Secretory Expression Research Articles

Suitable carrier protein and linker peptide significantly increase the secretory expression of human lysozyme in Aspergillus niger.

Suitable carrier protein and linker peptide significantly increase the secretory expression of human lysozyme in Aspergillus niger.

Secretome profiling of Artemisia absinthium extract-loaded polymeric nanoparticle-treated MCF-7 and MDA-MB-231 revealed perturbation in microtubule assembly and cell migration.

Artemisia absinthium (wormwood) exhibits anticancer properties by inhibiting proliferation and causing cell death in breast cancer. Targeted drug delivery of A. absinthium nanoformulation using N-isopropyl acrylamide, N-vinyl pyrrolidone, and acrylic acid-based polymeric nanoparticles (NVA-AA NPs) was ensured by utilizing features of the tumor microenvironment, although their mechanism of action involved in cytotoxicity remains unknown. The present study employed nano LC-MS/MS to identify differences in secretory protein expression associated with the treatment of breast cancer cell lines (MCF-7; MDA-MB-231) by NVA-AA NPs for the determination of affected pathways and easily accessible therapeutic targets. Different bioinformatics tools were used to identify signature differentially expressed proteins (DEPs) using survival analysis by GENT2 and correlation analysis between their mRNA expressions and sensitivity toward small-molecule drugs as well as immune cell infiltration by GSCA. Analysis by GENT2 revealed 22 signature DEPs with the most significant change in their expression regulation, namely, gelsolin, alpha-fetoprotein, complement component C3, C7, histone H2B type 1-K, histone H2A.Z, H2AX, heat shock cognate 71 kDa protein, heat shock 70 kDa protein 1-like, cytochrome c somatic, GTP-binding nuclear protein Ran, tubulin beta chain, tubulin alpha-1B chain, tubulin alpha-1C chain, phosphoglycerate mutase 1, kininogen 1, carboxypeptidase N catalytic chain, fibulin-1, peroxiredoxins 4, lactate dehydrogenase C, SPARC, and SPARC-like protein 1. Correlation analysis between their mRNA expressions versus immune cell infiltrates showed a positive correlation with antitumor immune response elicited by these NPs as well as a correlation with drug response shown by the GDSC and CTRP drugs in different cancer cells. Our results suggest that NVA-AA NPs were able to invade the tumor microenvironment; transformed the communication network between the cancer cells; affected potential drivers of microtubular integrity, nucleosome assembly, and cell cycle; and eventually caused cell death.

Secretory expression and fermentation optimization for extracellular production of pullulanase in Vibrio natriegens

Pullulanase is a starch debranching enzyme, which is difficult in secretory expression due to its large molecular weight. Vibrio natriegens is a novel expression host with excellent efficiency in protein synthesis. In this study, we achieved secretory expression of the full-length pullulanase PulA and its truncated mutant PulN2 using V. natriegens VnDX strain. Subsequently, we investigated the effects of signal peptide, fermentation temperature, inducer concentration, glycine concentration and fermentation time on the secretory expression. Moreover, the extracellular enzyme activities of the two pullulanases produced in V. natriegens VnDX and E. coli BL21(DE3) were compared. The highest extracellular enzyme activity of PulA and PulN2 in V. natriegens VnDX were 61.6 U/mL and 64.3 U/mL, which were 110% and 62% that of those in E. coli BL21(DE3), respectively. The results indicated that V. natriegens VnDX can be used for secretory expression of the full-length PulA with large molecular weight, which may provide a reference for the secretory expression of other large molecular weight proteins in V. natriegens VnDX.

Trehalose Production Using Three Extracellular Enzymes Produced via One-Step Fermentation of an Engineered Bacillus subtilis Strain.

At present, the double-enzyme catalyzed method using maltooligosyltrehalose synthase (MTSase) and maltooligosyltrehalose trehalohydrolase (MTHase) is the mainstream technology for industrial trehalose production. However, MTSase and MTHase are prepared mainly using the heterologous expression in the engineered Escherichia coli strains so far. In this study, we first proved that the addition of 3 U/g neutral pullulanase PulA could enhance the trehalose conversion rate by 2.46 times in the double-enzyme catalyzed system. Then, a CBM68 domain was used to successfully assist the secretory expression of MTSase and MTHase from Arthrobacter ramosus S34 in Bacillus subtilis SCK6. At the basis, an engineered strain B. subtilis PSH02 (amyE::pulA/pHT43-C68-ARS/pMC68-ARH), which co-expressed MTSase, MTHase, and PulA, was constructed. After the 24 h fermentation of B. subtilis PSH02, the optimum ratio of the extracellular multi-enzymes was obtained to make the highest trehalose conversion rate of 80% from 100 g/L maltodextrin. The high passage stability and multi-enzyme preservation stability made B. subtilis PSH02 an excellent industrial production strain. Moreover, trehalose production using these extracellular enzymes produced via the one-step fermentation of B. subtilis PSH02 would greatly simplify the procedure for multi-enzyme preparation and be expected to reduce production costs.

