Production Of Secreted Proteins Research Articles

Multiple strategies to improve the secretory expression of prolyl endopeptidase in Aspergillus niger

Prolyl Endopeptidase (PEP) has the unique ability to hydrolyze the carboxyl terminal peptide bonds of Proline. This enzyme holds great value in various fields including medical research, disease treatment, human diet, and food production. Aspergillus niger is a significant food-grade protein expression system renowned for its high safety. It is considered an ideal system for producing Aspergillus-derived PEP due to its ability to secrete a large amount of endogenous proteins. Our research group successfully constructed the Aspergillus niger PEP engineering strain in the early stages by eliminating the background protein AsaA, thereby increasing secretion protein production. In this article, we explore new solutions to further enhance production. Experimental investigations were conducted to regulate secretory protein production at different levels by modifying the 5′UTR and reconstructing the secretion pathways. The results demonstrate that the strain modified by the 5′UTR and Kozak sequence (glaA5'UTR-GCCACC, glaA::protA) exhibited a 1.88-fold increase in gene transcription and a 105% increase in extracellular PEP activity compared to the non-modified strain. Co-expression of the ERdjs gene AnScj1 further increased extracellular PEP activity by 168%. However, deletion of derA or vps10, key genes in the Aspergillus niger secretory protein degradation pathway, resulted in reduced levels of gene transcription and secretory expression. This article holds practical significance in enhancing the secretion and expression of PEP, obtaining higher-yield strains, and exploring optimization strategies for highly expressed proteins.

Exploring the secretome of Corynebacterium glutamicum ATCC 13032.

The demand for alternative sources of food proteins is increasing due to the limitations and challenges associated with conventional food production. Advances in biotechnology have enabled the production of proteins using microorganisms, thus prompting the exploration of attractive microbial hosts capable of producing functional proteins in high titers. Corynebacterium glutamicum is widely used in industry for the production of amino acids and has many advantages as a host organism for recombinant protein production. However, its performance in this area is limited by low yields of target proteins and high levels of native protein secretion. Despite representing a challenge for heterologous protein production, the C. glutamicum secretome has not been fully characterized. In this study, state-of-the-art mass spectrometry-based proteomics was used to identify and analyze the proteins secreted by C. glutamicum. Both the wild-type strain and a strain that produced and secreted a recombinant β-lactoglobulin protein were analyzed. A total of 427 proteins were identified in the culture supernatants, with 148 predicted to possess a secretion signal peptide. MS-based proteomics on the secretome enabled a comprehensive characterization and quantification (based on abundance) of the secreted proteins through label-free quantification (LFQ). The top 12 most abundant proteins accounted for almost 80% of the secretome. These are uncharacterized proteins of unknown function, resuscitation promoting factors, protein PS1, Porin B, ABC-type transporter protein and hypothetical membrane protein. The data can be leveraged for protein production by, e.g., utilizing the signal peptides of the most abundant proteins to improve secretion of heterologous proteins. In addition, secretory stress can potentially be alleviated by inactivating non-essential secreted proteins. Here we provide targets by identifying the most abundant, secreted proteins of which majority are of unknown function. The data from this study can thus provide valuable insight for researchers looking to improve protein secretion and optimize C. glutamicum as a host for secretory protein production.

Expression and purification of the receptor-binding domain of SARS-CoV-2 spike protein in mammalian cells for immunological assays.

The receptor-binding domain (RBD) of the spike glycoprotein of SARS-CoV-2 virus mediates the interaction with the host cell and is required for virus internalization. It is, therefore, the primary target of neutralizing antibodies. The receptor-binding domain soon became the major target for COVID-19 research and the development of diagnostic tools and new-generation vaccines. Here, we provide a detailed protocol for high-yield expression and one-step affinity purification of recombinant RBD from transiently transfected Expi293F cells. Expi293F mammalian cells can be grown to extremely high densities in a specially formulated serum-free medium in suspension cultures, which makes them an excellent tool for secreted protein production. The highly purified RBD is glycosylated, structurally intact, and forms homomeric complexes. With this quick and easy method, we are able to produce large quantities of RBD (80 mg·L-1 culture) that we have successfully used in immunological assays to examine antibody titers and seroconversion after mRNA-based vaccination of mice.

