Bioresource Technology

The increasing accumulation of polyethylene terephthalate (PET) waste has prompted the development of sustainable biotechnological strategies for its degradation and valorisation. This study presents an integrated approach combining enzymatic PET depolymerization by Yarrowia lipolytica, engineered to express and secrete the cutinase HiC and the lipase CalB, with the microbial valorization of PET-derived monomers, terephthalic acid (TPA) and ethylene glycol (EG), by Rhodococcus jostii RHA1. Y. lipolytica was further engineered for xylose metabolism, enabling enzyme production from low-cost lignocellulose-derived substrates. Enzymatic assays with HiC and CalB crudes effectively hydrolysate PET to TPA and EG, demonstrating functional enzymatic activity without purification steps. In addition, R. jostii RHA1 was able to use as substrate the released monomers and accumulated intracellular lipids. Overall, this work demonstrates the feasibility of coupling the production of PET degrading enzymes and microbial lipid, using an abundant monosaccharide, with the assimilation of the PET degradation products to also produce microbial lipids. This modular system provides a promising framework for the sustainable upcycling of plastic waste into value-added bioproducts within a circular economy.

Engineering heterologous utilization of substrates requires selection of catabolic pathways that balance strain performance and product biosynthesis. Here, we compare the oxidative and isomerase arabinose utilization pathways in Pseudomonas putida strains engineered for cis,cis-muconic acid production from glucose and xylose. Based on the point of entry into central carbon metabolism, we hypothesized that the oxidative arabinose pathway would enable higher productivity while the arabinose isomerase pathway would enable higher muconate yield. In both strains, additional modifications were engineered to improve muconic acid production including sugar transporter tuning, catechol 1,2-dioxygenase overexpression, a feedback-resistant DAHP synthase, and a flux-stabilizing gltA variant. Consistent with our hypothesis, the oxidative arabinose pathway supported faster growth and higher productivity (0.58 g/L/h), whereas the arabinose isomerase pathway improved carbon efficiency, achieving muconate yields of up to 50 C-mol% in fed-batch bioreactors. Process modeling indicates that these performance metrics can reduce the minimum selling price of muconate-derived adipic acid to $2.74/kg and greenhouse gas emissions to 1.31 kg CO2e/kg, approaching cost parity and reducing emissions by 86% relative to fossil carbon-derived adipic acid. Overall, this study presents a systematic comparison of sugar catabolic pathways that enabled development of strains suited for the tradeoffs between rate and yield.

Cellobiose is an important disaccharide used in food, health, and biorefinery applications, but its efficient enzymatic production from crystalline cellulose remains challenging. In this study, crystalline cellulose was dissolved in ionic liquids and regenerated by dilution, and subsequently hydrolyzed at 80{degrees}C using a highly thermostable cellobiohydrolase, HmCel6A-3SNP. The enzyme retained activity in the presence of low concentrations of ionic liquids. Among the pretreatment conditions tested, cellulose treated with 1-butyl-3-methylimidazolium chloride ([Bmim]Cl) showed the highest enzymatic digestibility. After washing to remove residual ionic liquid, the reaction produced reducing sugars at levels 1.5-fold higher than those obtained in the presence of 10% [Bmim]Cl, with cellobiose accounting for approximately 96% of the products. Under the optimized conditions, the hydrolysis yield reached [~]36% after 48 hr. Structural analyses using birefringence imaging, electron microscopy, and Fourier transform infrared spectroscopy indicated that higher-order structural changes in regenerated cellulose strongly influence enzymatic accessibility. These results demonstrate the potential of combining ionic-liquid pretreatment with thermostable enzymes for selective cellobiose production from cellulose.