Slow hydrogel matrix degradation enhances salivary gland mimetic phenotype.

We recently developed a salivary gland tissue mimetic (SGm), comprised of salivary gland cells encapsulated in matrix metalloproteinase (MMP)-degradable poly(ethylene glycol) hydrogels within arrays of ∼320 µm diameter spherical cavities molded in PDMS. The SGm provides a functional and physiologically relevant platform well-suited to high-throughput drug screening for radioprotective compounds. However, the utility of the SGm would benefit from improved retention of acinar cell phenotype and function. We hypothesized that tuning biochemical cues presented within the PEG hydrogel matrix would improve maintenance of acinar cell phenotype and function by mimicking the natural extracellular matrix microenvironment of the intact gland. Hydrogels formed using slower-degrading MMP-sensitive peptide crosslinkers showed >2-fold increase in sphere number formed at 48 h, increased expression of acinar cell markers, and more robust response to calcium stimulation by the secretory agonist, carbachol, with reduced SGm tissue cluster disruption and outgrowth during prolonged culture. The incorporation of adhesive peptides containing RGD or IKVAV improved calcium flux response to secretory agonists at 14 days of culture. Tuning the hydrogel matrix improved cell aggregation, and promoted acinar cell phenotype, and stability of the SGm over 14 days of culture. Furthermore, combining this matrix with optimized media conditions synergistically prolonged the retention of the acinar cell phenotype in SGm. STATEMENT OF SIGNIFICANCE: Salivary gland (SG) dysfunction occurs due to off-target radiation due to head and neck cancer treatments. Progress in understanding gland dysfunction and developing therapeutic strategies for the SG are hampered by the lack of in vitro models, as salivary gland cells rapidly lose critical secretory function within 24 hours in vitro. Herein, we identify properties of poly(ethylene glycol) hydrogel matrices that enhance the secretory phenotype of SG tissue mimetics within the previously-described SG-microbubble tissue chip environment. Combining slow-degrading hydrogels with media conditions optimized for secretory marker expression further enhanced functional secretory response and secretory marker expression.

A Genetically Engineered Bivalent Vaccine Coexpressing a Molecular Adjuvant against Classical Swine Fever and Porcine Epidemic Diarrhea.

Classical swine fever (CSF) and porcine epidemic diarrhea (PED) are highly contagious viral diseases that pose a significant threat to piglets and cause substantial economic losses in the global swine industry. Therefore, the development of a bivalent vaccine capable of targeting both CSF and PED simultaneously is crucial. In this study, we genetically engineered a recombinant classical swine fever virus (rCSFV) expressing the antigenic domains of the porcine epidemic diarrhea virus (PEDV) based on the modified infectious cDNA clone of the vaccine strain C-strain. The S1N and COE domains of PEDV were inserted into C-strain cDNA clone harboring the mutated 136th residue of Npro and substituted 3'UTR to generate the recombinant chimeric virus vC/SM3'UTRN-S1NCOE. To improve the efficacy of the vaccine, we introduced the tissue plasminogen activator signal (tPAs) and CARD domain of the signaling molecule VISA into vC/SM3'UTRN-S1NCOE to obtain vC/SM3'UTRN-tPAsS1NCOE and vC/SM3'UTRN-CARD/tPAsS1NCOE, respectively. We characterized three vaccine candidates in vitro and investigated their immune responses in rabbits and pigs. The NproD136N mutant exhibited normal autoprotease activity and mitigated the inhibition of IFN-β induction. The introduction of tPAs and the CARD domain led to the secretory expression of the S1NCOE protein and upregulated IFN-β induction in infected cells. Immunization with recombinant CSFVs expressing secretory S1NCOE resulted in a significantly increased in PEDV-specific antibody production, and coexpression of the CARD domain of VISA upregulated the PEDV-specific IFN-γ level in the serum of vaccinated animals. Notably, vaccination with vC/SM3'UTRN-CARD/tPAsS1NCOE conferred protection against virulent CSFV and PEDV challenge in pigs. Collectively, these findings demonstrate that the engineered vC/SM3'UTRN-CARD/tPAsS1NCOE is a promising bivalent vaccine candidate against both CSFV and PEDV infections.