Taste papilla cell differentiation requires the regulation of secretory protein production by ALK3-BMP signaling in the tongue mesenchyme.

Taste papillae are specialized organs, each of which comprises an epithelial wall hosting taste buds and a core of mesenchymal tissue. In the present study, we report that during early taste papilla development in mouse embryos, bone morphogenetic protein (BMP) signaling mediated by type 1 receptor ALK3 in the tongue mesenchyme is required for epithelial Wnt/β-catenin activity and taste papilla differentiation. Mesenchyme-specific knockout (cKO) of Alk3 using Wnt1-Cre and Sox10-Cre resulted in an absence of taste papillae at E12.0. Biochemical and cell differentiation analyses demonstrated that mesenchymal ALK3-BMP signaling governed the production of previously unappreciated secretory proteins, i.e. it suppressed those that inhibit and facilitated those that promote taste papilla differentiation. Bulk RNA-sequencing analysis revealed many more differentially expressed genes (DEGs) in the tongue epithelium than in the mesenchyme in Alk3 cKO versus control. Moreover, we detected downregulated epithelial Wnt/β-catenin signaling and found that taste papilla development in the Alk3 cKO was rescued by the GSK3β inhibitor LiCl, but not by Wnt3a. Our findings demonstrate for the first time the requirement of tongue mesenchyme in taste papilla cell differentiation.

Biochemical analysis of Komagataella phaffii oxidative folding proposes novel regulatory mechanisms of disulfide bond formation in yeast

Oxidative protein folding in the endoplasmic reticulum (ER) is driven mainly by protein disulfide isomerase PDI and oxidoreductin Ero1. Their activity is tightly regulated and interconnected with the unfolded protein response (UPR). The mechanisms of disulfide bond formation have mainly been studied in human or in the yeast Saccharomyces cerevisiae. Here we analyze the kinetics of disulfide bond formation in the non-conventional yeast Komagataella phaffii, a common host for the production of recombinant secretory proteins. Surprisingly, we found significant differences with both the human and S. cerevisiae systems. Specifically, we report an inactive disulfide linked complex formed by K. phaffii Ero1 and Pdi1, similarly to the human orthologs, but not described in yeast before. Furthermore, we show how the interaction between K. phaffii Pdi1 and Ero1 is unaffected by the introduction of unfolded substrate into the system. This is drastically opposed to the previously observed behavior of the human pathway, suggesting a different regulation of the UPR and/or possibly different interaction mechanics between K. phaffii Pdi1 and Ero1.

The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris

Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously identified mutant strain, bgs13, was found to efficiently secrete most recombinant proteins tested, raising the possibility that this bgs13 mutant is a universal super secreter. In this study, we used a reporter protein, β-lactoglobulin (b-LG), to perform structural analysis of the protein secreted from wild type and mutant bgs13 strains to investigate the secretory mechanism. Primary, secondary, and tertiary structures of b-LG were examined using Edman sequencing, circular dichroism, tryptophan fluorescence, and temperature induced aggregation analysis. Our results demonstrate that the bgs13 produced more b-LG than the wt strain and that this protein was functionally folded similar to the wt. Surprisingly, we also found that the bgs13 b-LG was more resistant to aggregation, providing another example of the superior qualities of this strain for enhanced secreted protein production.

Imprinted Dlk1 dosage as a size determinant of the mammalian pituitary gland.

Co-regulated genes of the Imprinted Gene Network are involved in the control of growth and body size, and imprinted gene dysfunction underlies human paediatric disorders involving the endocrine system. Imprinted genes are highly expressed in the pituitary gland, among them, Dlk1, a paternally expressed gene whose membrane-bound and secreted protein products can regulate proliferation and differentiation of multiple stem cell populations. Dosage of circulating DLK1 has been previously implicated in the control of growth through unknown molecular mechanisms. Here we generate a series of mouse genetic models to modify levels of Dlk1 expression in the pituitary gland and demonstrate that the dosage of DLK1 modulates the process of stem cell commitment with lifelong impact on pituitary gland size. We establish that stem cells are a critical source of DLK1, where embryonic disruption alters proliferation in the anterior pituitary, leading to long-lasting consequences on growth hormone secretion later in life.

Co-feeding strategy alleviates hypoxic stress in large-scale bioreactor for optimal production of secretory recombinant proteins in Pichia pastoris.

