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Efficient secretion of heterologous proteins is crucial for applications in industrial and biomedical fields. Selecting appropriate signal peptides and bacterial strains is critical for successful protein expression and export. Bacillus thuringiensis , known for its robust secretion capabilities within the Bacillus genus, shows promise as an ideal host for this purpose. We performed genome-based bioinformatic analysis of B . thuringiensis HD73. A total of 525 proteins were predicted to contain signal peptides, exceeding those in other Bacillus species. The extracellular proteome of B . thuringiensis HD73 was analyzed via LC-MS/MS, identifying 100 secreted proteins. A library of 30 signal peptides was constructed by integrating genome-based predictions with experimental secretome data. Using this library, green fluorescent protein secretory expression systems were developed in the acrystalliferous mutant strain B . thuringiensis HD73 − , and the strain carrying signal peptide S17 showed the highest secretion efficiency. Additionally, the top 10 performing signal peptides were used to express and secrete the convenient enzyme cutinase, with the S20 fusion strain exhibiting the highest cutinase activity (3.65 U/mL in the culture supernatant). This study provides the first combined bioinformatic and experimental characterization of the B . thuringiensis secretome. The developed secreted protein expression system and signal peptide library demonstrate effectiveness and offer potential for future heterologous protein secretion in B . thuringiensis . Key points • Genome-based secretome and experimental secretome of B. thuringiensis were characterized . • A SP library comprising 30 SPs derived from B. thuringiensis was constructed . • GFP and cutinase were successfully secreted by B. thuringiensis .

Anti-lipopolysaccharide factor 3 (ALFPm3) possesses a wide antimicrobial spectrum and high antibacterial and viral activities for broad application prospects in the aquaculture industry. However, the application of ALFPm3 is limited by its low production in nature, as well as its low activity when expressed in Escherichia coli and yeast. Although it has been proven that its secretory expression can be used to produce antimicrobial peptides with strong antimicrobial activity, there is no study on the high-efficiency secretory expression of ALFPm3 in Chlamydomonas reinhardtii. In this study, signal peptides ARS1 and CAH1 were fused with ALFPm3 and inserted into the pESVH vector to construct pH-aALF and pH-cALF plasmids, respectively, that were transformed to C. reinhardtii JUV using the glass bead method. Subsequently, through antibiotic screening, DNA-PCR, and RT-PCR, transformants expressing ALFPm3 were confirmed and named T-JaA and T-JcA, respectively. The peptide ALFPm3 could be detected in algal cells and culture medium by immunoblot, meaning that ALFPm3 was successfully expressed in C. reinhardtii and secreted into the extracellular environment. Moreover, ALFPm3 extracts from the culture media of T-JaA and T-JcA showed significant inhibitory effects on the growth of V. harveyi, V. alginolyticus, V. anguillarum, and V. parahaemolyticus within 24 h. Interestingly, the inhibitory rate of c-ALFPm3 from T-JcA against four Vibrio was 2.77 to 6.23 times greater than that of a-ALFPm3 from T-JaA, indicating that the CAH1 signal peptide was more helpful in enhancing the secreted expression of the ALFPm3 peptide. Our results provided a new strategy for the secretory production of ALFPm3 with high antibacterial activity in C. reinhardtii, which could improve the application potentiality of ALFPm3 in the aquaculture industry.

Filamentous fungi are able to produce a wide range of valuable proteins and enzymes for many industrial applications. Recent advances in fungal genomics and experimental technologies are rapidly changing the approaches for the development and use of filamentous fungi as hosts for the production of both homologous and heterologous proteins. In this review, we highlight the benefits and challenges of using filamentous fungi for the production of heterologous proteins. We review various techniques commonly employed to improve the heterologous protein production in filamentous fungi, such as strong and inducible promoters, codon optimization, more efficient signal peptides for secretion, carrier proteins, engineering of glycosylation sites, regulation of the unfolded protein response and endoplasmic reticulum associated protein degradation, optimization of the intracellular transport process, regulation of unconventional protein secretion, and construction of protease-deficient strains.Key points• This review updates the knowledge on heterologous protein production in filamentous fungi.• Several fungal cell factories and potential candidates are discussed.• Insights into improving heterologous gene expression are given.