Muconic acid is a versatile platform chemical that can be biologically produced from lignocellulosic substrates, including from lignin-related aromatic compounds. Pseudomonas putida has been previously engineered to convert lignin-related aromatic compounds to muconate at quantitative molar yields. This high atom efficiency requires a supplemental carbon and energy source to support bacterial growth, and central carbon metabolic efficiency and its interaction with aromatic catabolism are underexplored. Here, we applied proteomics, metabolomics, and 13C-fluxomics to quantitatively compare central carbon and energy metabolism in wild-type P. putida KT2440 and a muconate-producing strain, P. putida CJ781. During cultivation on glucose and 4-hydroxybenzoate, CJ781 showed increased glucose uptake, reconfigured central fluxes, and increased extracellular leakage of aliphatic acids relative to wild type. These altered fluxes supported a 3-fold higher ATP pool, in excess of demand. Pyruvate and acetate secretion in CJ781 was mitigated by debottlenecking TCA-cycle entry via citrate synthase overexpression. Furthermore, tuned expression of the catechol dioxygenase and protocatechuate decarboxylase enabled the production of 36.3 g L-1 muconate at 1.1 g L-1 h-1. Overall, this work reveals how P. putida redirects carbon and energy fluxes to support aromatic bioconversion for improved bioproduction from renewable feedstocks.

Plastic pollution from fossil-based materials is a major global environmental challenge. Microbial-derived bioplastics, such as polyhydroxybutyrate (PHB), offer a promising biodegradable alternative. However, the high substrate and operational costs of PHB production remain a major barrier to large-scale deployment. Optimizing PHB synthesis requires navigating a multidimensional design space of metabolic, nutritional, and operational variables that is impractical to explore experimentally. Here, we developed an integrated computational framework that combines a genome-scale metabolic model (GEM) of Rhodopseudomonas palustris, machine-learning surrogate modeling, Pareto multi-objective optimization, and thermodynamics-based flux analysis (TFA) to identify cost-efficient and biologically feasible PHB production strategies. Experimental and literature-derived medium compositions were translated into mechanistic constraints, enabling the GEM to generate metabolically coherent synthetic datasets that augmented sparse experimental observations. CatBoost surrogate models trained on this hybrid dataset accurately predicted PHB synthesis across thousands of hypothetical conditions, and Pareto optimization revealed operating regimes that balance PHB productivity with nutrient cost. TFA validated the thermodynamic feasibility of these strategies and refined pathway usage, reinforcing thiolase-initiated routing into PHB biosynthesis and suppressing infeasible {beta}-oxidation-like redox loops. Overall, this hybrid GEM-ML-TFA framework identifies metabolic bottlenecks, engineering targets, and cost-optimal nutrient regimes for phototrophic PHB production, providing a scalable blueprint for rational process and strain design.

Acetate metabolism drives mixotrophic and heterotrophic growth in some microalgae. Acetyl-CoA synthetase (ACS) and acetate kinase (ACK) are often considered the main enzymes involved in acetate catabolism in microalgae; however, their contributions to metabolic flux and carbon allocation are not fully understood. In this study, the functions of cytosolic ACS1 and mitochondrial ACK2 were characterized using two knockout mutants of the model microalga Chlamydomonas reinhardtii. The acs1 mutant exhibited a growth-oriented phenotype, characterized by 29.8% faster cell growth at 96 h and up to a 15.5% higher acetate depletion rate, yet showed a 38.3% lower triacylglycerol (TAG) content at 48 h under heterotrophic conditions. By contrast, the ack2 mutant exhibited an altered carbon-allocation phenotype under heterotrophic conditions. Despite an up to 32.4% lower respiratory oxygen consumption rate and a 27.7% reduction in cell density, ack2 exhibited a 39.3% higher biomass concentration and a 90.4% greater dry weight per cell than the wild type at 96 h. Biochemical analysis revealed that ack2 accumulated 23.3% more carbohydrate than the wild type at 120 h under heterotrophic conditions, whereas its TAG level remained comparable to that of the wild type. These findings suggest that, under heterotrophic conditions, the loss of cytosolic ACS1 facilitates cell growth and division at the expense of TAG biosynthesis, whereas the loss of mitochondrial ACK2 regulates growth by affecting carbon flux toward biomass and carbohydrate accumulation. This work provides insight into acetate catabolism in C. reinhardtii and suggests targets for engineering microalgae for production of biomass and bioproducts.

Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of few cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium Cyanothece PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO3) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (cax3-cax4, gor, and sodB) acting in metabolism and/or response to stresses, using Cas12a mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of{Delta} sodB mutant further confirmed its targeted deletion. Overall, CRISRPR/Cas12a provides a rapid and efficient system for genome editing in Cyanothece PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity and moreover, the still poorly known mechanism(s) of intracellular CaCO3 biomineralization. Key PointsO_LIRapid and efficient CRISPR/Cas12a editing established in Cyanothece PCC 7425. C_LIO_LIFully segregated knockout mutants obtained after single selection round. C_LIO_LIPlatform for nuclear waste bioremediation and other biotechnological applications. C_LI

Biotechnological applications of oxygenic photosynthetic organisms depend on conversion of light energy into chemical energy through photosystems (PS). This energy can then be used to drive engineered metabolic pathways that are designed as strong electron sinks. For optimal performance, the engineered host metabolism must also be balanced with the native photoprotective electron transfer network. This includes the energy-consuming function of flavodiiron (Flv) proteins, which are universal to cyanobacteria and all other oxygenic photosynthetic organisms except angiosperms. In the cyanobacterium Synechocystis sp. PCC 6803, four different Flv proteins have been shown to function in a Mehler-like reaction within two heterodimeric forms (Flv1/Flv3 and Flv2/Flv4), donating electrons to O2 without generating oxidative stress. Previously, deleting Flv3 in the Synechocystis sucrose-producing (S02) strain was shown to cause drastic metabolic changes in S02{Delta}flv3, shifting it from photoautotrophic to mixotrophic growth (Muth-Pawlak, et al., 2024). In this study, we took an opposite approach by complementing S02 with Flv3 overexpression at different levels using RBS tuning. Interestingly, this resulted in S02oeFlv3 strains with significantly increased overall photosynthetic activity and sucrose production, enhanced cell growth, and storage compound accumulation. However, these outcomes are shown not to be due to conventional O2 photoreduction activity catalysed by Flv1/Flv3. Instead, we postulate that the observed changes are linked to the previously unidentified function of homomeric Flv3/Flv3 and the strongly increased sulphate redox metabolism. Based on extensive proteomic and metabolite analyses, we hypothesise that the Flv3 homooligomer uses sulfate metabolites directly or indirectly as the final electron acceptor instead of O2. This would also explain the upregulation of sulfate-related enzymes, as well as SQR, which passes the electrons back to the PQ pool in the Flv3 overexpression strain.