The loss of mixing efficiency inherent to bioreactor process operated at large-scale yields to the formation of concentration gradient and thus to heterogeneous culture conditions. For processes operated with methanol feeding, P. pastoris faces oscillatory culture conditions that significantly affect the cell ability to produce secretory recombinant proteins at high yield. Extended cell residence time in microenvironments of high methanol concentration and low oxygen availability that are typically found in the upper part of the bioreactor near the feeding point, triggers the unfolded protein response (UPR) and thus impairs proper protein secretion. Methanol co-feeding with sorbitol was shown herein to reduce the UPR response and to restore productivity of secreted protein.

The endoplasmic reticulum acetyl-CoA transporter AT-1 plays a role in the protective unfolded protein response in the exocrine pancreas

Pancreatic acinar cells are responsible for the synthesis and secretion of digestive enzymes. In order to sustain high rates of secretory protein production, acinar cells rely on adaptive mechanisms to mitigate endoplasmic reticulum (ER) stress and maintain secretory homeostasis. The adaptive unfolded protein response (UPR), mediated by the IRE1-XBP1s axis, is essential for acinar function and protects against cellular injury. Recent work has demonstrated that the ER acetyl-CoA transporter, AT-1, is necessary for nascent secretory protein acetylation and export out of the ER and is regulated by IRE1-XBP1s. Furthermore, AT-1 is rapidly downregulated during experimental pancreatitis. To investigate the role of AT-1 in exocrine pancreas function, we generated inducible, acinar-specific AT-1 knockout mice (Ela-Cre AT-1-/-). Loss of AT-1 produced a chronic pancreatitis phenotype characterized by inflammation, immune cell infiltration, tissue fibrosis, and aberrant intracellular activation of the proteolytic digestive enzyme trypsin. Despite the histological and biochemical dysfunction observed, Ela-Cre AT-1-/- mice grow normally, have typical lifespans, and do not lose pancreatic mass. Interestingly, XBP1s expression is significantly upregulated in Ela-Cre AT-1-/- and may confer a protective effect with loss of AT-1. Our data suggests a role for AT-1 in the IRE1-XBP1s axis and highlights the previously undescribed role of protein acetylation in exocrine pancreas physiology. Given the numerous potential downstream effects of dysregulated acetyl-CoA transport, this model will advance our understanding of pancreatic function and disease. NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Whole genome sequence analysis enabled affirmation of the probiotic potential of marine sporulater Bacillus amyloliquefaciens BTSS3 isolated from Centroscyllium fabricii

Probiotics are microorganisms when administered in adequate amounts, confer health benefits on the host. Many probiotics find application in various industries however, probiotic bacteria linked to marine environments are less explored.Although Bifidobacteria, Lactobacilli, and Streptococcus thermophilus are the most frequently used probiotics, Bacillus spp. have acquired much acceptance in human functional foods due to their increased tolerance and enduring competence in harsh environments like the gastrointestinal (GI) tract. In this study, the 4 Mbp genome sequence of Bacillus amyloliquefaciens strain BTSS3, a marine spore former isolated from deep-sea shark Centroscyllium fabricii, with antimicrobial and probiotic properties was sequenced, assembled, and annotated. Analysis revealed the presence of numerous genes presenting probiotic traits like production of vitamins, secondary metabolites, amino acids, secretory proteins, enzymes and other proteins that allow survival in GI tract as well as adhesion to intestinal mucosa. Adhesion by colonization in the gut was studied in vivo in zebrafish (Danio rerio) using FITC labelled B.amyloliquefaciens BTSS3. Preliminary study revealed the ability of the marine Bacillus to attach to the intestinal mucosa of the fish gut. The genomic data and the in vivo experiment affirms that this marine spore former is a promising probiotic candidate with potential biotechnological applications.

Golgi Stress Response: New Insights into the Pathogenesis and Therapeutic Targets of Human Diseases.

