Search

Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application. [ABSTRACT FROM AUTHOR]

BACKGROUND: Isomaltulose is a 'generally recognized as safe' ingredient and is widely used in the food, pharmaceutical and chemical industries. The exploration and development of efficient technologies is essential for enhancing isomaltulose yield. RESULTS: In the present study, a simple and efficient surface display platform mediated by a non‐yeast signal peptide was developed in Yarrowia lipolytica and utilized to efficiently produce isomaltulose from sucrose. We discovered that the signal peptide SP1 of sucrose isomerase from Pantoea dispersa UQ68J (PdSI) could guide SIs anchoring to the cell surface of Y. lipolytica, demonstrating a novel and simple cell surface display strategy. Furthermore, the PdSI expression level was significantly increased through optimizing the promoters and multi‐site integrating genes into chromosome. The final strain gained 451.7 g L−1 isomaltulose with a conversion rate of 90.3% and a space–time yield of 50.2 g L−1 h−1. CONCLUSION: The present study provides an efficient way for manufacturing isomaltulose with a high space–time yield. This heterogenous signal peptide‐mediated cell surface display strategy featured with small fusion tag (approximately 2.2 kDa of SP1), absence of enzyme leakage in fermentation broth and ample room for optimization, providing a convenient way to construct whole‐cell biocatalysts to synthesize other products and broadening the array of molecular toolboxes accessible for engineering Y. lipolytica. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS. Synopsis: Toxins delivered by the extracellular contractile injection system (eCIS) of 950 microbial genomes were identified using XGBoost classifier algorithm, four of which were experimentally confirmed and functionally characterized. eCIS is a poorly studied microbial toxin delivery system encoded by many environmental microbes. The first algorithm for predicting eCIS-associated toxins (EATs) was developed using genomic and biochemical features, and was used to identify ~2,200 EATs from 950 genomes. Features characterizing the N terminal signal peptide of EATs were identified and combined into a signal peptide score. Four new EATs genes (EAT14-17) were experimentally validated as being toxins, and critical EAT residues within the predicted catalytic sites were identified. EAT14 inhibits SRE-dependent mitogenic signaling in HEK293T cells. Toxins delivered by the extracellular contractile injection system (eCIS) of 950 microbial genomes were identified using XGBoost classifier algorithm, four of which were experimentally confirmed and functionally characterized. [ABSTRACT FROM AUTHOR]

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites". [ABSTRACT FROM AUTHOR]

D-psicose-3-epimerase (DPEase), a key enzyme for D-psicose production, has been successfully expressed in Escherichia coli with high yield. However, intracellular expression results in high downstream processing costs and greater risk of lipopolysaccharide (LPS) contamination during cell disruption. The secretory expression of DPEase could minimize the number of purification steps and prevent LPS contamination, but achieving the secretion expression of DPEase in E. coli is challenging and has not been reported due to certain limitations. This study addresses these challenges by enhancing the secretion of DPEase in E. coli through computational predictions and structural analyses. Signal peptide prediction identified PelB as the most effective signal peptide for DPEase localization and enhanced solubility. Supplementary strategies included the addition of 0.1% (v/v) Triton X-100 to promote protein secretion, resulting in higher extracellular DPEase (0.5 unit/mL). Low-temperature expression (20 °C) mitigated the formation of inclusion bodies, thus enhancing DPEase solubility. Our findings highlight the pivotal role of signal peptide selection in modulating DPEase solubility and activity, offering valuable insights for protein expression and secretion studies, especially for rare sugar production. Ongoing exploration of alternative signal peptides and refinement of secretion strategies promise further enhancement in enzyme secretion efficiency and process safety, paving the way for broader applications in biotechnology. [ABSTRACT FROM AUTHOR]

BACKGROUND: Acidic lipases with high catalytic activities under acidic conditions have important application values in the food, feed and pharmaceutical industries. However, the availability of acidic lipases is still the main obstacle to their industrial applications. Although a novel acidic lipase Rasamsonia emersonii (LIPR) was heterologously expressed in Escherichia coli, the expression level was unsatisfactory. RESULTS: To achieve the high‐efficiency expression and secretion of LIPR in Pichia pastoris GS115, the combinatorial optimization strategy was adopted including gene codon preference, signal peptide, molecular chaperone co‐expression and disruption of vacuolar sorting receptor VPS10. The activity of the combinatorial optimization engineered strain in a shake flask reached 1480 U mL−1, which was 8.13 times greater than the P. pastoris GS115 parental strain. After high‐density fermentation in a 5‐L bioreactor, the highest enzyme activity reached as high as 11 820 U mL−1. LIPR showed the highest activity at 40 °C and pH 4.0 in the presence of Ca2+ ion. LIPR exhibited strong tolerance to methanol, indicating its potential application in biodiesel biosynthesis. Moreover, the gastrointestinal digestion simulation results demonstrated that LIPR was tolerant to pepsin and trypsin, but its activity was inhibited by sodium taurodeoxycholate. CONCLUSION: This study provided an effective approach for the high expression of acidic lipase LIPR. LIPR was more appropriate for lipid digestion in the stomach than in intestine according to the gastrointestinal digestion simulation results. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Anderson–Fabry disease (AFD) is an X-linked multisystemic disorder with a heterogeneous phenotype, resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) and leading to globotriaosylceramide systemic accumulation. Lysosomal storage is not the unique player in organ failure and different mechanisms could drive tissue damage, including endoplasmic reticulum (ER) stress and its related signaling pathway's activation. We identified a new missense variant in the signal peptide of α-GLA gene, c.13 A/G, in a 55-year-old woman affected by chronic kidney disease, acroparesthesia, hypohidrosis, and deafness and exhibiting normal values of lysoGb3 and αGLA activity. The functional study of the new variant performed by its overexpression in HEK293T cells showed an increased protein expression of a key ER stress marker, GRP78, the pro-apoptotic BAX, the negative regulator of cell cycle p21, the pro-inflammatory cytokine, IL1β, together with pNFkB, and the pro-fibrotic marker, N-cadherin. Transmission electron microscopy showed signs of ER injury and intra-lysosomal inclusions. The proband's PBMC exhibited higher expression of TGFβ 1 and pNFkB compared to control. Our findings suggest that the new variant, although it did not affect enzymatic activity, could cause cellular damage by affecting ER homeostasis and promoting apoptosis, inflammation, and fibrosis. Further studies are needed to demonstrate the variant's contribution to cellular and tissue damage. [ABSTRACT FROM AUTHOR]