The accumulation of plastic waste necessitates innovative strategies that convert polymer carbon into value-added products. Here, we present a sequential yeast-bacterial workflow for the integrated valorisation of glycerol and amorphous polyethylene terephthalate (amPET) into triacyl glyceride (TAGs) and polyhydroxyalkanoates (PHAs). First, an engineered obese strain of Yarrowia lipolytica was cultivated on glycerol, for the simultaneous production of intracellular lipids, up to 42.9% of its cell dry weight, and secretion of a PET-depolymerizing enzymatic cocktail, composed of the cutinase from Mycothermus thermophilus (HiC) and the lipase B from Moesziomyces antarcticus (CALB). The resulting enzymatic crude hydrolysed amPET, and the released degradation products--terephthalic acid (TPA) and ethylene glycol (EG)-- served as feedstocks for a bacterial consortium composed of Comamonas testosteroni RW31 and Pseudomonas putida JM37, which naturally assimilate TPA and EG, respectively. This consortium successfully upcycled the released monomers into intracellular polyhydroxybutyrate (PHB) and medium-chain-length PHAs. Furthermore, fluorescent strains of both bacteria enabled the development of a semi-quantitative method for monitoring the consortium population dynamics. Overall, this study provides a robust proof-of-concept for a circular bioeconomy approach, successfully coupling glycerol-based enzyme and lipid production with the downstream biological conversion of PET-derived monomers into valuable bioplastics. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=142 SRC="FIGDIR/small/729029v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1575eedorg.highwire.dtl.DTLVardef@1195aeborg.highwire.dtl.DTLVardef@1a01bb0org.highwire.dtl.DTLVardef@79f4f6_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lignocellulosic biomass represents a promising renewable feedstock for sustainable biorefinery applications, yet efficient enzymatic saccharification remains challenging due to the recalcitrant structure of plant cell walls. This study presents a comprehensive comparative analysis of enzymatic activities, saccharification performance, and secretome composition of six fungal species cultivated under solid-state fermentation (SSF) and submerged fermentation (SmF) conditions using untreated flax shives as substrate. While SmF yielded approximately 4-fold higher total protein concentrations (0.38 {+/-} 0.13 g.L-1 vs. 0.08 {+/-} 0.02 g.L-1), SSF-derived enzymes demonstrated superior specific enzymatic activities, particularly for endo-xylanase and endo-cellulase, resulting in more efficient biomass saccharification. Proteomics analysis revealed distinct secretome profiles between fermentation modes, with SSF showing higher proportions of polysaccharide metabolism proteins (71.0%) compared to SmF (49.3%), while SmF exhibited greater enzyme diversity including more lytic polysaccharide monooxygenases (LPMOs) and auxiliary activity enzymes. Trichoderma species consistently demonstrated the highest saccharification efficiency, with glucose yields reaching 2.37 mM under SSF conditions. A Scheffe simplex-lattice mixture design comprising 65 enzyme cocktail combinations revealed significant synergistic interactions between several cocktails, with the binary mixture of Trichoderma 2SA21 and P. chrysogenum achieving 54% synergy - in terms of higher sugar release above expectations - and the highest total monosaccharide release (1.80 mM). These findings provide practical guidance for developing cost-effective enzyme cocktails for lignocellulosic biorefinery applications, emphasizing the importance of fermentation mode selection and strategic strain combination over enzyme supplementation complexity. The methodology established here, combining systematic screening, comparative proteomics, and statistical mixture design, offers a robust framework for optimizing fungal enzyme systems across diverse biomass substrates. BULLET POINTSSuperior enzymatic activity (xylanase, cellulase) and saccharification in solid-state fermentation Superior total protein content and diversity in submerged fermentation Specific enzyme cocktails combination can lead to synergistic effects, justifying a combinatorial approach GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/729743v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1f3704dorg.highwire.dtl.DTLVardef@151e4bforg.highwire.dtl.DTLVardef@180ced7org.highwire.dtl.DTLVardef@18bb42f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Engineering the ethanologenic Gram-negative bacterium Z. mobilis for the secretion of hydrolytic enzymes is a key step towards establishing a biofuel cell factory that uses complex waste material as feedstock. Secretion strategies in Z. mobilis have exclusively relied on signal peptides, which limit protein transport to the periplasm. To achieve single-step secretion across the Z. mobilis double-layered membrane, we sought to identify a native Type I Secretion System (T1SS) tag for fusion to proteins of interest. While a T1SS operon had been identified in the Z. mobilis genome, its native cargo had remained unknown and the use of T1SS secretion tags had so far been unexplored. Here, bioinformatic analysis identified the Major Intrinsic Protein (MIP) as a putative T1SS cargo, and its role validated through fusion of C-terminal sequences of two lengths (61 and 141 amino acids) to a heterologous {beta}-galactosidase from Bacteroides thetaiotaomicron, expressed in Z. mobilis. The 141 amino acid tag, including two RTX domains, resulted in significantly higher secretion efficiency than the 61 amino acid tag lacking RTX repeats, consistent with the established role of RTX domains in preventing premature cytoplasmic folding, thus improving secretion. As extracellular secretion of hydrolytic enzymes has remained a major bottleneck in the development of Z. mobilis as a sustainable cell factory, the identification of a native T1SS secretion tag directly addresses this limitation, introducing a novel tool for enzyme delivery.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant protein on Earth, is an attractive and sustainable food ingredient owing to its favourable nutritional and techno-functional properties. Leafy vegetables are particularly rich sources of RuBisCO; however, large-scale vegetable production generates substantial quantities of residual biomass throughout agri-food supply chains. Drying is widely used to stabilise this biomass and facilitate storage, transport, and handling, yet most reported RuBisCO extraction methods have been developed for fresh material and are poorly suited to dried feedstocks. Here, we present a simple, scalable, and cost-effective process for the recovery of food-grade RuBisCO from dried leafy biomass. Using spinach, rocket, and kale as model systems, efficient protein extraction was achieved from both freshly dried leaves and commercially available leaf powders without the need for resource-intensive processing. Application of the method to spinach yielded approximately 75 mg of high-purity RuBisCO per 100 g fresh-leaf equivalent, corresponding to an extraction efficiency of [~]70%, which increased to [~]90% following supplementation with 20 mM CaCl2. The recovered protein fraction also exhibited favourable foaming capacity and foam stability, demonstrating its potential as a functional food ingredient. This work provides a practical route for the valorisation of dried vegetable residues and supports the development of circular, waste-to-value supply chains for sustainable plant protein production.

