represents a strategic frontier for biotechnological applications. This study screened, identified, and evaluated the potential of yeasts isolated from Amazonian environments to bioconvert sugarcane bagasse hydrolysate into ethanol and xylitol. The lignocellulosic hydrolysate was obtained by thermochemical treatment using H₂SO₄ (0.78% v/v) at 121 °C for 68 min, with a 20% solid loading. The yeast-like isolates were identified by sequencing the internal transcribed spacer (ITS) region (5.8S rDNA) and the D1/D2 domain of the 26S rDNA. Fifteen strains belonging to the genera Pichia, Debaryomyces, Meyerozyma, and Cyberlindnera exhibited the native capacity to convert xylose into xylitol and/or ethanol, achieving product yields (Y_P/S) of up to 0.20 g g^− 1 for ethanol and 0.25 g g^− 1 for xylitol in synthetic medium. Although initial fermentation in the acid hydrolysate (28.9 g L^− 1 xylose, 5.0 g L^− 1 arabinose, 1.2 g L^− 1 glucose, and 5.9 g L^− 1 acetic acid) supported cell growth, product synthesis was strongly inhibited by the high concentration of byproducts. The use of a biomass deacetylation strategy reduced acetic acid levels to 1.0 g L^− 1, enabling the conversion of sugars into the desired alcohols. Cyberlindnera saturnus strains achieved Y_P/S of up to 0.10 g g^− 1 for ethanol and 0.14 g g^− 1 for xylitol on deacetylated hydrolysate, while simultaneously performing the bioabatement of furans and residual acetic acid. These findings highlight unconventional Amazonian yeasts that, pending bioprocess optimization, exhibit promising metabolic traits for lignocellulosic biorefineries, including potential new yeast taxa.

The integrated cyanobacterial biorefineries are emerging as versatile microbial cell factories, where waste management can be coupled with simultaneous resource recovery to align with the circular economy approach. This review emphasizes the use of domestic or industrial wastewaters for cultivation of these photosynthetic prokaryotes due to their ability to simultaneously recover nutrients, capture CO₂ and produce multiple high-value bioproducts, including pigments, exopolysaccharides, bioplastics, nanomaterials and/ or biofuels. However, the major technical bottlenecks, such as low biomass productivity, wastewater variability, instability of engineered strains, cost-effective harvesting and downstream processing and insufficient pilot-scale validation remain the critical constraints for commercial success. Therefore, this review emphasizes the use of recent advancements, such as metabolic engineering, CRISPR-based genome editing, metabolic flux analysis, multi-omics strategies to improve productivity. Additionally, the techno-economic and life cycle assessment approaches are also discussed, which may help to evaluate the possible environmental impacts and industrial feasibility of the cyanobacterial biorefineries. Thus, this review proposes transformation of wastewater treatment systems into near zero-waste, scalable and low-cost multiproduct biorefineries aimed at achieving both economic viability and environmental sustainability.

The industrial production of nicotinic acid (vitamin B3) primarily relies on chemical synthesis, which suffers from harsh reaction conditions and severe environmental pollution. Although biocatalysis using nitrilase offers a greener alternative, the practical application of free enzymes is constrained by poor operational stability and difficulties in recovery. We developed a biomimetic hybrid immobilization platform that combines a zeolite core with poly(ethylene glycol) (PEG200) crosslinking, and a TEOS-derived silica shell to encapsulate whole cells of Corynebacterium glutamicum engineered to express a mutant nitrilase from Pseudomonas putida. This core–shell architecture is designed to balance mechanical robustness (from the silica shell) with biocompatibility and mass transfer (from PEG200 crosslinking). Systematic optimization of the immobilization conditions (6 g/L zeolite, 50 g/L cell loading, 3 g/L PEG200, 7 g/L TEOS) yielded a molar conversion rate of 88.78% for 50 g/L 3-cyanopyridine, significantly outperforming free cells (62.83%). Scanning electron microscopy (SEM) confirmed the formation of a dense yet porous core–shell structure. Compared with free cells, the immobilized biocatalyst exhibited optimal activity at 40℃ and pH 7.0, with a broader pH tolerance range (7.0–9.0). In repeated batch reactions, the immobilized cells achieved 14 reuse cycles with a loading of 700 g/L, producing 603.56 g/L nicotinic acid at a total molar conversion of 90.2%, whereas free cells were limited to 12 cycles and 86.7% conversion. This work establishes a sustainable biocatalytic platform for nicotinic acid production and demonstrates a generalizable immobilization strategy for whole-cell catalysis based on rationally designed organic–inorganic hybrid shells.