The N-terminal sequences of proteins and their corresponding encoding sequences may play crucial roles in the heterologous expression. In this study, the secretory expression of alkaline pectin lyase APL in B. subtilis was investigated to explore the effects of the N-terminal 5–7 amino acid sequences of different signal peptides on the protein expression and secretion. It was identified for the first time that the first five amino acid sequences of the N-terminal of the signal peptide (SP-LipA) from Bacillus subtilis lipase A play an important role in promoting the expression of APL. Furthermore, it was revealed that SP-LipA resulted in higher secretory expression compared to other signal peptides in this study primarily due to its encoding of N-terminal amino acids with relatively higher transcription levels and its efficient secretion capacity. Based on this foundation, the recombinant strain constructed in this work achieved a new record for the highest extracellular yields of APL in B. subtilis, reaching 12,295 U/mL, which was 1.9-times higher than that expressed in the recombinant Escherichia coli strain previously reported. The novel theories uncovered in this study are expected to play significant roles in enhancing the expression of foreign proteins both inside and outside of cells. [ABSTRACT FROM AUTHOR]

Abstract Maltotetraose amylase (Mta) catalyzes the hydrolysis of amylaceous polysaccharides into maltotetraose, which is an important functional sugar used in the food industry. However, the lack of efficient expression systems for recombinant Mta has hindered its scale-up production and application. In this study, a codon-optimized mta gene from Pseudomonas saccharophila was efficiently produced in Bacillus subtilis by optimizing the regulatory elements. First, a plasmid library containing 173 different signal peptide sequences placed upstream of mta gene was constructed, and transformed into B. subtilis strain WB800N(amyEΔ1) for high-throughput screening. The signal peptide yhcR was found to significantly enhance the secretion of Mta, reaching an activity of 75.4 U/mL in the culture medium. After optimization of the promoters, the Mta activity was further increased to 100.3 U/mL using a dual-promoter PHpaIIPamyE. Finally, the carbon sources and nitrogen sources for recombinant Mta production were optimized, yielding a highest Mta activity of 288.9 U/mL under the optimal culture conditions. The crude enzyme solution containing recombinant Mta produced a highest maltotetraose yield of 70.3% with 200 g/L of maltodextrin as the substrate. Therefore, the present study have demonstrated a high yield of Mta produced in B. subtilis, laying the foundation for large-scale Mta production and application.

Abstract Secretion systems play a crucial role in microbe-microbe or host-microbe interactions. Among these systems, the extracellular contractile injection system (eCIS) is a unique bacterial and archaeal extracellular secretion system that injects protein toxins into target organisms. However, the specific proteins that eCISs inject into target cells and their functions remain largely unknown. Here, we developed a machine learning classifier to identify eCIS-associated toxins (EATs). The classifier combines genetic and biochemical features to identify EATs. We also developed a score for the eCIS N-terminal signal peptide to predict EAT loading. Using the classifier we classified 2,194 genes from 950 genomes as putative EATs. We validated four new EATs, EAT14-17, showing toxicity in bacterial and eukaryotic cells, and identified residues of their respective active sites that are critical for toxicity. Finally, we show that EAT14 inhibits mitogenic signaling in human cells. Our study provides insights into the diversity and functions of EATs and demonstrates machine learning capability of identifying novel toxins. The toxins can be employed in various applications dependently or independently of eCIS.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop's classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a "snow-capped" appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype–phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5′ truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3′ truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by "toxic" cellular effects of the mutant proteins. [ABSTRACT FROM AUTHOR]

Bacillus subtilis is regarded as a promising microbial expression system in bioengineering due to its high stress resistance, nontoxic, low codon preference and grow fast. The strain has a relatively efficient expression system, as it has at least three protein secretion pathways and abundant molecular chaperones, which guarantee its expression ability and compatibility. Currently, many proteins are expressed in Bacillus subtilis, and their application prospects are broad. Although Bacillus subtilis has great advantages compared with other prokaryotes related to protein expression and secretion, it still faces deficiencies, such as low wild-type expression, low product activity, and easy gene loss, which limit its large-scale application. Over the years, many researchers have achieved abundant results in the modification of Bacillus subtilis expression systems, especially the optimization of promoters, expression vectors, signal peptides, transport pathways and molecular chaperones. An optimal vector with a suitable promoter strength and other regulatory elements could increase protein synthesis and secretion, increasing industrial profits. This review highlights the research status of optimization strategies related to the expression system of Bacillus subtilis. Moreover, research progress on its application as a food-grade expression system is also presented, along with some future modification and application directions. [ABSTRACT FROM AUTHOR]

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N‐terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro‐alpha‐factor. The unbinding parameters including off‐rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko–Hummer–Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off‐rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor. [ABSTRACT FROM AUTHOR]

β-Glucosidases are widely applied to biofuel production, food industry, and improvement in flavors. The novel β-glucosidase gene debgl was identified from the Arctic bacterium Devosia sp. Arc12 and expressed in Escherichia coli BL21 (DE3). The putative gene, belonging to the glycoside hydrolase family 1, consists of 1311 nucleotides and encodes 437 amino acid residues. The optimal temperature and pH of the recombinant DeBgl were 55 °C and 7, respectively. The recombinant DeBgl displayed salt and ethanol tolerance, and the V max , K m , and k cat for the substrate cellobiose were 754 μmol min−1 mg−1, 12.8 mM, and 661.4 s−1, respectively. The concentration of glucose was reached 51.6 g L−1 after incubation at 40 °C for 8 h in 100 g L−1 cellobiose solution. It suggests that the recombinant DeBgl is a good candidate enzyme to degrade complex celluloses. The signal peptide (SP) citH has been proven to be the most suitable for the secretory expression of β-glucosidase by screening the signal peptide library in Bacillus subtilis. The secretory activity of the SP citH fused DeBgl in the vector pMA5 was 9-fold higher than that of the DeBgl without signal peptide. This secretory expression system of β-glucosidase may benefit its industrial application. [Display omitted] • A novel β-glucosidase was isolated from the genus Devosia. • The recombinant DeBgl was highly tolerant to many types of chemicals. • The recombinant DeBgl is a good candidate to degrade complex celluloses. • DeBgl was expressed in Bacillus subtilis WB600 and could be secreted. • Signal peptide citH was the best in DeBgl in WB600. [ABSTRACT FROM AUTHOR]