Extremophilic red microalgae are promising platforms for sustainable biotechnology, combining robust growth under selective thermoacidophilic conditions with production of thermostable phycobiliproteins and carbon-rich biomass. However, reactor-dependent effects on growth, product formation and biomass composition remain insufficiently resolved. Here, we systematically evaluated the extremophilic red microalga Cyanidioschyzon merolae across cultivation scales and reactor formats and benchmarked its performance against the well-established Galdieria javensis and Limnospira platensis. In small-scale multi-cultivator photobioreactors and microfluidic growth chambers, C. merolae showed superior growth, reaching a maximum growth rate of 0.034 {+/-} 0.001 h-1 and 8.3 {+/-} 0.3 g l-1 cell dry weight. Microfluidic cultivation enabled growth analysis at single-cell resolution and matched growth rates obtained in photobioreactors. To identify scalable production strategies, C. merolae was further cultivated in a flat-panel photobioreactor and a custom-designed internally illuminated photobioreactor. The custom-designed photobioreactor delivered the highest biomass concentration and productivity, yielding 11.5 {+/-} 0.6 g l-1 cell dry weight and 1.07 {+/-} 0.06 g l-1 d-1, and comparable yields with regard to R-phycocyanin and R-allophycocyanin. Biomass analysis revealed substantial carbon and nitrogen contents, starch accumulation up to > 20 % of cell dry weight, and fatty acids dominated by palmitic, linoleic and oleic acids. Despite its reduced cell wall fraction, C. merolae contained structurally diverse, cultivation-dependent polysaccharides. These results establish C. merolae as a versatile chassis for thermostable pigment production and renewable feedstock generation, highlighting photobioreactor design as a key determinant of productivity and biomass quality.

Microorganisms are the key drivers of carbon cycling in coastal marine sediment ecosystems, significantly influencing carbon storage and release during Gracilariopsis lemaneiformis cultivation. This study employed 16S rRNA sequencing, a high-throughput qPCR chip, and carbon isotope labeling to assess the impact of G. lemaneiformis cultivation on carbon cycling processes in coastal sediments. A comparative analysis was conducted between cultivated zones (GZ) of G. lemaneiformis and adjacent control zones (CZ). The results indicated that macroalgae cultivation significantly modified sediment-seawater exchange dynamics and accelerated carbon cycling within coastal marine sediment ecosystems. Furthermore, G. lemaneiformis cultivation increased the abundance of genes linked to polysaccharide degradation and carbon fixation pathways, thereby enhancing carbon cycling efficiency. The ecosystem multifunctional index, calculated based on carbon fixation gene abundance, was significantly higher in GZ compared to CZ. Incubation experiments using 13C-NaHCO3 demonstrated that cultivation markedly elevated the carbon fixation rate of sediment, emphasizing a higher potential for carbon sequestration in sedimentary environments cultivated with macroalgae. Additionally, cultivation significantly altered sediment microbial communities, simplifying their structural complexity. Key microbial taxa identified via k-core species analysis--including Subgroup10 of Desulfobacterota and MBNT15, correlated strongly with carbon fixation rates, indicating their pivotal roles in sediment carbon cycling processes. This study provides critical insights into how large-scale macroalgae cultivation influences coastal carbon dynamics and informs strategies for optimizing carbon management in aquaculture ecosystems.