The Golgi apparatus modifies and transports secretory and membrane proteins. In some instances, the production of secretory and membrane proteins exceeds the capacity of the Golgi apparatus, including vesicle trafficking and the post-translational modification of macromolecules. These proteins are not modified or delivered appropriately due to insufficiency in the Golgi function. These conditions disturb Golgi homeostasis and induce a cellular condition known as Golgi stress, causing cells to activate the 'Golgi stress response,' which is a homeostatic process to increase the capacity of the Golgi based on cellular requirements. Since the Golgi functions are diverse, several response pathways involving TFE3, HSP47, CREB3, proteoglycan, mucin, MAPK/ETS, and PERK regulate the capacity of each Golgi function separately. Understanding the Golgi stress response is crucial for revealing the mechanisms underlying Golgi dynamics and its effect on human health because many signaling molecules are related to diseases, ranging from viral infections to fatal neurodegenerative diseases. Therefore, it is valuable to summarize and investigate the mechanisms underlying Golgi stress response in disease pathogenesis, as they may contribute to developing novel therapeutic strategies. In this review, we investigate the perturbations and stress signaling of the Golgi, as well as the therapeutic potentials of new strategies for treating Golgi stress-associated diseases.

LBODP078 Role Of Ire1a-xbp1 Pathway In Rodent Functional Pituitary Tumor Cells.

Abstract Introduction Pituitary tumors (PTs) occur in almost 17% of the population and they represent about 10% of all intracranial tumors. At present, many patients with pituitary tumors suffer from symptoms and complications caused by abnormal hormone production and/or mass effects due to limited treatment options. Therefore, the development of novel therapies is an urgent issue. The inositol requiring enzyme 1a (IRE1a)-X-box binding protein 1 (XBP1) pathway is an important component for the endoplasmic reticulum (ER) stress response. This pathway is activated by immature protein overload in the ER and elimination or repair of such protein can reduce ER stress. This pathway has been reported to regulate cancer cell growth and secretory protein production. The detection of potential XBP1 binding sites in the promoter region of rat GH and mouse POMC similar to those of human genes suggests the involvement of the IRE1a-XBP1 pathway in the production of these hormones and pituitary tumor cell growth. In this study, we aimed to clarify the role of the IRE1a-XBP1 pathway in functional pituitary tumor cells. Methods and Results We conducted in vitro experiments by using rat GH-producing pituitary GH4 cells and mouse ACTH-producing AtT-20 cells as the models of GH- and ACTH-producing pituitary adenomas, respectively. Cell proliferation was measured by WST-8 assay, gene expression levels by qPCR, and protein expression by western blotting, respectively. In GH4 cells, STF08310 (40µM), an IRE1a inhibitor, reduced levels of GH and XBP1 spliced form (XBP1s) mRNA and GH protein with suppressed cell proliferation. In AtT-20 cells, STF08310 (40µM) also suppressed protein expression of proopiomelanocortin (POMC) and cell proliferation. Gene suppression of IRE1a by siRNA and over-expression of XBP1s showed marked reduction and increase of POMC protein, respectively. Conclusion Our findings suggest that the IRE1a-XBP1s pathway would be a possible therapeutic target that has both anti-tumoral and hormone control effects against functional pituitary tumor. Presentation: No date and time listed

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica).

The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing (HLB; Citrus greening). This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible 'Valencia' sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, β-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected 'Valencia' sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and 'Valencia' suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ 'Valencia' sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.

Potential of Hibiscus sabdariffa L. and Hibiscus Acid to Reverse Skin Aging.

Hibiscus sabdariffa L. (HS) has a long history of edible and medicinal uses. In this study, the biological activities of the extracts, chromatographic fractions, and hibiscus acid obtained from HS were evaluated for their potential bioactivities. Their ability to promote extracellular matrix synthesis in skin fibroblasts was evaluated by enzyme-linked immunosorbent assays. Their anti-inflammatory activity was evaluated in a nitric oxide (NO)–Griess inflammatory experiment. Furthermore, hibiscus acid was found to have a strong anti-oxidative stress effect through the establishment of an oxidative stress model induced by hydrogen peroxide. Several assays indicated that hibiscus acid treatment can effectively reduce extracellular adenosine triphosphate (ATP) secretion and carbonyl protein production, as well as maintain a high level of reduced/oxidized glutathione (GSH/GSSG) in skin cells, thus providing a possible mechanism by which hibiscus acid can counter antioxidative stress. The present study is the first to explore the reversing skin aging potential and the contributory component of HS.

