Biosynthesis of cyanophycin by high-cell-density cultivation of recombinant Escherichia coli for corrosion-resistant biomaterial applications.

Enhanced Trehalose Production Through Integrated Chassis and Expression Engineering in Bacillus subtilis

Trehalose is a functional disaccharide widely used in the food, pharmaceutical, and cosmetic industries. It is industrially produced via a dual-enzyme process involving maltoligosaccharide trehalose synthase (MTSase) and maltoligosaccharide trehalose hydrolase (MTHase), with Escherichia coli (E. coli) serving as the expression host. Bacillus subtilis (B. subtilis) is an ideal host for industrial trehalose production due to its generally recognized as safe (GRAS) status and low phage susceptibility. However, engineered B. subtilis strains often exhibit slow growth, low heterologous protein expression, and high fermentation costs, thereby limiting their industrial application. To address these challenges, this study employed a synergistic strategy that combined chassis modification, expression element optimization, and knockout of substrate-competition pathways. First, a tryptophan-independent strain was constructed by reverting the trpC2 mutation to shorten the growth cycle. Next, knockout of flgD, yueB, and integration of E. coli-derived glutamate dehydrogenase (gdhA) significantly enhanced biomass accumulation. Expression of MTSase and MTHase was markedly improved through tandem strong promoters (PHpaII-P36) and ribosome-binding site (RBS) optimization (RBS1), achieving a 10.87-fold and 4.22-fold increase in enzyme activity, respectively. Finally, disruption of the amyE gene reduced non-specific substrate degradation. Using maltodextrin as substrate, the final trehalose conversion rate reached 76%. This study constructed B. subtilis chassis cells that highly express MTHase and MTSase respectively, laying a foundation for subsequent industrial trehalose production.

Evaluation of Monascus ruber Inoculum Preparation Strategies and Surfactant Supplementation to Enhance Biopigment Production in a Xylose-Based Medium Derived from Ethanol Biorefinery By-Products

Bioenergy biorefineries generate lignocellulosic by-products rich in fermentable sugars that can serve as renewable feedstocks for the production of high-value bioproducts, including microbial pigments with promising bioactive properties (antioxidant, antimicrobial, and anticancer). Therefore, this study aimed to identify a suitable inoculum preparation strategy, to optimize the culture medium, and to evaluate biopigment production using xylose-based media derived from sugarcane bagasse hemicellulosic hydrolysate (SBHH). Different inoculation strategies were evaluated (cell suspension, whole mycelial discs, and fractionated mycelial discs) and supplementation with Tween 80 (TW80). The medium composition was optimized using a Box–Behnken design, with xylose, yeast extract, and TW80 as variables, and fermentations were then conducted under selected inoculum conditions in semi-defined media and SBHH. The mycelial disc inoculation strategy was selected due to its high biopigment production and lower operational complexity, yielding 8.99, 8.48, and 11.78 AU of yellow, orange, and red biopigments, respectively. The optimized culture composition consisted of 55.65 g/L of xylose, 4.18 g/L of yeast extract, and 15.38 g/L of Tween 80. The cultivation of M. ruber in SBHH resulted in 12.73, 10.75, and 14.56 AU of yellow, orange, and red biopigments, respectively. Thus, the strategy of inoculum preparation associated with non-ionic surfactant proved promising for application in bioenergy biorefineries.

Bacillus subtilis GB Shows High Polyglutamic Acid Bioconversion Efficiency in Low-Glutamic-Acid Monosodium Glutamate Wastewater

Low-glutamic-acid monosodium glutamate wastewater (L-MSGW), characterized by high (NH4)2SO4 concentrations, presents significant challenges for conventional treatment. γ-Polyglutamic acid (γ-PGA) production using industrial wastewater is an economical and environmentally friendly strategy. In the current study, we isolated and identified Bacillus subtilis GB, which exhibited exceptional tolerance to (NH4)2SO4, and capability for the high-efficiency biosynthesis of γ-PGA using untreated L-MSGW. Fermentation conditions were optimized using single-factor experiments coupled with response surface methodology, followed by scale-up validation in a 5 L fermenter. Under optimal conditions, the maximum γ-PGA yield reached 16.57 g/L with a minimal glutamate consumption of only 4.9 g/L. The study validated the feasibility of efficient γ-PGA production from L-MSGW by B. subtilis GB, providing a novel technical approach and theoretical basis for low-cost treatment and high-value resource utilization of L-MSGW. This study not only demonstrates the low-cost L-MSGW can be used for the high-value γ-PGA by B. subtilis GB but also provides a sustainable and economically viable solution for industrial wastewater treatment.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol production

Niacin Synthesis from 3-Cyanopyridine by Recombinant Escherichia coli: Optimization and Immobilization

Niacin (vitamin B3) is a high-value chemical widely used in the pharmaceutical and food industries. In this study, a recombinant Escherichia coli strain expressing nitrilase from Pseudomonas putida CGMCC3830 was evaluated for the whole-cell biotransformation of high-concentration 3-cyanopyridine to niacin. Molecular docking predicted important substrate-interacting residues, including T135, K131, F202, and W166. Under optimized conditions (160 g/L wet cells, pH 8.5), free whole cells completely hydrolyzed 3.0 M 3-cyanopyridine within 8 h. To enhance operational stability, the cells were immobilized in sodium alginate beads containing green zeolite. The resulting catalyst achieved complete conversion of 1.4 M 3-cyanopyridine within 4 h and maintained 96.5% conversion after six cycles. These findings demonstrate the potential of this system for future industrial applications.

Plant chloroplasts enable high-yield and cost-effective production of the engineered bacterial enzyme CueO-MacHis as a technical protein via transient expression in Nicotiana benthamiana

Laccases are versatile oxidoreductases found in bacteria, fungi, and plants., which have a wide range of industrial applications, including the removal of pharmaceutical micropollutants from wastewater. Bacterial laccases are particularly attractive because of their high catalytic activity and stability over broad temperature and pH ranges. One example is the copper efflux oxidase (CueO) from Escherichia coli , which has been fused to the adhesion-promoting peptide Macaque Histatin (MacHis) because immobilized enzymes have proven especially effective for solid-phase wastewater treatment. However, the practical application of this enzyme is constrained by the relatively low production yields of 25–50 mg/L when expressed in recombinant E. coli or Pichia pastoris . Here, we used transient expression in Nicotiana benthamiana to demonstrate that the engineered CueO-MacHis can be produced at substantially higher levels, reaching 250–320 (286 ± 35) mg/kg biomass in the prokaryotic-like environment of chloroplasts in 6-week-old plants, which are typically used for production purposes. Subsequent systematic optimization of the transient expression process based on statistical experimental designs increased the accumulation of recombinant CueO-MacHis approximately threefold to 846 ± 57 mg/kg biomass. Furthermore, we established a simplified and cost-effective purification strategy that leverages the enzyme’s temperature and pH stability to clarify extracts before anion exchange chromatography, achieving 76% recovery and 92% purity. Moreover, the plant-derived CueO-MacHis had a specific activity of 7.22 ± 1.55 U/mg, which is in the same range as that reported for other CueO enzymes. Our approach paves the way for the large-scale production of CueO-MacHis as a technical enzyme for industrial applications.