System reconstruction of Bacillus licheniformis for efficient expression of alkaline protease

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.

Advances in Key Genetic Elements and Strategies for High-Yield Heterologous Protein Expression in Komagataella phaffii

This review summarizes recent advances in engineering key expression elements underlying heterologous protein production in K. phaffii, with particular emphasis on promoter architecture redesign, signal peptide replacement and sequence engineering, molecular chaperone co-expression, and quantitative regulation of the unfolded protein response.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol production

Development of a bifunctional dynamic control system for effective product synthesis in Bacillus licheniformis DW2.

Bacillus licheniformis DW2 has emerged as a promising microbial chassis for biosynthesis of industrial enzymes, lipopeptides and biochemicals. However, the lack of dynamic control tools to date has limited its expansion in basic research and industrial applications. Here, a well- performing bifunctional dynamic control system was developed for the dynamic up- and down- regulation of genes, as well as the effective biosynthesis of poly-γ-glutamic acid (γ-PGA). Firstly, a dynamic turn-on module was developed in B. licheniformis DW2, including an artificially designed broad-range regulated xylose-inducible expression system with a regulation range of 1.03 ∼ 352.37-fold, and an artificially designed highly sensitive IPTG-inducible expression system that can respond to 0.001 mM IPTG. Subsequently, a dynamic turn-off module based on the ClpXP protease was developed, which could regulate the degradation efficiency of target protein by selecting different ssrA tags. Then, a bifunctional dynamic control system based on the coupling of LacI and ClpXP was established in B. licheniformis for the first time, through combining the dynamic turn- on and off modules, which realized the dynamic up- and down- regulation of the expression of rfp and gfp genes simultaneously. Finally, it was successfully applied to dynamically optimize the metabolic flux of γ-PGA, increasing the yields of γ-PGA by 33.51 %. This work not only developed a well-performance bifunctional dynamic control tool for gene expression regulation, but also offered a strong support for the efficient production of diversified products by B. licheniformis.

Heterologous Expression of a Neurospora crassa Catalase Reprograms the Cellulase System and Enhances β-Glucosidase Production in Trichoderma reesei

Efficient saccharification of lignocellulose, the most abundant renewable carbon reservoir resource, is of great industrial importance. Trichoderma reesei is a premier cellulase producer, but its fermentation efficiency is often constrained by dual challenges: dissolved oxygen limitation and intrinsic oxidative stress. To address this, we engineered T. reesei to heterologously express a robust catalase gene (cat-3) from Neurospora crassa. The recombinant strain Tr-cNcat3 exhibited a 7.4-fold increase in extracellular catalase activity. Tr-cNcat3 showed an increase in total extracellular protein, resulting in markedly enhanced filter paper activity (FPA) and β-glucosidase activity compared to the control. Strikingly, this intervention specifically triggered a significantly higher expression of β-glucosidase, a known bottleneck in T. reesei’s cellulase system, particularly on bagasse and straw as the carbon source. Moreover, the ability of the supernatant to degrade cellulose substrates was improved. Our results reveal that overexpression of cat-3 in T. reesei could modify the cellulase cocktail by triggering a higher level of β-glucosidase. This study provides a novel and effective genetic engineering strategy to unlock the full industrial potential of T. reesei for cost-effective lignocellulosic biorefining.

Multistrategy metabolic engineering of Talaromyces pinophilus for α-amylase production from lignocellulosic biomass

Metabolic engineering of Talaromyces pinophilus through promoter optimization, multicopy integration, and protease deletion enables efficient α-amylase production from lignocellulosic biomass, achieving 26 712 U/mL in bioreactor fermentation.