Effects of signal peptides on the secreted green fluorescent protein (GFP) expression by recombinant Saccharomyces cerevisiae

Recombinant proteins secreted by Saccharomyces cerevisiae have been widely applied in the food industry and biopharmaceuticals because of their valuable features. However, low-secreted protein production in S. cerevisiae is a pending challenge that has hampered applications based on S. cerevisiae. To improve the secreted protein expression in S. cerevisiae, it is important to develop an efficient cassette for the secretory expression of recombinant protein by S. cerevisiae. In this study, three cassettes were constructed in which a gene of interest was fused with genes coding for secretory signal peptides from AGA2, MFα and α-glucoamylase. Green fluorescent protein (GFP) was employed as a model protein to investigate the secretion of recombinant protein mediated by these signal peptides in S. cerevisiae. The secreted expression of GFP was detected by Western Blot and dot-blot analyses using a specific antibody against GFP which showed the strongest GFP levels from MFα signal-related cassette. Therefore, their cassettes could be employed for following studies on the secretion of some other proteins.

Efficient secretion of mussel adhesion proteins using a chaperone protein Spy as fusion tag in Bacillus subtilis.

Mussel foot proteins (Mfps) are considered as remarkable materials due to their extraordinary adhesive capability. Recombinant expression is an ideal way to synthesis these proteins at large scale. However, secretory expression of Mfps into culture medium has not been achieved in a heterologous host. Here, to realize the secretion of Mfp3 and Mfp5 in Bacillus subtilis, signal peptide screening was first performed. Minimal Mfp3-6×His was targeted into the growth medium with AmyE signal peptide. We found that a small chaperone protein Spy was secreted efficiently in B. subtilis, and the fusion proteins Spy-Mfp3-6×His and Spy-Mfp5-6×His could also be delivered into growth medium well. The yield of Spy-Mfp3-6×His and Spy-Mfp5-6×His reached 255 and 119mgL-1 at shake flask conditions, respectively. Mfp3-6×His and Mfp5-6×His were finally purified via TEV protease cleavage and NTA affinity chromatography. Mfp3-6×His and Mfp5-6×His could be efficiently secreted using a chaperone protein Spy as fusion tag in B. subtilis.

Comparing the Ability of Secretory Signal Peptides for Heterologous Expression of Anti-Lipopolysaccharide Factor 3 in Chlamydomonas reinhardtii

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

7-Ketocholesterol accelerates pancreatic β-cell senescence by inhibiting the SIRT1/CDK4–Rb–E2F1 signaling pathway

ABSTRACT Pancreatic β-cell dysfunction is a key factor in the development of type 2 diabetes. Pancreatic β-cell senescence accelerates abnormal glucose metabolism, which decreases insulin secretion and cell regeneration ability, eventually leading to diabetes. A cholesterol oxidation product, 7-ketocholesterol (7-KC) can affect pancreatic β-cell function. However, its role in pancreatic β-cell senescence has not been reported. We investigated the role of 7-KC in pancreatic β-cell senescence and its underlying molecular mechanism in MIN6 cells. MIN6 cells were treated with 25 μmol/L 7-KC for 24 h and the proportion of senescent cells was detected based on senescence-associated β-galactosidase (SA-β-gal) activity. The cell cycle, DNA damage, and the senescence-associate secretory phenotype (SASP) and protein expression were detected by flow cytometry, immunofluorescence, and western blotting, respectively. 7-KC can significantly increase SA-β-gal activity, promoted G0/G1 arrest, DNA damage, and interleukin-1β expression in MIN6 cells and significantly inhibited insulin synthesis. Further studies indicated that 7-KC induced β-cell senescence by inhibiting the SIRT1/CDK4–Rb – E2F1 signaling pathway.

Effect of pregnancy and hypertension on kidney function in female rats: Modeling and functional implications.

