Biosynthesis of Lycopene and Ergothioneine in Escherichia coli Using Temperature-Responsive Biosensors

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.

Psychrophilic Quorum Sensing Genes Enable Unimodal, Adjustable Protein Expression Across the Entire Escherichia coli Population

A previous study demonstrated that quorum sensing (QS) genes from the psychrophilic bacterium Aliivibrio logei can be used in Escherichia coli to obtain bacterial cultures with controlled amounts of a target protein. However, the QS system may be induced non-uniformly across the cell population. In this study, we used an expression vector containing luxR/luxI regulatory genes from A. logei carrying sfGFP as a reporter gene. Reporter expression was regulated by the autoinducer 3OxoC6-HSL, activated at 22 °C, and terminated at 37 °C. Flow cytometry was used to assess the GFP fluorescence distribution at the single-cell level. The system provided dose-dependent unimodal expression lacking formation of distinct ON/OFF subpopulations, while the robust coefficient of variation decreased with increasing autoinducer concentration. The autoinducer synthase LuxI enabled autoinduction, but under the tested conditions, no substantial effect on expression homogeneity was detected. Raising the temperature to 37 °C effectively halted expression, allowing intermediate target protein values to be fixed at the single-cell level. Overall, the developed system represents a promising tool for biotechnological applications requiring precise and uniform control of expression.

A novel arabinose-inducible promoter in the carotenogenic yeast Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycetous yeast capable of synthesizing astaxanthin, a powerful antioxidant carotenoid pigment. Despite appreciable research efforts, molecular tools for the genetic manipulation of this yeast remain limited. Inducible promoters are essential tools to control gene expression in response to external stimuli, such as molecules or stress signals. This regulation enables precise control of gene expression in synthetic biological systems, minimizing unwanted effects caused by constant gene expression. In the present work inducible promoters from the L-arabinitol dehydrogenase ( LAD ), isocitrate lyase ( ICL ), alpha glucosidase ( AG ), glycerol kinase ( GUT1 ), and xylitol dehydrogenase ( XDH ) genes were screened and evaluated. The promoter sequences were cloned into a plasmid containing the encoding sequence for Enhanced Yellow Fluorescent Protein ( EYFP ) for functional assessment. Promoter activity was quantified by fluorescence under induction and repression conditions in the engineered yeast cells. Among the promoters analyzed, only the LAD promoter led to high fluorescence under induction conditions, and low under repression. Yeast cells harboring the LAD promoter showed mostly an increasing fluorescence with culture time. Moreover, dose-response analyses with arabinose from 0.05 to 30 g/L indicated that only 2.5 g/L arabinose is needed to achieve maximum fluorescence from the yeast cells. Finally, we performed a two-stage culture of X. dendrorhous harboring the LAD promoter and arabinose as inducer for prospective novel practical applications. Overall, we believe that the LAD promoter is a promising tool for fine-tuning of protein expression in X. dendrorhous . A native arabinose-inducible promoter is disclosed in the yeast. LAD promoter exhibits a sensitive response to the inducer. A two-stage culture of X. dendrorhous allows first the accumulation biomass, and then the expression of EYFP.

An expression framework for production of recombinant methionine aminopeptidase in Escherichia coli: a case study of upstream process.

Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.

A recombinant xylose reductase enzyme with biotechnological potential for xylitol 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.