Periplasmic expression of antimicrobial peptides is one of the most important issues in cloning and protein expression systems. In the present study, bioinformatics methods were used to predict the best signal peptides for periplasmic expression of the melittin peptide in Escherichia coli. Therefore, the sequence of 12 signal peptides was prepared from signal peptide databases. In order to choose the best signal peptide, periplasmic expression, sub-cellular localization site, solubility, physical and chemical properties of signal peptides were analyzed by Signalp6, Psort, Protein-sol, PRED-TAT, and Portparam servers. Six of the 12 signal peptides passed the Signalp6 filter, and only two passed Psort. After examining the solubility of signal peptides and investigating their physical and chemical properties, it was determined that zraP has the most favorable characteristics. Finally, zraP could be considered the best signal peptide for the melittin expression. Our results can be used for periplasmic expression of melittin in Escherichia coli host. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV) gene therapy for neurological disease has gained traction due to stunning advances in capsid evolution for CNS targeting. With AAV brain delivery now in focus, conventional improvements in viral expression vectors offer a complementary route for optimizing gene delivery. We previously introduced a novel AAV gene therapy to slow amyloid aggregation in the brain based on neuronal release of an Aβ sequence variant that inhibited fibrilization of wild-type Aβ. Here we explore three coding elements of the virally delivered DNA plasmid in an effort to maximize the production of therapeutic peptide in the brain. We demonstrate that simply replacing the Gaussia luciferase signal peptide with the mouse immunoglobulin heavy chain signal peptide increased release of variant Aβ by ∼5-fold. Sequence modifications within the expressed minigene further increased peptide release by promoting γ-secretase cleavage. Addition of a cytosolic fusion tag compatible with γ-secretase interaction allowed viral transduction to be tracked by immunostaining, independent from the variant Aβ peptide. Collectively these construct modifications increased neuronal production of therapeutic peptide by 10-fold upon intracranial AAV injection of neonatal mice. These findings demonstrate that modest changes in expression vector design can yield substantial gains in AAV efficiency for therapeutic applications.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD‐hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild‐type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7‐fold higher kcat/KM. Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high‐producing strain, 8GM1, containing eight copies of AOX1 promoter‐GAS1 signal peptide‐MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co‐expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2‐fold. In the 5‐L bioreactor, conventional fed‐batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3‐fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high‐producing strains for other industrial enzymes. A novel strategy combining directed enzyme evolution, optimizing signal peptide and gene dosage, and engineering vesicle trafficking was adopted to improve secretory production of glucose oxidase (GOD) in Pichia pastoris, generating an industrial hyper‐production strain. In the 5‐L bioreactor, an extraorinary high GOD activity and protein titer were obtain in culture supernatants with almost on other proteins. The EES gene, a putative component COPII‐coated vesicles, was found to facilitate both protein secretion and production for the first time. [ABSTRACT FROM AUTHOR]

Small proteins (<50 amino acids) are emerging as ubiquitous and important regulators in organisms ranging from bacteria to humans, where they commonly bind to and regulate larger proteins during stress responses. However, fundamental aspects of small proteins, such as their molecular mechanism of action, downregulation after they are no longer needed, and their evolutionary provenance, are poorly understood. Here, we show that the MntS small protein involved in manganese (Mn) homeostasis binds and inhibits the MntP Mn transporter. Mn is crucial for bacterial survival in stressful environments but is toxic in excess. Thus, Mn transport is tightly controlled at multiple levels to maintain optimal Mn levels. The small protein MntS adds a new level of regulation for Mn transporters, beyond the known transcriptional and post‐transcriptional control. We also found that MntS binds to itself in the presence of Mn, providing a possible mechanism of downregulating MntS activity to terminate its inhibition of MntP Mn export. MntS is homologous to the signal peptide of SitA, the periplasmic metal‐binding subunit of a Mn importer. Remarkably, the homologous signal peptide regions can substitute for MntS, demonstrating a functional relationship between MntS and these signal peptides. Conserved gene neighborhoods support that MntS evolved from the signal peptide of an ancestral SitA protein, acquiring a life of its own with a distinct function in Mn homeostasis. [ABSTRACT FROM AUTHOR]

Most of the active proteins and industrial enzymes commonly used in industry, agriculture and medicine are obtained through heterologous expression systems. Pichia pastoris is one of the excellent hosts for expressing heterologous proteins. This expression system has the advantages including good genetic stability, high protein expression and secretory level, post-translational modification system, and low cost etc. However, excessive aggregation of heterologous proteins usually leads to incorrect fold and ineffective secretion of the target proteins, thus decling protein expression level. This paper reviewed the progress in the study on exogenous protein expression in Pichia pastoris by engineering the secretion and folding pathways such as signal peptide optimization, chaperone optimization and fusion protein expression. [ABSTRACT FROM AUTHOR]

Abstract Background Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. Results In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5’UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5’UTR also has significant effects on protein expression and secretion. The best 5’UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5’UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. Conclusions An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

The chimeric signal peptides superior to natural signal peptides were constructed based on the N, H and C regions of different natural signal peptides to improve the α-amylase secretion in Bacillus subtilis. Through screening the natural signal peptides library, several optimal natural signal peptides were selected for Bacillus amyloliquefaciens α-amylase secretion in Bsubtilis. The H region of signal peptide SPsacB were replaced by H region of four natural signal peptides (SPyvcE, SPyoqM, SPBglS and SPyobB) to construct four different chimeric signal peptides (RSP1 to RSP4) applied in α-amylase genetic engineering strain. The results show that the most suitable natural and chimeric signal peptides for α-amylase were SPyoqM and RSP1, respectively. The α-amylase secretion efficiency guided by RSP1 was 27% higher than that by SPyoqM, 36 times higher than that by SPyvcE which has the same H region with RSP1, and 207 times higher than that by SPsacB which has the same N and C region but different in H region. The chimeric signal peptide construction strategy is effective for α-amylase secretion in Bacillus subtilis, which provides guidance for efficient secretion of other proteins.

