Bioresource Technology最新文献

Escherichia coli is a dominant host for recombinant protein production due to its well-characterized genetics and low cultivation cost, yet high-level intracellular expression frequently leads to insoluble inclusion bodies. Targeting proteins to the periplasm represents an effective strategy for improving soluble expression, but the efficiency of conventional signal peptides is highly protein-specific, restricting their universal utility. This study investigates an artificially engineered peptide, Vesicle Nucleating peptide 6 (VNp6), and systematically evaluates its capacity to mediate recombinant protein expression with green fluorescent protein (GFP) as a reporter. Results revealed that VNp6 markedly enhanced intracellular soluble GFP expression at 37 ℃, 25 ℃, and 16 ℃. Its intracellular fluorescence intensity was 128-fold and 220-fold higher than that of PelB and OmpA at 37 ℃, 82-fold and 113-fold at 25 ℃, and 107-fold and 160-fold at 16 ℃, respectively. Subcellular localization confirmed predominant periplasmic localization and improved solubility of the protein. Relative to the native periplasmic signal peptides FimH and PhoA, VNp6 improved solubility by 4‑fold and 14‑fold, respectively. For SacB, TVE, and amylase, solubility was enhanced by 27%, 30% and 17% at 16 ℃, respectively. Although extracellular protein accumulation was partially attributable to cell lysis, VNp6 consistently promoted periplasmic protein accumulation and enhanced soluble protein production across multiple recombinant proteins. Collectively, these results demonstrate that VNp6 serves as a powerful tool for improving periplasmic protein accumulation and soluble expression in E. coli expression systems.

The polar environment harbors extremely rich and valuable natural product resources. In this study, a strain of Antarctic fungus, Geomyces sp. wnf-18c, in Antarctic soil was isolated in a laboratory. This fungus produces a purplish-red pigment whose hue closely resembles that of the high-end pigment carminic acid. However, approximately 50 % of the pigment remains within the fungal hyphae, restricting subsequent separation and extraction. In this study, the deletion of the key cell wall protein-encoding genes CHS3 and AGS1 enhanced pigment secretion. It also considerably inhibited the strain's growth, making it difficult to substantially increase the total pigment yield. To overcome these hurdles, the xylose-inducible system from Trichoderma reesei was heterologously expressed in Antarctic fungi, and the CRISPR-Cas9 system was placed under the control of this induction system. The exogenous addition of xylose as a signal to initiate genetic editing achieved the spatiotemporal regulation of the target gene-activating gene editing that restricts growth only after the completion of the strain's growth phase. The spatiotemporal regulation of key cell wall proteins significantly mitigated growth inhibition, greatly enhanced pigment secretion, and raised the total pigment yield. This discovery advances our understanding of the spatiotemporal specificity and dynamic regulation of microorganisms, offering novel strategies for exploiting polar microbial resources and reducing industrial production costs.

Iron salt-based chemically enhanced primary treatment effectively captures organic carbon from municipal wastewater, providing a concentrated substrate for methane recovery through anaerobic digestion. However, methane production from iron salt-based chemically enhanced primary sedimentation (Fe-CEPS) sludge remains substantially lower than expected, and the mechanisms responsible for this reduction remain poorly understood. Here, the relationship between iron-bound organic carbon (Fe-OC) formation and methane production was investigated by combining anaerobic digestion experiments with iron mineral-glucose model systems. Increasing Fe dosage reduced methane yield from 221.1 to 74.7 mL CH₄ g^-1 VS while promoting Fe-OC accumulation. A strong negative correlation was observed between Fe-OC content and methane production. Fresh Fe-CEPS sludge contained only limited Fe-OC (<10% of total organic carbon), whereas Fe-OC accounted for approximately 26% of total organic carbon after anaerobic digestion, indicating that Fe-OC formed predominantly during digestion rather than during coagulation. Iron speciation analyses revealed extensive Fe(III) reduction accompanied by secondary iron mineral formation. Model experiments further demonstrated that Fe(II)-induced mineral transformation increased mineral crystallinity, enhanced organic carbon stabilization, reduced carbon bioavailability, and consequently suppressed methane production. Scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy further revealed progressively stronger spatial associations between organic carbon and iron minerals as mineral crystallinity increased. These findings reveal a previously unrecognized mechanism whereby Fe(II)-induced secondary iron mineral formation promotes Fe-OC accumulation and reduces organic carbon bioavailability during anaerobic digestion. Regulating iron mineral transformation may therefore represent an effective strategy for improving methane recovery from iron-enhanced primary sludge.

Systematic mining of heat stress factors is pivotal for constructing robust microbial cell factories. However, the genetic basis underlying thermotolerance remains poorly understood in C1 gas-utilizing chassis. Here, we identified LexA as a key regulator governing thermotolerance and glycogen accumulation in the methanotroph Methylotuvimicrobium buryatense 5GB1C. Through ultraviolet mutagenesis coupled with adaptive laboratory evolution, we developed a thermotolerant evolved strain 5C27. Whole-genome resequencing and transcriptomic analysis revealed global transcriptional reprogramming underlying the heat tolerance phenotype. Notably, LexA was sharply downregulated at 40 °C and identified as a core regulatory factor for thermoadaptation. Transcriptomic analysis indicated that the acquired thermotolerance arises from coordinated regulation of oxidative stress response, energy metabolism, and cellular homeostasis. Subsequent validation experiments demonstrated that lexA deficiency simultaneously conferred enhanced thermotolerance and redirected intracellular carbon flux, resulting in increased glycogen production that exceeded wild-type levels even at the optimal growth temperature of 30 °C. Collectively, our work demonstrated for the first time that LexA plays a critical regulatory role in thermotolerance of methanotrophs. This discovery also provides a foundation for advancing the development of thermotolerant cell factories and promotes the deployment of C1-gas industrial biomanufacturing.

