Combined strategies for improving the heterologous expression of a novel xylanase from Fusarium oxysporum Fo47 in Pichia pastoris.

Xylanase, an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat, has been found to improve the organizational structure of dough and thus increase its volume. In our past work, one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P. pastoris. It has shown significant potential in improving the quality of whole wheat bread, making it become a candidate for development as a new flour improver. After optimization of expression elements and gene dose, the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P. pastoris. In addition, 12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain, and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL. Nevertheless, combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone. To further evaluate the industrial potential, the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter. These engineering strategies improved the expression of xylanase FXYL by more than 104-fold, providing valuable insights for the cost-effective industrial application of FXYL in the baking field.

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.

The superposition anti-viral activity of porcine tri-subtype interferon expressed by Saccharomyces cerevisiae.

Interferon (IFN)-mediated antiviral responses are central to host defense against viral infection. Porcine viral infection has emerged as a serious hazard for the pig industry. The construction of an engineered Saccharomyces cerevisiae strain that efficiently produces porcine IFN has demonstrated several advantages. It can be easily fed to pigs, which helps in reducing antibiotic residues in pork and improve meat quality. In this study, the stable expression of several porcine IFN molecules (pIFN-α1, pIFN-ß, pIFN-λ1, pIFN-λ1-ß, pIFN-λ1-ß-α1) were determined using an engineered S. cerevisiae system. With the YeastFab assembly method, the complete transcriptional units containing promoter (GPD), secretory peptide (α-mating factor), target gene (IFN) and terminator (ADH1) were successfully constructed using the characteristics of type II restriction endonuclease, and then integrated into the chromosomes Ⅳ and XVI of ST1814 yeast host strain, respectively. The expression kinetics of recombinant pIFNs were further analyzed. Synergism in the expression level of IFN receptor, antiviral protein, and viral loading was observed in viral-cell infection model treated with different porcine IFN subtypes. The porcine reproductive and respiratory syndrome viral load and antibody titer in serum decreased significantly after oral administration of IFN expression yeast fermentation broth. These findings indicate the potential efficacy of multi-valent pIFNs expressing S. cerevisiae as a potent feed material to prevent viral infections of pigs.

Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase

Background: Protein glutaminase specifically deamidates glutamine residue in protein and therefore significantly improves protein solubility and colloidal stability of protein solution. In order to improve its preparation efficiency, we exploited the possibility for its secretory expression mediated by twin-arginine translocation (Tat) pathway in Bacillus licheniformis. Results: The B. licheniformis genome-wide twin-arginine signal peptides were analyzed. Of which, eleven candidates were cloned for construction of expression vectors to mediate the expression of Chryseobacterium proteolyticum protein glutaminase (PGA). The signal peptide of GlmU was confirmed that it significantly mediated PGA secretion into media with the maximum activity of 0.16 U/ml in Bacillus subtilis WB600. A mutant GlmU-R, being replaced the third residue aspartic acid of GlmU twin-arginine signal peptide with arginine by site-directed mutagenesis, mediated the improved secretion of PGA with about 40% increased (0.23 U/ml). In B. licheniformis CBBD302, GlmU-R mediated PGA expression in active form with the maximum yield of 6.8 U/ml in a 25-l bioreactor. Conclusions: PGA can be produced and secreted efficiently in active form via Tat pathway of B. licheniformis, an alternative expression system for the industrial-scale production of PGA.

Mechanism-Guided Design of Highly Efficient Protein Secretion and Lipid Conversion for Biomanufacturing and Biorefining.

Bacterial protein secretion represents a significant challenge in biotechnology, which is essential for the cost-effective production of therapeutics, enzymes, and other functional proteins. Here, it is demonstrated that proteomics-guided engineering of transcription, translation, secretion, and folding of ligninolytic laccase balances the process, minimizes the toxicity, and enables efficient heterologous secretion with a total protein yield of 13.7 g L-1. The secretory laccase complements the biochemical limits on lignin depolymerization well in Rhodococcus opacus PD630. Further proteomics analysis reveals the mechanisms for the oleaginous phenotype of R. opacus PD630, where a distinct multiunit fatty acid synthase I drives the carbon partition to storage lipid. The discovery guides the design of efficient lipid conversion from lignin and carbohydrate. The proteomics-guided integration of laccase-secretion and lipid production modules enables a high titer in converting lignin-enriched biorefinery waste to lipid. The fundamental mechanisms, engineering components, and design principle can empower transformative platforms for biomanufacturing and biorefining.

Enhanced Bioactivity of the Anti-LOX-1 scFv Engineered by Multimerization Strategy.