Microalgae are a promising platform for sustainable biorefinery applications due to their ability to produce a diverse array of high-value metabolites. Among these, astaxanthin has garnered significant interest for its potent antioxidant properties and extensive industrial relevance. However, large-scale production is limited by low biomass productivity, inefficient bioprocess strategies, suboptimal astaxanthin accumulation conditions, and energy-intensive harvesting techniques, leading to high operational costs. This review presents a comprehensive and advanced perspective on astaxanthin biorefinery by integrating cultivation optimization, bioprocess intensification, and innovative downstream processing. The study explores advancements in bioprocess engineering, metabolic regulation, and novel extraction techniques to enhance astaxanthin yield while minimizing resource inputs. Furthermore, the valorization of residual biomass post-extraction for sustainable fish feed and/or biofuel development is examined, offering a circular approach to waste reduction and resource efficiency. By adopting an integrated systems approach, this review addresses scale-up challenges and provides sustainable solutions for efficient astaxanthin production while contributing to aquaculture sustainability. The findings contribute to the development of a next-generation microalgal biorefinery that maximizes product recovery, minimizes waste, and strengthens the role of microalgae in a green bioeconomy. The subject aligns with multiple United Nations' Sustainable Development Goals by promoting sustainable astaxanthin production, resource efficiency, and circular bioeconomy principles: SDG 9 (Industry, Innovation, and Infrastructure), SDG 12 (Sustainable Production), and SDG 13 (Climate Action).

The Crabtree effect in Saccharomyces cerevisiae involves rapid metabolic switching between respiratory and fermentative states under dynamic conditions, yet its regulatory principles across different perturbation timescales remain poorly understood. Here, we imposed short-term glucose pulses and long-term dilution-rate perturbations on steady-state yeast cultures with extracellular physiological analysis, time-resolved intra- and extracellular metabolomics, proteomics, and constraint-based flux analysis to investigate dynamic Crabtree regulation. During short-term glucose pulses, ethanol accumulation scaled linearly with perturbation intensity, while approximately 33% of the incoming glucose carbon was consistently redirected to fermentation, revealing a conserved carbon-partitioning strategy. Under long-term dilution rate perturbations, abrupt shift-up triggered rapid overflow metabolism, respiratory imbalance, and growth suppression, whereas gradual ramp-up enabled coordinated respiratory adaptation and delayed fermentation onset. These results demonstrate that ethanol overflow does not require complete respiratory inhibition, but instead arises from a timescale mismatch between rapid glycolytic activation and slower mitochondrial adaptation. Furthermore, metabolite profiling identified fructose−1,6-bisphosphate (FBP), trehalose metabolism, and the glutamate/glutamine (Glu/Gln) ratio as key regulatory nodes coordinating carbon flux, nitrogen assimilation, energy metabolism, and phosphate homeostasis. Together, these findings establish a hierarchical and timescale-dependent framework for understanding dynamic Crabtree regulation and provide insights for engineering robust yeast cell factories under fluctuating industrial environments.

Engineered mycelial lines hold promise for enhanced production of valuable metabolites, serving as efficient biofactories for target compounds. However, in Alternaria alternata, the optimization of taxane biosynthesis remains constrained by an incomplete understanding of culture medium requirements, environmental factors, and the synergistic interactions among medium components that collectively influence metabolic pathways. In this investigation, the effects of critical culture factors—pH (4.0–8.0), sucrose (10.0–50.0 g L^− 1), (NH₄)₂HPO₄ (1.25-5.0 mmol L^− 1), and pectin (0.7–2.1 mg mL^− 1) as a biotic elicitor—on the biomass rate and total taxane yield were assessed using Response Surface Methodology (RSM). Among the evaluated factors, 50.0 g L^− 1 sucrose independently increased biomass and taxane yield by up to 1.86-fold and 71.74 µg gFW^− 1, respectively. Taxane biosynthesis was maximized (1.9-fold) under acidic conditions (pH 4.0), while biomass accumulation peaked at pH ~ 6.0. Notably, the highest biomass production was observed for the pH–sucrose interaction, reaching approximately 9.1 g flask^− 1whereas the sucrose–(NH₄)₂HPO₄ interaction exhibited the strongest statistical synergy for the taxane content, yielding up to approximately 210 µg gFW^− 1. Desirability function identified the optimal condition at pH 6.2, sucrose 50.0 g L^− 1, (NH₄)₂HPO₄ 3.75 mmol L^− 1,pectin ~ 1.5 mg mL^− 1 and overall desirability = 1.0. Importantly, extracts obtained under high-desirability conditions exhibited a significantly enriched taxane profile and pronounced cytotoxic activity against MCF-7 cells. Collectively, these findings offer a robust and potentially scalable fermentation strategy for enhancing taxane biosynthesis in A. alternata, and underscore the potential of engineered mycelial lines as high-yielding biofactories under optimized conditions.