Isobutene is an important platform chemical that is still predominantly produced from petroleum-derived feedstocks. The ferulic acid decarboxylase (Fdc) catalyzed decarboxylation of 3-methylcrotonic acid provides a green and sustainable route to bio-based isobutene. However, the poor activity of native Fdc toward this non-natural substrate severely limits its practical application. Here, we developed an integrated enzyme-engineering strategy that combines sequence-cluster mining with distal-site engineering to improve catalytic performance. The engineered triple-site mutant V132C/Y298F/S484A (3MUT) exhibited 5.3-fold the isobutene production of WT. Coupling this mutant with reaction-process optimization and cofactor engineering increased the isobutene yield to 67.9%, representing the highest reported yield to date. Mechanistic studies revealed that distal-site mutations in 3MUT reshaped the active site by disrupting the M293-mediated hydrogen bond that blocks the substrate to active site, then further promoting favorable hydrogen-bond interactions with the substrate via R183 and E292, respectively. These findings establish an efficient route for sustainable bio-based isobutene production and demonstrate the potential of distal-site engineering for improving UbiD-family decarboxylases.

Phosphorus (P) and nitrogen (N) are critical nutrients increasingly lost to wastewater streams. Existing methods focus on removal rather than recycling, and are poorly equipped for valorization. Building on our group's prior work engineering Citrobacter freundii (CPP) overexpressing ppk for enhanced P removal, we demonstrate here that CPP simultaneously valorizes both P and N from real municipal wastewater into intracellular polyphosphate (polyP) and spermidine (Spd). Under optimized conditions, CPP accumulated intracellular polyP and Spd at 247.56 mg/g and 102.71 mg/g, representing 2.44- and 2.99-fold increases over pre-optimization levels, with corresponding P and N valorization rates reaching 76.76% and 51.16%, respectively, substantially exceeding the productivity of conventional polyphosphate-accumulating organisms. Driven by the co-accumulation, polyP and Spd phase-separated into insoluble granules termed stabilisomes with diameters of up to 181 nm and a composition of 44.6% polyP and 20.5% Spd, which maintained intracellular homeostasis and sustaining their continuous co-production. Leveraging the sustained co-production of polyP and Spd, the product value far exceeds that of conventional single-nutrient recovery techniques. Calculations indicate that the heat-inactivated CPP-derived biostimulant (CPPB) has a unit production cost of approximately ¥1.97/g. Applied as a foliar spray at 1.8 g/L, increased Brassica chinensis dry weight by 121.14% and plant height by 31.08%, outperforming commercial microbial fertilizers tested. This work establishes a practical biorefinery route for simultaneous P and N valorization from municipal wastewater, advancing the transition toward a circular bioeconomy.

Nitrous oxide (N_2O) emissions from biological wastewater treatment represent a significant challenge for climate-responsible operation due to their intermittency and occurrence as short-lived emission hot moments. Effective mitigation therefore requires accurate prediction and systematic identification of causal pathways and operational risk conditions. This study develops a probabilistic causal machine learning framework based on long-term online monitoring data from a full-scale wastewater treatment plant for low-emission process control and operational management. An optimal predictive model is first established to characterize N_2O dynamics under varying operational regimes. Building upon this foundation, cohort-based SHapley Additive exPlanations are used to identify regime-dependent nonlinear effects of key operational variables, including dissolved oxygen, ammonium, nitrate, and temperature. Linear Non-Gaussian Acyclic Model-based causal discovery is then used to characterize causal pathways associated with N_2O emission hot moments under different operational regimes. Copula-based joint probability analysis is conducted to quantify the likelihood of N_2O emission hot moments under interacting process conditions. Results demonstrate that N_2O emission hot moments are not triggered by single-factor thresholds but emerge from specific combinations of operational states, revealing interaction-dominated and regime-sensitive causal pathways. By converting interpretable machine learning outputs into probabilistic risk-decision rules, the proposed framework provides actionable guidance for adaptive aeration control, substrate load regulation, and proactive emission mitigation. This study establishes a data-driven framework for interpreting and managing N_2O emissions in biological wastewater treatment. Overall, the proposed framework provides practical support for proactive N_2O emission mitigation while maintaining process stability in full-scale wastewater treatment plants.

Achieving efficient delignification while preserving the native structure of lignin remains a critical challenge for integrated bamboo biorefineries. In this study, a ternary deep eutectic solvent (DES) composed of (Benzyltriethylammonium chloride) TEBAC, ethylene glycol (EG), and p-toluenesulfonic acid (p-TsOH) was developed for bamboo fractionation. The optimized TEBAC/EG/p-TsOH system removed up to 97.94% of lignin and 85.99% of hemicellulose, while retaining 84.46% of cellulose. The pretreated bamboo showed significantly improved enzymatic saccharification, achieving a glucose yield of 78.98% and an ethanol yield of 73.16% following fermentation. The recovered lignin, characterized by a low molecular weight and well-preserved β-O-4 linkages, self-assembled into uniform lignin nanoparticles (LNPs) with approximately 318 nm. These LNPs efficiently encapsulated difenoconazole (Dif@LNP-140°C) with an encapsulation efficiency of 70.02%, exhibiting excellent photostability and pH-responsive release and full antifungal activity against Fusarium spp. Molecular dynamics simulations revealed that synergistic non-covalent interactions, particularly hydrogen bonding with G-units and π-π stacking with S-units, govern the stable encapsulation of the drug. This work presents a sustainable lignin-first strategy for integrated bamboo valorization, enabling the co-production of biofuels and lignin-based nanomaterials while maximizing resource efficiency.