A genetic toolkit for efficient production of secretory protein in Bacillus subtilis

Bacillus subtilis is a microbial cell factory widely used to produce recombinant proteins, but the expression of heterologous proteins is often severely hampered. This study constructed a genetic toolkit for improving the secretory efficiency of heterologous proteins in Bacillus subtilis. First, the protease-deficient hosts were reconstructed. Then, two endogenous constitutive promoters, Phag and PspovG, were screened. Next, a method called systemic combinatorial optimization of ribosome binding site (RBS) equipped with signal peptide (SCORES) was designed for optimizing the secretion and translation of the heterologous protein. Finally, Serratia marcescens nonspecific endonuclease (SMNE), which causes cell death by degrading nucleic acids, was expressed. The enzyme activity in the shake flask reached 7.5 × 106 U/L, which was 7.5-times that of the control RBS and signal peptide combination (RS0). This study not only expanded on the synthetic biology toolbox in B. subtilis but also provided strategies to create a prokaryotic protein expression system.

Mechanism of Protein Translocation by the Sec61 Translocon Complex.

The endoplasmic reticulum (ER) is a major site for protein synthesis, folding, and maturation in eukaryotic cells, responsible for production of secretory proteins and most integral membrane proteins. The universally conserved protein-conducting channel Sec61 complex mediates core steps in these processes by translocating hydrophilic polypeptide segments of client proteins across the ER membrane and integrating hydrophobic transmembrane segments into the membrane. The Sec61 complex associates with several other molecular machines and enzymes to enable substrate engagement with the channel and coordination of protein translocation with translation, protein folding, and/or post-translational modifications. Recent cryo-electron microscopy and functional studies of these translocon complexes have greatly advanced our mechanistic understanding of Sec61-dependent protein biogenesis at the ER. Here, we will review the current models for how the Sec61 channel performs its functions in coordination with partner complexes.

Balancing energy and protein homeostasis at ER-mitochondria contact sites.

The endoplasmic reticulum (ER) is the largest organelle of the cell and participates in multiple essential functions, including the production of secretory proteins, lipid synthesis, and calcium storage. Sustaining proteostasis requires an intimate coupling with energy production. Mitochondrial respiration evolved to be functionally connected to ER physiology through a physical interface between both organelles known as mitochondria-associated membranes. This quasi-synaptic structure acts as a signaling hub that tunes the function of both organelles in a bidirectional manner and controls proteostasis, cell death pathways, and mitochondrial bioenergetics. Here, we discuss the main signaling mechanisms governing interorganellar communication and their putative role in diseases including cancer and neurodegeneration.

Sec61 Inhibitor Apratoxin S4 Potently Inhibits SARS-CoV-2 and Exhibits Broad-Spectrum Antiviral Activity.

There is a pressing need for host-directed therapeutics that elicit broad-spectrum antiviral activities to potentially address current and future viral pandemics. Apratoxin S4 (Apra S4) is a potent Sec61 inhibitor that prevents cotranslational translocation of secretory proteins into the endoplasmic reticulum (ER), leading to anticancer and antiangiogenic activity both in vitro and in vivo. Since Sec61 has been shown to be an essential host factor for viral proteostasis, we tested Apra S4 in cellular models of viral infection, including SARS-CoV-2, influenza A virus, and flaviviruses (Zika, West Nile, and Dengue virus). Apra S4 inhibited viral replication in a concentration-dependent manner and had high potency particularly against SARS-CoV-2 and influenza A virus, with subnanomolar activity in human cells. Characterization studies focused on SARS-CoV-2 revealed that Apra S4 impacted a post-entry stage of the viral life-cycle. Transmission electron microscopy revealed that Apra S4 blocked formation of stacked double-membrane vesicles, the sites of viral replication. Apra S4 reduced dsRNA formation and prevented viral protein production and trafficking of secretory proteins, especially the spike protein. Given the potent and broad-spectrum activity of Apra S4, further preclinical evaluation of Apra S4 and other Sec61 inhibitors as antivirals is warranted.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in Escherichia coli.

Main reasons to produce recombinant proteins in the periplasm of E. coli rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production. Here, we discuss these strategies using notable examples as a thread.

The iSplit GFP assay detects intracellular recombinant proteins in Bacillus subtilis

BackgroundBacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP.ResultsAs proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4–12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity.ConclusionOur results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level.Graphic abstract