D-psicose-3-epimerase (DPEase), a key enzyme for D-psicose production, has been successfully expressed in Escherichia coli with high yield. However, intracellular expression results in high downstream processing costs and greater risk of lipopolysaccharide (LPS) contamination during cell disruption. The secretory expression of DPEase could minimize the number of purification steps and prevent LPS contamination, but achieving the secretion expression of DPEase in E. coli is challenging and has not been reported due to certain limitations. This study addresses these challenges by enhancing the secretion of DPEase in E. coli through computational predictions and structural analyses. Signal peptide prediction identified PelB as the most effective signal peptide for DPEase localization and enhanced solubility. Supplementary strategies included the addition of 0.1% (v/v) Triton X-100 to promote protein secretion, resulting in higher extracellular DPEase (0.5 unit/mL). Low-temperature expression (20 °C) mitigated the formation of inclusion bodies, thus enhancing DPEase solubility. Our findings highlight the pivotal role of signal peptide selection in modulating DPEase solubility and activity, offering valuable insights for protein expression and secretion studies, especially for rare sugar production. Ongoing exploration of alternative signal peptides and refinement of secretion strategies promise further enhancement in enzyme secretion efficiency and process safety, paving the way for broader applications in biotechnology.

Anderson–Fabry disease (AFD) is an X-linked multisystemic disorder with a heterogeneous phenotype, resulting from deficiency of the lysosomal enzyme α-galactosidase A (α-Gal A) and leading to globotriaosylceramide systemic accumulation. Lysosomal storage is not the unique player in organ failure and different mechanisms could drive tissue damage, including endoplasmic reticulum (ER) stress and its related signaling pathway’s activation. We identified a new missense variant in the signal peptide of α-GLA gene, c.13 A/G, in a 55-year-old woman affected by chronic kidney disease, acroparesthesia, hypohidrosis, and deafness and exhibiting normal values of lysoGb3 and αGLA activity. The functional study of the new variant performed by its overexpression in HEK293T cells showed an increased protein expression of a key ER stress marker, GRP78, the pro-apoptotic BAX, the negative regulator of cell cycle p21, the pro-inflammatory cytokine, IL1β, together with pNFkB, and the pro-fibrotic marker, N-cadherin. Transmission electron microscopy showed signs of ER injury and intra-lysosomal inclusions. The proband’s PBMC exhibited higher expression of TGFβ 1 and pNFkB compared to control. Our findings suggest that the new variant, although it did not affect enzymatic activity, could cause cellular damage by affecting ER homeostasis and promoting apoptosis, inflammation, and fibrosis. Further studies are needed to demonstrate the variant’s contribution to cellular and tissue damage.

Background: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. Results: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. Conclusions: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications. [ABSTRACT FROM AUTHOR]

New SARS-CoV-2 variants continue to prevail worldwide, and effective vaccines are needed to prevent an epidemic. mRNA vaccines are gradually being applied to the prevention and control of infectious diseases with significant safety and effectiveness. The spike (S) protein of SARS-CoV-2 is the main target of mRNA vaccine design, but the impact of the signal peptide (SP), transmembrane region (TM), and cytoplasmic tail (CT) on mRNA vaccine remains unclear. In this study, we constructed three forms of mRNA vaccines related to the S protein: full-length, deletion of the TM and CT, and simultaneous deletion of the SP, TM and CT, and compared their immunogenicity. Our experimental data show that full-length S protein and deletion of the TM and CT could effectively induce neutralizing antibody production in mice, while S protein without the SP and TM could not. This indicates that the S protein SP is necessary for the design of mRNA vaccine. [ABSTRACT FROM AUTHOR]

Nattokinase, traditionally produced by the fermentation of Bacillus natto , has a high-efficiency and safe thrombolytic effect. However, low activity limits its industrial application as a thrombolytic agent. In this study, the nattokinase encoding gene from Bacillus natto was heterologously expressed in B. subtilis WB800 to realize the high-efficiency expression. At first, the gene was amplified and connected to the Escherichia coli-B. subtilis shuttle plasmid pP43NMK to construct the recombinant plasmid pP43NMK-NKF. The enzyme activity increased to 75.3 FU/mL after the signal peptide starting codon was optimized as ATG from GTG. A total of 19 signal peptides were screened to construct a series of recombinants. The recombinant PSP7 showed the highest nattokinase enzyme activity, 235.2 FU/mL. Furthermore, constructing the promoter tandem expression vector proved that the dual-promoter recombinant PSP7C43 has the highest enzyme activity of 247.1 FU/mL. After the optimization of fermentation conditions, the nattokinase enzyme activity of recombinant PSP7C43 finally increased to 325.4 FU/mL. The combinatorial strategy adopted in this study realizes the substantial improvement of nattokinase expression level, which lays a foundation for the industrial development of nattokinase as a thrombolytic reagent. [Display omitted] • Signal peptide with high expression of nattokinase was screened out. • Dual-promoter systems were constructed to improve the enzymatic activity. • Combinatorial strategy was adopted to realize high-efficiency expression. [ABSTRACT FROM AUTHOR]