Intramembrane proteolysis describes the cleavage of substrate proteins within their hydrophobic transmembrane segments. Several families of intramembrane proteases have been identified including the aspartyl proteases Signal peptide peptidase (SPP) and its homologues, the SPP-like (SPPL) proteases SPPL2a, SPPL2b, SPPL2c and SPPL3. As presenilin homologues, they employ a similar catalytic mechanism as the well-studied γ-secretase. However, SPP/SPPL proteases cleave transmembrane proteins with a type II topology. The characterisation of SPP/SPPL-deficient mouse models has highlighted a still growing spectrum of biological functions and also promoted the substrate discovery of these proteases. In this review, we will summarise the current hypotheses how phenotypes of these mouse models are linked to the molecular function of the enzymes. At the cellular level, SPP/SPPL-mediated cleavage events rather provide specific regulatory switches than unspecific bulk proteolysis. By this means, a plethora of different cell biological pathways is influenced including signal transduction, membrane trafficking and protein glycosylation.

Hybrid seed production technology (SPT) is achieved through the utilization of a recessive nuclear male-sterile mutant transformed with a transgenic cassette comprising three essential components: the wild-type gene to restore the fertility of the male-sterile mutant, an α-amylase gene to disrupt transgenic pollen grains, and red fluorescence protein gene DsRed to distinguish the transgenic seeds from the nontransgenic male sterile seeds. In rice, we establish the pollen disruption system by introducing an amyloplast targeting signal peptide (ASP) at the N-terminus of maize α-amylase protein ZM-AA1 ΔSP (ZM-AA1 with the N-terminal signal peptide removed). The ASP facilitates the transport of ZM-AA1 ΔSP protein into amyloplast where it degrades starch, resulting in disruption of the pollen fertility. To obtain such signal peptides for rice, we searched the rice proteins homologous to the defined wheat amyloplast proteins followed by protein–protein interaction network predictions and targeting signal peptides prediction. These analyses enabled the identification of four candidate ASPs in rice, which were designated as ASP1, ASP2, ASP3, and ASP4, respectively. ASP1 and ASP2, when linked with ZM-AA1 ΔSP , exhibited the capability to disrupt transgenic pollen grains, whereas ASP3 and ASP4 did not produce this effect. Interestingly, the localization experiments showed that ASP3 and ASP4 were able to target the proteins into chloroplast. The ASP1 and ASP2 sequences provide valuable tools for genetic engineering of the rice male-sterile system, which will contribute to the hybrid rice breeding and production.

Although aspartic intramembrane-cleaving proteases (I-CLIPs) are crucial switches of multiple signaling pathways and involved in several devastating diseases, little is known about their physiological regulation. We have recently identified Frey regulator of sperm-oocyte fusion 1 (Frey1) as an inhibitory protein of Signal Peptide Peptidase-like 2c (SPPL2c), a member of this protease family. Employing structure modeling along with cell-based inhibition and interaction studies, we identify a short motif within the Frey1 transmembrane domain essential for inhibition of SPPL2c. Intriguingly, this motif can be transplanted to the SPPL2c substrate PLN, thereby transforming it into an inhibitor of this enzyme. It can be adopted for the generation of Notch1-based γ-Secretase inhibitors demonstrating its versatile use among aspartic I-CLIPs. In summary, we describe a mechanism of aspartic I-CLIP inhibition which allows the targeted generation of specific inhibitors of these enzymes and might enable the identification of endogenous negative regulators of these enzymes.

Extracellular vesicles, such as microvesicles (LEV) and exosomes (SEV), play an important role in intercellular signaling by encapsulating functional molecules and delivering them to specific cells. Recent studies showed that signal peptides (SPs), which are derived from sequences at the N-terminal of newly synthesized proteins, exhibited biological activity in the extracellular fluid. We previously reported that SPs were secreted into the extracellular fluid via SEV; however, it remains unclear whether the release of SPs occurs via LEV. In the present study, we demonstrated that SP fragments from human placental secreted alkaline phosphatase (SEAP) were present in LEV as well as SEV released from RAW-Blue cells, which stably express an NF-κB-inducible SEAP reporter. When RAW-Blue cells were treated with LPS at 0–10,000 ng/mL, SEAP SP fragments per particle were more abundant in LEV than in SEV, with fragments in LEV and SEV reaching a maximum at 1000 and 100 ng/mL, respectively. The content of SEAP SP fragments in LEV from IFNγ-stimulated RAW-Blue cells was higher than those from TNFα-stimulated cells, whereas that in SEV from TNFα-stimulated RAW-Blue cells was higher than those from IFNγ−stimulated cells. Moreover, the content of SEAP SP fragments in LEV and SEV decreased in the presence of W13, a calmodulin inhibitor. Collectively, these results indicate that the transportation of SP fragments to extracellular vesicles was changed by cellular activation, and calmodulin was involved in their transportation to LEV and SEV.