Throughout pregnancy, the kidneys undergo significant adaptations in morphology, hemodynamics, and transport to achieve the volume and electrolyte retention required to support a healthy pregnancy. Additionally, during pregnancies complicated by chronic hypertension, altered renal function from normal pregnancy occurs. The goal of this study is to analyze how inhibition of critical transporters affects gestational kidney function as well as how renal function is affected during chronic hypertension in pregnancy. To do this, we developed epithelial cell-based multi-nephron computational models of solute and water transport in the kidneys of a female rat in mid- and late pregnancy. We simulated the effects of key individual pregnancy-induced changes on renal Na+ and K+ transport: proximal tubule length, Na+/H+ exchanger isoform 3 (NHE3) activity, epithelial Na+ channel activity (ENaC), K+ secretory channel expression, and H+-K+-ATPase activity. Additionally, we conducted simulations to predict the effects of inhibition and knockout of the ENaC and H+-K+-ATPase transporters on virgin and pregnant rat kidneys. Our simulation results predicted that the ENaC and H+-K+-ATPase transporters are essential for sufficient Na+ and K+ reabsorption during pregnancy. Last, we developed models to capture changes made during hypertension in female rats and considered what may occur when a rat with chronic hypertension becomes pregnant. Model simulations predicted that in hypertension for a pregnant rat there is a similar shift in Na+ transport from the proximal tubules to the distal tubules as in a virgin rat.

Expression element optimization and molecular modification to enhance the secretory expression of chitinase from Bacillus licheniformis in Bacillus subtilis

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.

Expression of β-Glucosidases from the Yak Rumen in Lactic Acid Bacteria: A Genetic Engineering Approach.

β-glucosidase derived from microorganisms has wide industrial applications. In order to generate genetically engineered bacteria with high-efficiency β-glucosidase, in this study two subunits (bglA and bglB) of β-glucosidase obtained from the yak rumen were expressed as independent proteins and fused proteins in lactic acid bacteria (Lactobacillus lactis NZ9000). The engineered strains L. lactis NZ9000/pMG36e-usp45-bglA, L. lactis NZ9000/pMG36e-usp45-bglB, and L. lactis NZ9000/pMG36e-usp45-bglA-usp45-bglB were successfully constructed. These bacteria showed the secretory expression of BglA, BglB, and Bgl, respectively. The molecular weights of BglA, BglB, and Bgl were about 55 kDa, 55 kDa, and 75 kDa, respectively. The enzyme activity of Bgl was significantly higher (p < 0.05) than that of BglA and BglB for substrates such as regenerated amorphous cellulose (RAC), sodium carboxymethyl cellulose (CMC-Na), desiccated cotton, microcrystalline cellulose, filter paper, and 1% salicin. Moreover, 1% salicin appeared to be the most suitable substrate for these three recombinant proteins. The optimum reaction temperatures and pH values for these three recombinant enzymes were 50 °C and 7.0, respectively. In subsequent studies using 1% salicin as the substrate, the enzymatic activities of BglA, BglB, and Bgl were found to be 2.09 U/mL, 2.36 U/mL, and 9.4 U/mL, respectively. The enzyme kinetic parameters (Vmax, Km, Kcat, and Kcat/Km) of the three recombinant strains were analyzed using 1% salicin as the substrate at 50 °C and pH 7.0, respectively. Under conditions of increased K+ and Fe2+ concentrations, the Bgl enzyme activity was significantly higher (p < 0.05) than the BglA and BglB enzyme activity. However, under conditions of increased Zn2+, Hg2+, and Tween20 concentrations, the Bgl enzyme activity was significantly lower (p < 0.05) than the BglA and BglB enzyme activity. Overall, the engineered lactic acid bacteria strains generated in this study could efficiently hydrolyze cellulose, laying the foundation for the industrial application of β-glucosidase.

Exploration of the Strategy for Improving the Expression of Heterologous Sweet Protein Monellin in Aspergillus niger

Aspergillus niger is a primary cell factory for food-grade protein (enzyme) production due to its strong protein secretion capacity and unique safety characteristics. The bottleneck issue for the current A. niger expression system is the difference in expression yield of heterologous proteins of non-fungal origin compared to those of fungal origin, which is about three orders of magnitude. The sweet protein monellin, derived from West African plants, has the potential to become a food-grade sweetener due to its high sweetness and the benefit of not containing sugar itself, but it is extremely difficult to establish a research model for heterologous expression in A. niger, owing to extremely low expression, a small molecular weight, and being undetectable with conventional protein electrophoresis. HiBiT-Tag was fused with low-expressing monellin in this work to create a research model for heterologous protein expression in A. niger at ultra-low levels. We increased monellin expression by increasing the monellin copy number, fusing monellin with the endogenous highly expressed glycosylase glaA, and eliminating extracellular protease degradation, among other strategies. In addition, we investigated the effects of overexpression of molecular chaperones, inhibiting the ERAD pathway, and enhancing the synthesis of phosphatidylinositol, phosphatidylcholine, and diglycerides in the biomembrane system. Using medium optimization, we finally obtained 0.284 mg/L of monellin in the supernatant of the shake flask. This is the first time recombinant monellin has been expressed in A. niger, with the goal of investigating ways to improve the secretory expression of heterologous proteins at ultra-low levels, which can serve as a model for the expression of other heterologous proteins in A. niger.