African swine fever virus (ASFV) is the cause of African swine fever (ASF), a devastating disease that affects domestic pigs and wild boar and is currently responsible for the largest animal epidemic. One of the characteristics of ASFV infection, but absent in naturally attenuated strains, is hemadsorption (HAD), a phenomenon that has been linked to virulence. In this study, we have shown that ASFV HAD depends exclusively on the Nt domain of the ASFV CD2v protein during infection. CD2v is a highly glycosylated protein, and we found that glycosylation is essential for HAD. However, despite the higher degree of CD2v glycosylation, only simultaneous N-glycosylation of two Asp residues on the Nt region is the determinant for HAD. On the contrary, we have demonstrated that the presence of a specific signal peptide sequence on CD2v not only influences the degree of CD2v glycosylation but is also critical for HAD, although not for CD2v localization. Finally, we have shown that the CD2v expression during infection of the non-HAD NH/P68 strain is not sufficient for HAD, despite its glycosylation and cell surface localization. Complementation studies of CD2v from NH/P68 with CD2v signal peptides from HAD+ strains indicate (i) that different signal peptides from different genotypes are able to restore HAD and (ii) the existence of a HAD-inhibitory sequence in NH/P68-CD2v. This study lays the molecular basis for ASFV HAD, which could be key for the study of virulent and pathogenic aspects of the virus, as well as the rational development of new vaccines against ASFV. IMPORTANCE African swine fever virus (ASFV) is the cause of the current major animal epidemic worldwide. This disease affects domestic pigs and wild boars, has spread since 2007 through Russia, Eastern Europe, and more recently to Western European countries, and since 2018 emerged in China, from where it spread throughout Southeast Asia. Recently, outbreaks have appeared in the Caribbean, threatening the Americas. It is estimated that more than 900,000 animals have died directly or indirectly from ASFV since 2021 alone. One of the features of ASFV infection is hemoadsorption (HAD), which has been linked to virulence, although the molecular and pathological basis of this hypothesis remains largely unknown. In this study, we have analyzed and identified the key players responsible of HAD, contributing to the identification of new determinants of ASFV virulence, the understanding of ASFV pathogenesis, and the rational development of new vaccines. [ABSTRACT FROM AUTHOR]

Lipase B from Candida antarctica (CALB) is an important biocatalyst with many potential applications. However, original CALB is usually with lower enzyme activity and also costly to produce from Candida antarctica; hence, it is often necessary to prepare recombinant CALB through gene heterologous expression. In this research, seven promoters and five signal peptides were compared respectively for expressing codon-optimized CALB in Pichia pastoris, and then recombinant P. pastoris containing 3 copies of calb gene were obtained by screening with high concentrations of antibiotics under the condition of the optimal combination. In a 1.3-L bioreactor, the maximum CALB activity and total protein content reached 444.46 ± 18.81 U/mL and 5.41 ± 0.1 g/L, respectively, after about 9 days of incubation in FM22 medium, which were 34 times and 20 times higher than the initial strains, respectively. In addition, the obtained CALB was used to catalyze the transesterification of acidified gutter oil with methanol, suggesting a promising pathway to convert waste or low quality of bio-oil feedstocks with high amount of free fatty acids into biodiesel by using recombinant CALB as catalyst. The results can provide with a good reference for efficient expression of CALB and enhancing lipase production in P. pastoris. It is supposed to bring with new possibility for the bio-production of other valuable proteins. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Hebei University of Science & Technology is the property of Hebei University of Science & Technology, Journal of Hebei University of Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl transfer, and crosslinking reactions between Ƴ-carboxamide groups of peptides or protein-bound glutamine and the Ɛ-amino group of lysine. In this study, we demonstrated an efficient systematic engineering strategy to enhance the synthesis of TGase in a recombinant Streptomyces mobaraensis smL2020 strain in a 1000-L fermentor. Briefly, the enzymatic properties of the TGase TGL2020 from S. mobaraensis smL2020 and TGase TGLD from S. mobaraensis smLD were compared to obtain the TGase TGLD with perfected characteristics for heterologous expression in a recombinant S. mobaraensis smL2020ΔTG without the gene tgL 2020. Through multiple engineering strategies, including promoter engineering, optimizing the signal peptides and recombination sites, and increasing copies of the expression cassettes, the final TGLD activity in the recombinant S. mobaraensis smL2020ΔTG: (PL2020-spL2020-protgLD-tgLD)2 (tgL2020 and BT1) reached 56.43 U/mL and 63.18 U/mL in shake flask and 1000-L fermentor, respectively, which was the highest reported to date. With the improvement of expression level, the application scope of TGLD in the food industry will continue to expand. Moreover, the genetic stability of the recombinant strain maintained at more than 20 generations. These findings proved the feasibility of multiple systematic engineering strategies in synthetic biology and provided an emerging solution to improve biosynthesis of industrial enzymes.

Gene expression is a fundamental process that enables cells to produce specific proteins in a timely and spatially dependent manner. In eukaryotic cells, the complex organization of the cell body requires precise control of protein synthesis and localization. Certain mRNAs encode proteins with an N-terminal signal sequences that direct the translation apparatus toward a specific organelle. Here, we focus on the mechanisms governing the translation of mRNAs, which encode proteins with an endoplasmic reticulum (ER) signal in human cells. The binding of a signal-recognition particle (SRP) to the translation machinery halts protein synthesis until the mRNA–ribosome complex reaches the ER membrane. The commonly accepted model suggests that mRNA that encodes a protein that contains an ER signal peptide continuously repeats the cycle of SRP binding followed by association and dissociation with the ER. In contrast to the current view, we show that the long mRNAs remain on the ER while being translated. On the other hand, due to low ribosome occupancy, the short mRNAs continue the cycle, always facing a translation pause. Ultimately, this leads to a significant drop in the translation efficiency of small, ER-targeted proteins. The proposed mechanism advances our understanding of selective protein synthesis in eukaryotic cells and provides new avenues to enhance protein production in biotechnological settings.

