During riboflavin biosynthesis in Bacillus subtilis, GTP acts as a precursor supplied by the purine biosynthesis pathway. This study revealed that GTP biosynthesis is tightly regulated by intracellular guanine levels via a guanine-sensing riboswitch, limiting the final riboflavin yield. Using resting cell transformation assays, we confirmed that the transporters NupG and PbuO mediate guanine efflux and influx, respectively. Overexpression of nupG in the original strain BR reduced intracellular guanine levels, resulting in enhanced riboflavin production. These findings indicate that NupG-mediated intracellular guanine efflux could deregulate riboswitch-induced repression of the pur operon. This deregulation increased metabolic flux through the de novo purine biosynthesis pathway and elevated the synthesis of precursor GTP. As a result, engineered strain BR-02 achieved a riboflavin titer of 1,508.22 mg/L, a 15.3% increase over the original strain BR. This study highlights that transporter engineering is an effective metabolic engineering strategy to overcome purine feedback repression in riboflavin biomanufacturing, offering a new rationale for designing microbial cell factories.
Xylose is an abundant carbon source in lignocellulosic biomass, making it extremely important to construct microbial metabolic factories capable of efficiently utilizing xylose. Candida glycerinogenes is a strain with high stress tolerance, and it serves as an efficient platform for energy conversion via lignocellulosic feedstocks. Through transcriptional analysis of genes related to the xylose-highly assimilating strain C. glycerinogenes-cgS35P, it was found that CgGzf3, a transcription factor associated with amino acid metabolism, is involved in the regulation of xylose metabolism. The inhibition of CgGzf3 expression by antisense RNA increased the xylose consumption of the engineered strain by 33.7% and the ethanol yield by 30.4%. Yeast one-hybrid assays confirmed that CgGzf3 directly binds to the promoter regions of key xylose metabolism genes. Antisense RNA inhibits the expression of CgGzf3, which upregulates fermentation metabolism genes and thereby shifts xylose metabolism toward ethanol fermentation. Additionally, the key amino acid biosynthesis genes Arg1, Glt1, and Car2 were upregulated by 1.8-fold, 1.6-fold, and 1.3-fold, respectively. The intracellular amino acid content of the recombinant strain C. glycerinogenes-cgreGzf3 also increased. This study reveals for the first time that the amino acid metabolism transcription factor CgGzf3 is involved in the regulation of xylose metabolism, providing new experimental evidence for the intrinsic connection between xylose metabolism and amino acid metabolism.
The transcription factor CgGzf3 directly affects xylose metabolism by regulating the promoters of key genes, and can indirectly influence xylose assimilation by modulating amino acid anabolic metabolism. Inhibition of CgGzf3 by antisense RNA shifts the overall xylose assimilation toward ethanol fermentation metabolism
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Current production of polylactic acid (PLA) relies on agricultural feedstocks. Utilizing solar chemicals produced from CO2 by artificial photosynthesis offers a sustainable alternative for producing biodegradable plastics. However, the direct synthesis of PLA from CO2 within a cell factory remains challenging. Here, we report the direct production of PLA in engineered Escherichia coli from CO2 via ethanol or acetate, which are produced from CO2 electrolysis with solar energy. More importantly, we found that cofeeding ethanol and acetate synergistically enhanced PLA production. The PLA titer under cofeeding condition reached 5-folds and 53-folds of those obtained with ethanol or acetate alone, respectively. This cofeeding effect upregulated the glyoxylate shunt, the Entner-Doudoroff pathway, and serine anabolism, which facilitated efficient lactic acid generation from acetyl-CoA. By further integrating electroreduction and PLA bioproduction, we achieved PLA synthesis from CO2 with a titer of 241 mg/L. This work develops an agriculture-independent approach for bioplastic production from CO2, H2O, and renewable energy.