Efficient Microfluidic Screening Method Using a Fluorescent Immunosensor for Recombinant Protein Secretions.

Microbial secretory protein expression is widely used for biopharmaceutical protein production. However, establishing genetically modified industrial strains that secrete large amounts of a protein of interest is time-consuming. In this study, a simple and versatile high-throughput screening method for protein-secreting bacterial strains is developed. Different genotype variants induced by mutagens are encapsulated in microemulsions and cultured to secrete proteins inside the emulsions. The secreted protein of interest is detected as a fluorescence signal by the fluorescent immunosensor quenchbody (Q-body), and a cell sorter is used to select emulsions containing improved protein-secreting strains based on the fluorescence intensity. The concept of the screening method is demonstrated by culturing Corynebacterium glutamicum in emulsions and detecting the secreted proteins. Finally, productive strains of fibroblast growth factor 9 (FGF9) are screened, and the FGF9 secretion increased threefold compared to that of parent strain. This screening method can be applied to a wide range of proteins by fusing a small detection tag. This is a highly simple process that requires only the addition of a Q-body to the medium and does not require the addition of any substrates or chemical treatments. Furthermore, this method shortens the development period of industrial strains for biopharmaceutical protein production.

Secretory expression of recombinant small laccase genes in Gram-positive bacteria

BackgroundLaccases are multicopper enzymes that oxidize a wide range of aromatic and non-aromatic compounds in the presence of oxygen. The majority of industrially relevant laccases are derived from fungi and are produced in eukaryotic expression systems such as Pichia pastoris and Saccharomyces cerevisiae. Bacterial laccases for research purposes are mostly produced intracellularly in Escherichia coli, but secretory expression systems are needed for future applications. Bacterial laccases from Streptomyces spp. are of interest for potential industrial applications because of their lignin degrading activities.ResultsIn this study, we expressed small laccases genes from Streptomyces coelicolor, Streptomyces viridosporus and Amycolatopsis 75iv2 with their native signal sequences in Gram-positive Bacillus subtilis and Streptomyces lividans host organisms. The extracellular activities of ScLac, SvLac and AmLac expressed in S. lividans reached 1950 ± 99 U/l, 812 ± 57 U/l and 12 ± 1 U/l in the presence of copper supplementation. The secretion of the small laccases was irrespective of the copper supplementation; however, activities upon reconstitution with copper after expression were significantly lower, indicating the importance of copper during laccase production. The production of small laccases in B. subtilis resulted in extracellular activity that was significantly lower than in S. lividans. Unexpectedly, AmLac and ScLac were secreted without their native signal sequences in B. subtilis, indicating that B. subtilis secretes some heterologous proteins via an unknown pathway.ConclusionsSmall laccases from S. coelicolor, S. viridosporus and Amycolatopsis 75iv2 were secreted in both Gram-positive expression hosts B. subtilis and S. lividans, but the extracellular activities were significantly higher in the latter.

The Oral Delivery System of Modified GLP-1 by Probiotics for T2DM.

The glucagon-like peptide-1 (GLP-1) is a peptide with incretin activity and plays an important role in glycemic control as well as the improvement of insulin resistance in type 2 diabetes mellitus (T2DM). However, the short half-life of the native GLP-1 in circulation poses difficulties for clinical practice. To improve the proteolytic stability and delivery properties of GLP-1, a protease-resistant modified GLP-1 (mGLP-1) was constructed with added arginine to ensure the structural integrity of the released mGLP-1 in vivo. The model probiotic Lactobacillus plantarum WCFS1 was chosen as the oral delivery vehicle with controllable endogenous genetic tools driven for mGLP-1 secretory constitutive expression. The feasibility of our design was explored in db/db mice which showed an improvement in diabetic symptoms related to decreased pancreatic glucagon, elevated pancreatic β-cell proportion, and increased insulin sensitivity. In conclusion, this study provides a novel strategy for the oral delivery of mGLP-1 and further probiotic transformation.