The N-terminal sequences of proteins and their corresponding encoding sequences may play crucial roles in the heterologous expression. In this study, the secretory expression of alkaline pectin lyase APL in B. subtilis was investigated to explore the effects of the N-terminal 5–7 amino acid sequences of different signal peptides on the protein expression and secretion. It was identified for the first time that the first five amino acid sequences of the N-terminal of the signal peptide (SP-LipA) from Bacillus subtilis lipase A play an important role in promoting the expression of APL. Furthermore, it was revealed that SP-LipA resulted in higher secretory expression compared to other signal peptides in this study primarily due to its encoding of N-terminal amino acids with relatively higher transcription levels and its efficient secretion capacity. Based on this foundation, the recombinant strain constructed in this work achieved a new record for the highest extracellular yields of APL in B. subtilis, reaching 12,295 U/mL, which was 1.9-times higher than that expressed in the recombinant Escherichia coli strain previously reported. The novel theories uncovered in this study are expected to play significant roles in enhancing the expression of foreign proteins both inside and outside of cells.

AMELX mutations cause X-linked amelogenesis imperfecta (AI), known as AI types IE, IIB, and IIC in Witkop’s classification, characterized by hypoplastic (reduced thickness) and/or hypomaturation (reduced hardness) enamel defects. In this study, we conducted whole exome analyses to unravel the disease-causing mutations for six AI families. Splicing assays, immunoblotting, and quantitative RT-PCR were conducted to investigate the molecular and cellular effects of the mutations. Four AMELX pathogenic variants (NM_182680.1:c.2T>C; c.29T>C; c.77del; c.145-1G>A) and a whole gene deletion (NG_012494.2:g.307534_403773del) were identified. The affected individuals exhibited enamel malformations, ranging from thin, poorly mineralized enamel with a “snow-capped” appearance to severe hypoplastic defects with minimal enamel. The c.145-1G>A mutation caused a -1 frameshift (NP_001133.1:p.Val35Cysfs*5). Overexpression of c.2T>C and c.29T>C AMELX demonstrated that mutant amelogenin proteins failed to be secreted, causing elevated endoplasmic reticulum stress and potential cell apoptosis. This study reveals a genotype–phenotype relationship for AMELX-associated AI: While amorphic mutations, including large deletions and 5′ truncations, of AMELX cause hypoplastic-hypomaturation enamel with snow-capped teeth (AI types IIB and IIC) due to a complete loss of gene function, neomorphic variants, including signal peptide defects and 3′ truncations, lead to severe hypoplastic/aplastic enamel (AI type IE) probably caused by “toxic” cellular effects of the mutant proteins.

Abstract Background Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. Results In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter P veg -P ylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. Conclusions This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis.

Rice blast caused by Magnaporthe oryzae is one of the most destructive diseases and poses a growing threat to food security worldwide. Like many other filamentous pathogens, rice blast fungus releases multiple types of effector proteins to facilitate fungal infection and modulate host defence responses. However, most of the characterized effectors contain an N‐terminal signal peptide. Here, we report the results of the functional characterization of a nonclassically secreted nuclear targeting effector in M. oryzae (MoNte1). MoNte1 has no signal peptide, but can be secreted and translocated into plant nuclei driven by a nuclear targeting peptide. It could also induce hypersensitive cell death when transiently expressed in Nicotiana benthamiana. Deletion of the MoNTE1 gene caused a significant reduction of fungal growth and conidiogenesis, partially impaired appressorium formation and host colonization, and also dramatically attenuated the pathogenicity. Taken together, these findings reveal a novel effector secretion pathway and deepen our understanding of rice–M. oryzae interactions. [ABSTRACT FROM AUTHOR]

The red fluorescent protein mCherry is widely used as a tagged protein in intracellular protein positioning and dynamic tracing due to its stable characteristics in color and monomer molecule. In this study, mCherry was used as a tag to explore the cell wall-directed binding of the carbohydrate-binding module (CBM), in order to intuitively demonstrate that the fusion protein anchored on the cell wall. Two constitutive expression vectors harboring CBM-mCherry fusion gene were constructed for rice transformation. The results of fluorescent signal detection showed that both the ubiquitin promoter and the CaMV35S promoter could drive the expression of mCherry fusion gene in seeds, leaves, and roots. The results of subcellular localization by different methods, such as cytological observation, immunofluorescence detection and protoplast observation, displayed that the fluorescence signals were concentrated on the cell walls of the root, stem, leaf, and vascular bundle sheath in rice. It indicated that the CBM could mediate the red fluorescent protein anchoring on the cell wall, and the cell wall may be a good subcellular structure for accumulation of exogenous proteins in future. Key message: The fusion gene SP-CBM11-mCherry was transformed into rice, and the subcellular localization of fusion protein indicated the red fluorescent protein mCherry anchored on the cell wall. [ABSTRACT FROM AUTHOR]

Chitin is the second most abundant polysaccharide in nature after cellulose. The degradation products of chitin, amino-oligosaccharides, possess good biological activities and promising applications. Chitinase plays an important role in the degradation of chitin, and its highly efficient production has attracted wide attention. In this study, the Bacillus licheniformis -derived chitinase BlChiA was recombinantly expressed in Bacillus subtilis. The optimal promoter SpoVG and signal peptide YqxI were obtained by screening the endogenous promoters and signal peptides. The extracellular enzyme activity of the recombinant strain reached 1.96 U mL−1, 1.94-fold higher than that of the original, previously constructed strain. On this basis, mutant libraries were constructed using error-prone PCR, and mutants with enhanced activities were obtained using high-throughput activity assays. After combination, the mutant E336K/A472T exhibited 3.61-fold greater activity than the previous result, which represented the highest expression level of BlChiA in Bacillus subtilis. In addition, the mutant also exhibited greater acid adaptability. These results provided a foundation for further exploration of the function and industrial application of the enzyme. [Display omitted] • This is the inaugural effort to efficiently express BlChiA in Bacillus subtilis. • The promoter SpoVG and signal peptide YqxI facilitated BlChiA expression. • The E336K/A472T mutant with enhanced activity was obtained by directed evolution. • The expression level of BlChiA was increased by 3.61 times. • The chitinase BlChiA activity represented the highest level reported to date. [ABSTRACT FROM AUTHOR]