In this study, the lipid content produced by Thraustochytrium sp. in a medium prepared from orange pulp residue was compared with that obtained in a conventional medium. The pulp residue was subjected to freezing, blending, and filtration in seawater to prepare three treatments: conventional medium (T1), filtrate (T2), and filtrate supplemented with KNO3 (T3). A growth curve was performed over six days, after which biomass and lipid content were analyzed. The results showed that T2 exhibited the highest cell growth and biomass yield (5.24 g/L). However, lipid content was higher in the conventional medium (38.75%), whereas the treatments containing orange extract showed lower values. These findings suggest that the medium prepared from orange pulp residue is feasible for the growth of Thraustochytrium sp., but requires optimization to enhance lipid accumulation and its potential use in sustainable bioprocesses.

Muconic acid is an industrially valuable molecule that can be biologically produced from diverse biogenic and waste-derived feedstocks, including sugars and lignin- and plastic-derived aromatic compounds. However, accumulation of protocatechuate (PCA) has been observed in multiple microbes engineered for muconate production when the PCA decarboxylase, AroY, is used. This raises the question of whether PCA decarboxylation represents a rate-limiting step and how this bottleneck might be alleviated, especially given the toxicity and reactivity of PCA and catechol intermediates. To address this, we performed adaptive laboratory evolution (ALE) on a strain of Pseudomonas putida originally engineered for muconate production from aromatic compounds, but with catBC restored, to select for improved conversion of PCA and, in separate lineages, 4-hydroxybenzoate. Contrary to our expectations, the predominant beneficial mutations localized to the catA1 cassette encoding catechol 1,2-dioxygenase, rather than aroY or its associated cofactor biosynthesis genes. Transcriptomic analysis revealed elevated catA1 expression in evolved isolates from ALE, and introduction of these mutations improved productivity in strains designed for muconate production from both aromatic and sugar substrates. Quantitative proteomics and biochemical assays demonstrated that the mutations also led to increased CatA1 protein abundance and modest enhancements in catalytic efficiency, respectively, with strain phenotypes largely driven by high CatA1 levels and potentially synergistic kinetic improvements. Additional reverse-engineering studies identified variants with modest effects on muconate accumulation, including those with potential to enhance biosynthesis of the prenylated FMN cofactor of AroY. Collectively, these results indicate that catechol, not PCA, is the principal bottleneck in muconate production via the PCA decarboxylation route originally demonstrated by Draths et al., refining our understanding of pathway limitations and offering new strategies for improving rate, yield, and strain resilience in muconate bioproduction. HighlightsO_LIAccumulation of metabolic intermediates was alleviated by adaptive laboratory evolution C_LIO_LISequencing, proteomics, and enzyme kinetics revealed mechanisms for adaptation C_LIO_LIIncreased CatA1 expression reduced bottlenecks and improved muconate production C_LI

Insects offer promising opportunities for organic waste bioconversion; however, they cannot efficiently degrade synthetic polymers such as polyethylene terephthalate (PET). Here, we generated transgenic Drosophila melanogaster lines to express in vitro-evolved variants of two PET-degrading enzymes with distinct biochemical properties: an engineered Ideonella sakaiensis PETase variant (TS-{Delta}IsPET) and a leaf-branch compost cutinase variant (TA-{Delta}LCC). Both enzymes, fused to a Drosophila gut-derived secretory signal, were produced and secreted by both Drosophila cultured S2R+ cells and transgenic larvae. Both enzymes were glycosylated upon secretion, a post-translational modification that did not abolish their catalytic activity. Notably, TA-{Delta}LCC displayed [~]6-fold higher esterase activity than TS-{Delta}IsPET in larval extracts and TA-{Delta}LCC-containing extracts depolymerised PET nanoparticles in vitro under enzyme-favourable conditions. Transgenic flies showed normal development, fertility and survival. Morphological and biochemical analysis confirmed that TA-{Delta}LCC expression did not alter midgut structure and function. Together, these results establish Drosophila melanogaster as a model for functional expression and comparative evaluation of engineered PET-degrading enzymes and identify TA-{Delta}LCC as a promising candidate for exploitation in insect species relevant to plastic contaminated waste bioconversion. HighlightsO_LITransgenic D. melanogaster enables in vivo study of engineered PET enzymes C_LIO_LIEngineered TS-{Delta}IsPET and TA-{Delta}LCC are functional in larval extracts C_LIO_LITA-{Delta}LCC was selected for PET nanoparticle assays due to higher pNPA activity C_LIO_LID. melanogaster model enables comparative evaluation of PET-degrading enzymes C_LI