Breast cancer cell secretome analysis to decipher miRNA regulating the tumor microenvironment and discover potential biomarkers

MicroRNA (miRNA/miR) 526 b- and miR655-overexpressed tumor cell-free secretions regulate the breast cancer tumor microenvironment (TME) by promoting tumor-associated angiogenesis, oxidative stress, and hypoxic responses. Additionally, premature miRNA (pri-miR526b and pri-miR655) are established breast cancer blood biomarkers. However, the mechanisms of how these miRNAs regulate the TME has yet to be investigated. Mass spectrometry analysis of miRNA-overexpressed cell lines MCF7-miR526b, MCF7-miR655, and miRNA-low MCF7-Mock cell-free secretomes identified 34 differentially expressed proteins coded by eight genes. In both miRNA-high cell secretomes, four markers are upregulated: YWHAB, SFN, TXNDC12, and MYL6B, and four are downregulated: PEA15, PRDX4, PSMB6, and FN1. All upregulated marker transcripts are significantly high in both total cellular RNA pool and cell-free secretions of miRNA-high cell lines, validated with quantitative RT-PCR. Bioinformatics tools were used to investigate these markers' roles in breast cancer. These markers' top gene ontology functions are related to apoptosis, oxidative stress, membrane transport, and motility supporting oncogenic miR526b- and miR655-induced functions. Gene transcription factor analysis tools were used to show how these miRNAs regulate the expression of each secretory marker. Data extracted from the Human Protein Atlas showed that YWHAB, SFN, and TXNDC12 expression could distinguish early and late-stage breast cancer in various breast cancer subtypes and are associated with poor patient survival. Additionally, immunohistochemistry analysis showed the expression of each marker in breast tumors. A stronger correlation between miRNA clusters and upregulated secretory markers gene expression was found in the luminal A tumor subtype. YWHAB, SFN, and MYL6B are upregulated in breast cancer patient's blood, showing biomarker potential. Of these identified novel miRNA secretory markers, SFN and YWHAB successfully passed all validations and are the best candidates to further investigate their roles in miRNA associated TME regulation. Also, these markers show the potential to serve as blood-based breast cancer biomarkers, especially for luminal-A subtypes.

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin

Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in Bacillus subtilis with an activity around 100 U/mL. A secretory expression system containing the signal peptide SPEstA and the dual-promoter PHpaII-SrfA was established, and the extracellular PLD activity further reached 119.22 U/mL through scale-up fermentation, 191.30-fold higher than that of the control. Under optimum reaction conditions, a 61.61% conversion ratio and 21.07 g/L of phosphatidylserine production were achieved. Finally, the synthesis system of PL derivates was established, which could efficiently synthesis novel PL derivates. The results highlight that the secretory expression system constructed in this study provides a promising PLD producing strain in industrial application, and laid the foundation for the biosynthesis of phosphatidylserine and other PL derivates. As far as we know, this work reports the highest level of extracellular PLD expression to date and the enzymatic production of several PL derivates for the first time.

Secretory expression of β-1,3-glucomannanase in the oleaginous yeast Rhodosporidium toruloides for improved lipid extraction

Lipids produced by oleaginous yeasts are considered as sustainable sources for the production of biofuels and oleochemicals. The red yeast Rhodosporidium toruloides can accumulate lipids to over 70% of its dry cell mass. To facilitate lipid extraction, a recombinant β-1,3-glucomannanase, MAN5C, has been applied to partially breakdown R. toruloides cell wall. In this study, R. toruloides NP11 was engineered for secretory expression of MAN5C to simplify the lipid extraction process. Specifically, a cassette contained a codon-optimized gene MAN5C was integrated into the genome of R. toruloides by Agrobacterium-mediated transformation. The engineered strain NP11-MAN5C was found with proper expression and secretion of active MAN5C, yet no notable compromise in terms of cell growth and lipid production. When NP11-MAN5C cell cultures were extracted with ethyl acetate without any pretreatment, 20% of total lipids were recovered, 4.3-fold higher than that of the parental strain NP11. When the cells were heat-treated followed by extraction with ethyl acetate in the presence of the culture broth supernatants, up to 93% of total lipids were recovered, confirming beneficial effects of MAN5C produced in situ. This study provides a new strategy to engineer oleaginous yeasts for more viable lipid extraction and down-stream processes.Graphical