Rising global energy demands, together with the need for alternative, sustainable, and scalable energy production and storage systems, have attracted industry attention. One such potential solution is bioelectric systems, such as biofuel cells (BFCs) and bio-based batteries (BBBs). BFCs are systems that utilize redox-active biomolecules and biopolymers derived from renewable biological sources such as plants, algae, and bacteria to generate electricity. Generally categorized by their catalysts: microbial biofuel cells and enzymatic biofuel cells (EFCs). BBBs are a subclass of BFCs, which replace an active feeding system with an internal fuel that is discarded once depleted. The varying physical properties, biocompatibility, and operation on renewable biological fuels expanded their use across fields such as waste management, healthcare, agriculture, and robotics. Despite active research and development, regulatory bodies lack policies and regulations governing the production and commercial use of BFC. Efforts to commercialize the technology are held back by technical limitations, economic challenges, and the lack of solid policies surrounding green energy. The failure of previous attempts to commercialize biobatteries has highlighted the gap between experimental feasibility and real-world implementation. Future progress in the field is expected to rely on improved integration of biobatteries into existing hybrid energy systems, advancement in the stability of bio-based materials, and the development of supportive regulatory and market infrastructures.
Non-Saccharomyces yeasts significantly contribute to aroma complexity in fermented foods and beverages; however, the role of nutrient availability in constraining ester biosynthesis remains poorly understood. Here, an aroma-active non-Saccharomyces yeast, Cyberlindnera fabianii, isolated from a traditional fermented food, was used to investigate how carbon availability and carbon-to-nitrogen (C/N) ratio regulate ester biosynthesis. Five nutritional conditions were examined by integrating gas chromatography–mass spectrometry (GC–MS)–based volatile profiling, transcriptomics, and targeted intracellular metabolomics. High carbon and medium-to-low C/N ratios enhanced ester production, with phenethyl acetate and isoamyl acetate as major discriminant compounds. In contrast, low carbon and high C/N ratios favored organic acid accumulation and off-flavor formation. Multivariate analyses revealed ester-specific metabolic constraints: phenethyl acetate synthesis was limited by aromatic amino acid supply, whereas isoamyl acetate depended on branched-chain amino acid biosynthesis. Overall, these results demonstrate that carbon availability and C/N ratio reposition metabolic bottlenecks in ester formation, providing a nutrition-based framework for aroma modulation.
β-1,3-Glucosyltransferase is a key glycosyltransferase that affects the biosynthesis of polysaccharides in Ganoderma lucidum, an edible and medicinal fungal polysaccharide. Its isoenzymes, GL20535 and GL24465, serve dominant and auxiliary roles, respectively. Although the catalytic activity of β-1,3-glucosyltransferase is regulated by Rho GTPases, the mechanisms by which these regulators affect GL20535 and GL24465 and consequently influence polysaccharide synthesis remain unclear. In the present study, three members of the G. lucidum Rho GTPase (GLRho) family—GLRho1/GLRho2/GLRho3—were identified by gene mining. Through AlphaFold3 modeling and protein–protein interaction analysis, it was found that GLRho1 and GLRho3 possess similar structures, binding primarily to GL20535. However, GLRho exhibited stronger binding capacity to GL24465 due to differences in secondary structure by computational predictions. Polysaccharide metabolism and transcription analysis showed that GLRho1 and GLRho3 exerted positive effects on growth and polysaccharide synthesis in G. lucidum, with the overexpression of both genes upregulating the expression of gl20535, leading to significant increases of 27.65% and 24.26% in exopolysaccharide yield, respectively, compared with the wild-type strain. In contrast, glrho2 gene overexpression upregulated the expression of isoenzyme gene gl24465 but did not influence gl20535 expression. Consequently, the polysaccharide yield was unaffected. Moreover, glrho gene regulation did not exert significant influence on the intracellular polysaccharide yield or monosaccharide composition. Overall, this study revealed that the Rho GTPase family influences the molecular network of polysaccharide synthesis by specifically binding to isoenzymes. These results provide theoretical guidance for enhancing the biomanufacturing of polysaccharides in edible and medicinal fungi.