Single-chain variable fragment (scFv) antibodies as therapeutic agents have the potential to reduce the production cost and immunogenicity relative to monoclonal antibodies, but their monovalency and lack of a fragment crystallizable region can lead to reduced function. Multimerization is one strategy for recovering the function; however, their application is limited by the production of multimeric proteins. In our previous study, an anti-lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) scFv showed potential use in diagnosis and therapy of atherosclerotic diseases, but is limited by its inherent low antigen-binding activity. In this study, to improve the efficacy of the anti-LOX-1 scFv, we constructed the anti-LOX-1 scFv multimers by modifying the linker length between the variable domains of the scFv or by fusing the scFv with self-merization domains and expressed these scFv multimers in Brevibacillus choshinensis hosts. After optimization, all of the scFv multimers obtained efficient secretion expression. Compared with the scFv monomer, the multimers that are successfully fractionated displayed increased neutralization activity and showed elevated antigen-binding avidity, especially the tetramer, which improved the antigen avidity by two orders of magnitude. Moreover, the scFv dimer and the tetramer both displayed better stability and longer half-life in serum, which can be attractive candidates for the next-generation anti-LOX-1 therapeutic antibody.

Secreted expression of truncated capsid protein from porcine circovirus type 2 in Pichia pastoris.

OBJECTIVE: To achieve secreted expression of the truncated capsid protein from porcine circovirus type 2 (PCV2) in Pichia pastoris. RESULTS: A truncated cap gene (tcap) with a deleted N-terminal nuclear localization signal was optimized and synthesized. Effective secreted expression was achieved in P. pastoris GS115. The high-productive recombinant strain for tCap was grown in a 5 l bioreactor and the productivity of tCap in supernatant reached 250 µg/ml. Furthermore, serum antibody test demonstrated that adjuvant-assisting tCap induced a significant increase of specific PCV2-Cap antibody over time in mice and a similar antibody level in pigs compared with a commercial Cap-based subunit vaccine. CONCLUSION: This work establishes a secreted expression strategy in P. pastoris for the production of PCV2 Cap with superior bioactivity, and this strategy might provide potential uses in developing Cap-based subunit vaccine in the future.

Construction and characterization of three protein-targeting expression system in Lactobacillus casei.

We previously reported that the ß-1,4-Mannanase (manB) gene from Bacillus pumilus functions as a good reporter gene in Lactobacillus casei. Two vectors were constructed. One carries the signal peptide of secretion protein Usp45 (SPUsp45) from Lactococcus lactis (pELSH), and the other carries the full-length S-layer protein, SlpA, from L. acidophilus (pELWH). In this work, another vector, pELSPH, was constructed to include the signal peptide of protein SlpA (SPSlpA), and the capacity of all three vectors to drive expression of the manB gene in L. casei was evaluated. The results showed that SPUsp45 is functionally recognized and processed by the L. casei secretion machinery. The SPUsp45-mediated secretion efficiency was ∼87%, and SPSlpA drove the export of secreted ManB with ∼80% efficiency. SPSlpA secretion was highly efficient, and expressed SlpA was anchored to the cell wall by an unknown secretion mechanism. Full-length SlpA drove the cell wall-anchored expression of an SlpA-ManB fusion protein but at a much lower level than that of protein SlpA.

Construction of a novel secretion expression system guided by native signal peptide of PhoD in Zymomonas mobilis.

In the current study, three native signal peptides (SPs) from PhoC, PhoD, and ZMO0331were investigated and compared to construct novel secretion expression systems in Zymomonas mobilis. The secretion expression of target protein, α-amylase from Bacillus amyloliquefaciens (BAA), guided by PhoD's SP resulted in more hydrolysis of starch than that by the other two SPs. Extracellular and intracellular α-amylase activities of the strain containing PhoD's SP were also higher than the other two strains containing PhoC or ZMO0331's SP. In addition, the evidence by alcohol dehydrogenase activity assay further confirmed that the starch hydrolysis was resulted from the secretion expression of BAA rather than the breakage of cells. Our results indicated that the SP of PhoD is able to serve as a promising candidate to assist secretion expression of heterogeneous genes in Z. mobilis. This will contribute to development of engineered Z. mobilis strains converting starch into ethanol.

High-level Secretion Expression of Human ScFv Against Botulinum Neurotoxin A in Pichia pastoris* / 微生物学通报

The specific ScFv gene against botulinum neurotoxin A (BoNTa)was cloned into pPIC9k. Positive integrators were screened by increasing the dose of G418 in culture and expressed in Pichia pastoris GS115. As a result, engineered recombinant clone were obtained. 26 kD product of interest was seen easily in SDS-PAGE. Expression of human ScFv got the highest level 15% of total secreted proteins during 72～84 h after 1% methanol inducing. Purification of ScFv was finished by two steps: gel filter and ion exchange. Competing ELISA showed that recombinant ScFv could compete with antiserum to specific bind BoNTa.