Prokaryotic extracellular vesicles (EVs) are vesicles that bud from the cell membrane and are secreted by bacteria and archaea. EV cargo in Gram-negative bacteria includes mostly periplasmic and outer membrane proteins. EVs are clinically important as their cargo can include toxins associated with bacterial virulence and toxicity; additionally, they have been proposed as efficient vaccine agents and as the ancestors of the eukaryotic endomembrane system. However, the mechanistic details behind EV cargo selection and release are still poorly understood. In this study, we have performed bioinformatics analysis of published data on EV proteomes from 38 species of bacteria and 4 archaea. Focusing on clusters of orthologous genes (COGs) and using the EggNOG mapper function, we have identified cargo proteins that are commonly found in EVs across species. We discuss the putative role of these prominent proteins in EV biogenesis and function. We also analyzed the published EV proteomes for conserved signal sequences and discuss the potential role of these signal sequences for EV cargo selection. [ABSTRACT FROM AUTHOR]

ABSTRACT The intraerythrocytic malaria parasite Plasmodium falciparum exports hundreds of proteins into the host red blood cell (RBC). Most are targeted to the endoplasmic reticulum (ER) by a stretch of hydrophobic amino acids and cleaved further downstream at a conserved motif called the Plasmodium Export Element (PEXEL) by the ER protease plasmepsin V (PM V). The mature effectors then travel through the secretory pathway to the parasitophorous vacuole (PV) that surrounds the parasite. There, PEXEL proteins are somehow recognized as export-destined proteins, as opposed to PV-resident proteins, and are selectively translocated out into the RBC. The mature N-terminus appears to be important for export. There is conflicting data on whether PM V cleavage is needed for proper export or whether any means of generating the mature N-terminus would suffice. We replaced the PEXEL-containing N-terminal sequence of an exported GFP reporter with a signal peptide sequence and showed that precise cleavage by signal peptidase, generating the proper mature N-terminus, yields export competence. Expressing a construct with only the native ER targeting signal without the PM V cleavage site dramatically decreased the amount of a mature PEXEL reporter, indicating that the hydrophobic stretch lacks an efficient cleavage signal. Therefore, the PEXEL motif functions as a specialized signal cleavage site when appropriately located after an ER targeting sequence. Our data suggest that PM V cleavage and RBC export are two independent events for PEXEL proteins. We also tested and rejected the hypothesis that an alpha-helical mature N-terminus is necessary for export. IMPORTANCE Malaria parasites export hundreds of proteins to the cytoplasm of the host red blood cells for their survival. A five amino acid sequence, called the PEXEL motif, is conserved among many exported proteins and is thought to be a signal for export. However, the motif is cleaved inside the endoplasmic reticulum of the parasite, and mature proteins starting from the fourth PEXEL residue travel to the parasite periphery for export. We showed that the PEXEL motif is dispensable for export as long as identical mature proteins can be efficiently produced via alternative means in the ER. We also showed that the exported and non-exported proteins are differentiated at the parasite periphery based on their mature N-termini; however, any discernible export signal within that region remained cryptic. Our study resolves a longstanding paradox in PEXEL protein trafficking.

Soluble proteins sorted through the secretory pathway contain an N-terminal signal peptide that induces their translocation into the endoplasmic reticulum (ER) from the cytosol. However, a few proteins that lack a signal peptide are still translocated into the ER, such as SOD1. SOD1 is a causative gene of amyotrophic lateral sclerosis (ALS). A relationship has been suggested between the secretion of SOD1 and the pathogenesis of ALS; however, the transport mechanism of SOD1 remains unclear. We herein report that SOD1 was translocated into the ER lumen through the translocon Sec61 and was then secreted extracellularly. The present results indicate the potential of suppressing the secretion of SOD1 as a therapeutic target for ALS. • Recombinant human SOD1 is translocated into the endoplasmic reticulum in budding yeast. • Translocated human SOD1 into the ER is secreted via secretory pathway. • The present study suggests the potential of suppressing the secretion of SOD1 as a therapeutic target for ALS. [ABSTRACT FROM AUTHOR]

Purpose: The human somatropin is a single-chain polypeptide with a pivotal role in various biological processes. Although Escherichia coli is considered as a preferred host for the production of human somatropin, the high expression of this protein in E. coli results in the accumulation of protein as inclusion bodies. Periplasmic expression using signal peptides could be used to overcome the formation of inclusion bodies; still, the efficiency of each of the signal peptides in periplasmic transportation is varied and often is protein specific. The present study aimed to use in silico analysis to identify an appropriate signal peptide for the periplasmic expression of human somatropin in E. coli. Methods: A library containing 90 prokaryotic and eukaryotic signal peptides were collected from the signal peptide database, and each signal’s characteristics and efficiency in connection with the target protein were analyzed by different software. The prediction of the secretory pathway and the cleavage position was determined by the signalP5 server. Physicochemical properties, including molecular weight, instability index, gravity, and aliphatic index, were investigated by ProtParam software. Results: The results of the present study showed that among all the signal peptides studied, five signal peptides ynfB, sfaS, lolA, glnH, and malE displayed high scores for periplasmic expression of human somatropin in E. coli, respectively. Conclusion: In conclusion, the results indicated that in-silico analysis could be used for the identification of suitable signal peptides for the periplasmic expression of proteins. Further laboratory studies can evaluate the accuracy of the results of in silico analysis.