RNA helicases are a class of highly conserved RNA-binding proteins that unwind double-stranded RNA by hydrolyzing ATP, playing essential roles in processes such as ribosome biogenesis, pre-mRNA splicing, transport, translation, and decay of pre-mRNA. However, their functional roles in the stress responses of Candida glycerinogenes remain uncharacterized to date. Herein, CgDBP7 (C. glycerinogenes DEAD-box RNA helicase DBP7) and its downstream responsive gene CgMAK5 (C. glycerinogenes ATP-dependent RNA helicase MAK5) were first characterized as dual negative regulators of high-salinity tolerance in C. glycerinogenes. Mechanistically, the enhanced high-salinity tolerance of the engineered strain C. glycerinogenes-antiCgDBP7a-antiCgMAK5 was associated with intracellular reactive oxygen species (ROS) detoxification and glycerol biosynthesis. Glycerol acts as an osmoprotectant to balance osmotic pressure under high salt, while efficient ROS detoxification mitigates cellular oxidative damage, collectively boosting tolerance and metabolite production. Under high-salinity conditions, the stress-resistant engineered strain C. glycerinogenes-antiCgDBP7a-antiCgMAK5 achieved a 13.5% and 16.5% increase in glycerol and ethanol titers, respectively. In undetoxified lignocellulosic hydrolysate, this recombinant strain exhibited a further increase of 45.2% in ethanol titer and 13.4% in glycerol titer. Collectively, these findings demonstrate that CgDBP7 and CgMAK5 form a two-layered repressive circuit and serve as key genetic elements for salinity stress adaptation in C. glycerinogenes, providing programmable targets for engineering robust industrial yeast.
Keratinases offer a promising green strategy for valorizing recalcitrant keratin waste, yet their practical application is often hindered by low substrate specificity and limited catalytic efficiency. Herein, we report the successful engineering of a highly specific and robust keratinase variant, G209F/G235S, derived from KerBv via a semi-rational design strategy targeting the active pocket. By integrating homology modeling, molecular docking, and evolutionary conservation analysis, key residues within binding pocket were identified and optimized. The engineered mutant exhibited a doubled catalytic activity of 8,540 U/mL and a significantly enhanced keratin-to-casein hydrolytic ratio (K/C) of 1.64. Comprehensive characterization revealed that G209F/G235S possesses superior thermostability with the optimum reaction temperature of the G209F/G235S mutant shifted from 40 °C (WT) to 45 °C, and maintains robust activity across a broad alkaline pH range (7.0–11.0). Molecular dynamics simulations and structural analyses elucidated the underlying mechanisms: the mutations synergistically expanded the active pocket volume from 561 to 689 Å3, and optimized electrostatic complementarity for negatively charged keratin. In practical applications, the mutant achieved over 80% degradation of native feather waste within 2 h, outperforming the WT by 50% degradation ratio. This study not only delivers a potent biocatalyst for the efficient upcycling of keratinous by-products but also provides fundamental insights into the structure–function relationships governing keratinase specificity, establishing a robust strategy for the rational design of high-performance industrial enzymes.
3-dehydroshikimic acid (3-DHS), which is recognized as a key precursor of the shikimate pathway, has been regarded as an important platform compound for the biosynthesis of a wide range of aromatic products. However, microbial production is often constrained by imbalanced pathway expression and the accumulation of by-products, resulting in reduced cellular activity and loss of the target product. In this study, combinatorial metabolic engineering was applied for the production of 3-DHS. The downstream pathway was blocked through the deletion of aroE, after which engineering targets were screened and cumulatively integrated to enhance the basal metabolic flux. Subsequently, the biosynthetic pathway was optimized using a modular design strategy. On this basis, a multidimensional fine-tuning regulation strategy was implemented, by which the accumulation of the by-product gallic acid was reduced by 74.3%. Furthermore, carbon source utilization efficiency was enhanced through the combination of reinforced non-PTS glucose uptake and an optimized feeding strategy. As a result, a 3-DHS titer of 147.5 g/L was achieved in a 5 L bioreactor, with a yield of 0.6 g/g glucose and a productivity of 3.69 g/L/h, all of which represent the highest values reported to date. Overall, this study is expected to provide an important reference for the scalable biomanufacturing of 3-DHS and its high-value derivatives.