Background Our laboratory has constructed a Bacillus stearothermophilus α-amylase (AmyS) derivative with excellent enzymatic properties. Bacillus subtilis is generally regarded as safe and has excellent protein secretory capability, but heterologous extracellular production level of B. stearothermophilus α-amylase in B. subtilis is very low. Results In this study, the extracellular production level of B. stearothermophilus α-amylase in B. subtilis was enhanced by signal peptide optimization, chaperone overexpression and α-amylase mutant selection. The α-amylase optimal signal peptide (SP YojL ) was obtained by screening 173 B. subtilis signal peptides. Although the extracellular α-amylase activity that was produced by the resulting recombinant strain was 3.5-fold greater than that of the control, significant quantities of inclusion bodies were detected. Overexpressing intracellular molecular chaperones significantly reduced inclusion body formation and further increased α-amylase activity. Error-prone PCR produced an amylase mutant K82E/S405R (AmySA) with enzymatic activity superior to that of AmyS. Expression of the amySA gene with the SP YojL while overexpressing molecular chaperones resulted in a 7.1-fold improvement in α-amylase activity. When the final expression strain (WHS11YSA) was cultivated in a 3-L fermenter for 92 h, the α-amylase activity of the culture supernatant was 9201.1 U mL −1 , which is the highest level that has been reported to date. Conclusions This is the first report that describes an improvement of B. stearothermophilus α-amylase extracellular production levels in B. subtilis using these strategies, and this represents the highest extracellular production level ever reported for α-amylase from B. stearothermophilus in B. subtilis . This high-level production provides a basis for enhanced industrial production of α-amylase. These extracellular production level improvement approaches are also expected to be valuable in the expression of other enzymes in B. subtilis .

Protein secretion in yeast is a complex, multistep process heavily reliant on signal sequences to guide recombinant proteins through the secretory pathway. Among these, the mating factor alpha (MFα) signal sequence from Saccharomyces cerevisiae has emerged as a powerful tool for enhancing the extracellular production of heterologous proteins. This review provides a comprehensive overview of the MFα signal sequence, tracing its historical development and role in advancing our understanding of protein secretion mechanisms, including co- and post-translational secretory pathways. We highlight key studies focused on optimizing the MFα signal sequence for improved secretion efficiency, leading to the development of several highly effective variants. These optimized sequences have significantly increased recombinant protein yield and quality, with notable implications for both research and industrial applications. Additionally, we explore the challenges of MFα-based secretion, including issues of missorting, incorrect processing, and aggregation in the endoplasmic reticulum (ER). We discuss emerging strategies to overcome these bottlenecks, such as fusion with alternative signal sequences and strain engineering. Finally, the review highlights current efforts to develop more robust signal peptides, and underscores the importance of continued innovation in protein secretion systems to meet the growing demand for high-quality recombinant proteins in biotechnological and therapeutic applications. Key points • MFα remains the top choice for recombinant protein secretion in yeast • Challenges in secretion: ER aggregation, missorting, and processing errors • Mutated and hybrid signal peptides offer promising solutions

Bacillus subtilis, belonging to the type species of Bacillus, is a type of soil-derived, low %G+C, endospore-forming Gram-positive bacterium. After the discovery of B. subtilis 168 that displayed natural competence, this bacterium has been intensively considered to be an ideal model organism and a robust host to study several basic mechanisms, such as metabolism, gene regulation, bacterial differentiation, and application for industrial purposes, such as heterologous protein expression and the overproduction of an array of bioactive molecules. Since the first report of heterologous overproduction of recombinant proteins in this strain, the bulk production of a multitude of valuable enzymes, especially industrial enzymes, has been performed on a relatively large scale. Since B. subtilis can non-specifically secrete recombinant proteins using various signal peptides, it has tremendous advantages over Gram-negative bacterial hosts. Along with the report of the complete genome sequence of B. subtilis, a number of genetic tools, including diverse types of plasmids, bacterial promoters, regulatory elements, and signal peptides, have been developed and characterized. These novel genetic elements tremendously accelerated genetic engineering in B. subtilis recombinant systems. In addition, with the development of several complex gene expression systems, B. subtilis has performed a number of more complex functions. This ability enables it to be a substantial chassis in synthetic biology rather than just a workhorse for the overproduction of recombinant proteins. In this review, we review the progress in the development of B. subtilis as a universal platform to overproduce heterologous diverse high-value enzymes. This progress has occurred from the development of biological parts, including the characterization and utilization of native promoters, the fabrication of synthetic promoters and regulatory elements, and the assembly and optimization of genetic systems. Some important industrial enzymes that have been produced in large quantities in this host are also summarized in this review. Furthermore, the ability of B. subtilis to serve as a cellular tool was also briefly recapitulated and reviewed.