Background: Raw starch-degrading α-amylase (RSDA) can hydrolyze raw starch at moderate temperatures, thus contributing to savings in starch processing costs. However, the low production level of RSDA limits its industrial application. Therefore, improving the extracellular expression of RSDA in Bacillus subtilis, a commonly used industrial expression host, has great value. Results: In this study, the extracellular production level of Pontibacillus sp. ZY raw starch-degrading α-amylase (AmyZ1) in B. subtilis was enhanced by expression regulatory element modification and fermentation optimization. As an important regulatory element of gene expression, the promoter, signal peptide, and ribosome binding site (RBS) sequences upstream of the amyZ1 gene were sequentially optimized. Initially, based on five single promoters, the dual-promoter Pveg-PylB was constructed by tandem promoter engineering. Afterward, the optimal signal peptide SPNucB was obtained by screening 173 B. subtilis signal peptides. Then, the RBS sequence was optimized using the RBS Calculator to obtain the optimal RBS1. The resulting recombinant strain WBZ-VY-B-R1 showed an extracellular AmyZ1 activity of 4824.2 and 41251.3 U/mL during shake-flask cultivation and 3-L fermenter fermentation, which were 2.6- and 2.5-fold greater than those of the original strain WBZ-Y, respectively. Finally, the extracellular AmyZ1 activity of WBZ-VY-B-R1 was increased to 5733.5 U/mL in shake flask by optimizing the type and concentration of carbon source, nitrogen source, and metal ions in the fermentation medium. On this basis, its extracellular AmyZ1 activity was increased to 49082.1 U/mL in 3-L fermenter by optimizing the basic medium components as well as the ratio of carbon and nitrogen sources in the feed solution. This is the highest production level reported to date for recombinant RSDA production. Conclusions: This study represents a report on the extracellular production of AmyZ1 using B. subtilis as a host strain, and achieved the current highest expression level. The results of this study will lay a foundation for the industrial application of RSDA. In addition, the strategies employed here also provide a promising way for improving other protein production in B. subtilis. [ABSTRACT FROM AUTHOR]

Fungal effectors play a pivotal role in suppressing the host defence system, and their evolution is highly dynamic. By comparative sequence analysis of plant‐pathogenic fungi and Magnaporthe oryzae, we identified the small secreted C2H2 zinc finger protein MoHTR3. MoHTR3 exhibited high conservation in M. oryzae strains but low conservation among other plant‐pathogenic fungi, suggesting an emerging evolutionary selection process. MoHTR3 is exclusively expressed in the biotrophic stage of fungal invasion, and the encoded protein localizes to the biotrophic interfacial complex (BIC) and the host cell nucleus. The signal peptide crucial for MoHTR3′ secretion to the BIC and the protein section required for its translocation to the nucleus were both identified by a functional protein domain study. The host‐nuclear localization of MoHTR3 suggests a function as a transcriptional modulator of host defence gene induction. After ΔMohtr3 infection, the expression of jasmonic acid‐ and ethylene‐associated genes was diminished in rice, in contrast to when the MoHTR3‐overexpressing strain (MoHTR3ox) was applied. The transcript levels of salicylic acid‐ and defence‐related genes were also affected after ΔMohtr3 and MoHTR3ox application. In pathogenicity assays, ΔMohtr3 was indistinguishable from the wild type. However, MoHTR3ox‐infected plants showed diminished lesion formation and hydrogen peroxide accumulation, accompanied by a decrease in susceptibility, suggesting that the MoHTR3‐induced manipulation of host cells affects host–pathogen interaction. MoHTR3 emphasizes the role of the host nucleus as a critical target for the pathogen‐driven manipulation of host defence mechanisms and underscores the ongoing evolution of rice blast's arms race. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is often caused by mutations in the KRAS oncogene, making KRAS neoantigens a promising vaccine candidate for immunotherapy. Secreting KRAS antigens using live Generally Recognized as Safe (GRAS) vaccine delivery hosts such as Lactococcus lactis is deemed to be an effective strategy in inducing specific desired responses. Recently, through the engineering of a novel signal peptide SPK1 from Pediococcus pentosaceus, an optimized secretion system was developed in the L. lactis NZ9000 host. In this study, the potential of the L. lactis NZ9000 as a vaccine delivery host for the production of two KRAS oncopeptides (mutant 68V-DT and wild-type KRAS) through the use of the signal peptide SPK1 and its mutated derivative (SPKM19) was investigated. The expression and secretion efficiency analyses of KRAS peptides from L. lactis were performed in vitro and in vivo in BALB/c mice. Contradictory to our previous study using the reporter staphylococcal nuclease (NUC), the yield of secreted KRAS antigens mediated by the target mutant signal peptide SPKM19 was significantly lower (by ~1.3-folds) compared to the wild-type SPK1. Consistently, a superior elevation of IgA response against KRAS aided by SPK1 rather than mutant SPKM19 was observed. Despite the lower specific IgA response for SPKM19, a positive IgA immune response from mice intestinal washes was successfully triggered following immunization. Size and secondary conformation of the mature proteins are suggested to be the contributing factors for these discrepancies. This study proves the potential of L. lactis NZ9000 as a host for oral vaccine delivery due to its ability to evoke the desired mucosal immune response in the gastrointestinal tract of mice. [ABSTRACT FROM AUTHOR]

Pearl millet [Pennisetum glaucum (L.) R. Br.] is the essential food crop for over ninety million people living in drier parts of India and South Africa. Pearl millet crop production is harshly hindered by numerous biotic stresses. Sclerospora graminicola causes downy mildew disease in pearl millet. Effectors are the proteins secreted by several fungi and bacteria that manipulate the host cell structure and function. This current study aims to identify genes encoding effector proteins from the S. graminicola genome and validate them through molecular techniques. In silico analyses were employed for candidate effector prediction. A total of 845 secretory transmembrane proteins were predicted, out of which 35 proteins carrying LxLFLAK (Leucine–any amino acid–Phenylalanine–Leucine–Alanine–Lysine) motif were crinkler, 52 RxLR (Arginine, any amino acid, Leucine, Arginine), and 17 RxLR-dEER putative effector proteins. Gene validation analysis of 17 RxLR-dEER effector protein-producing genes was carried out, of which 5genes were amplified on the gel. These novel gene sequences were submitted to NCBI. This study is the first report on the identification and characterization of effector genes in Sclerospora graminicola. This dataset will aid in the integration of effector classes that act independently, paving the way to investigate how pearl millet responds to effector protein interactions. These results will assist in identifying functional effector proteins involving the omic approach using newer bioinformatics tools to protect pearl millet plants against downy mildew stress. Considered together, the identified effector protein-encoding functional genes can be utilized in screening oomycetes downy mildew diseases in other crops across the globe. [ABSTRACT FROM AUTHOR]