The growing demand for sustainable aquafeeds has intensified interest in alternative protein ingredients capable of reducing reliance on fishmeal without compromising fish performance. Here, we evaluated microbial community-based single-cell protein (SCP) as a fishmeal substitute in juvenile Asian seabass (Lates calcarifer) diets in two independent feeding trials of juvenile fish conducted over 49 and 56 days, respectively and compared them to a previous study that lasted 24 days. SCP was produced from nutrient-rich soybean-processing side streams by microbial communities in fermenters and incorporated into experimental diets at inclusion levels ranging from 10% to 100% fishmeal replacement. In the 24-day trial, a diet containing 50% fishmeal replacement with lab-scale produced SCP achieved 100% survival and a feed conversion ratio (FCR), specific growth rate (SGR), and weight gain comparable to the fishmeal control diet. In the 49-day trial using pilot-scale produced SCP, a 50% fishmeal replacement also maintained an FCR and feed intake comparable to the control, whereas complete replacement reduced feed intake and growth performance. In a 56-day pilot-scale trial that used 500-L fish tanks, diets containing up to 50% fishmeal replacement maintained comparable survival, weight gain, and SGR, although moderately higher FCR values were observed at higher SCP inclusion levels. Proximate composition and essential amino acid profiles of fish fed control or SCP-containing diets were comparable. Genome-resolved metagenomic analyses revealed diverse microbial taxa associated with the SCP. Collectively, these findings support microbial community-based SCP as a scalable and reproducible alternative protein platform for aquaculture feeds across independent trials and production scales.

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a {beta}-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 {micro}M and maximum binding capacity (Bmax) of 144 {micro}mol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 {degrees}C for ONPG and 50 {degrees}C for lactose, and CBE retained >80% activity after 390 min at 45 {degrees}C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 x106 M-1{middle dot}s-1 for ONPG and 4.40 x102 M-1{middle dot}s-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca2+ and Fe2+. These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.

The bacterium Zymomonas mobilis is widely studied for its potential as an industrial biofuel producer. Anoxic fermentation by Z. mobilis in lignocellulosic hydrolysate can generate bioethanol from renewable plant biomass. In this study, we deleted a gene from the Z. mobilis genome encoding a homolog of OxyR, a transcription factor that activates an oxidative stress response in bacteria to reduce reactive oxygen species (ROS). Deletion of this transcription factor inhibited growth of Z. mobilis in oxic, but not anoxic, conditions in laboratory media. A ROS probe revealed that the oxyR response is required to reduce intracellular ROS during oxic growth. Importantly for biofuel production, the absence of oxyR inhibited growth and delayed ethanol production during anoxic hydrolysate fermentation. To determine the source of oxidative stress in hydrolysate, we grew{Delta} oxyR in a synthetic hydrolysate containing known inhibitors found in hydrolysate. There was no growth defect in{Delta} oxyR in the synthetic hydrolysate, indicating that known inhibitory compounds are not the source of anoxic oxidative stress. We determined that Ammonia-Fiber Expansion (AFEX) switchgrass hydrolysate contains significant peroxide concentrations. Addition of catalase to hydrolysate improves growth of both{Delta} oxyR and wild-type Z. mobilis in hydrolysate. This study uncovers an important source of stress to Z. mobilis during biofuel fermentation. ImportanceFermentation of non-food biomass is a promising avenue for sustainable production of fuels and chemicals, but several challenges currently limit applicability of this technology. One major hurdle is that when biomass is deconstructed into a fermentable form, many byproducts are generated that inhibit microbial fermentation. Here, we investigated how a fermentative bacterium, Zymomonas mobilis, experiences oxidative stress during anoxic biomass fermentation, and identified genes important in this response. These findings provide a better understanding of the stresses faced by Z. mobilis during biofuel production. Fully understanding the effects of hydrolysate on biofuel-producing microbes is crucial for optimizing production and making carbon-neutral fuel a reality.