Heterologous production of active form of beta-lytic protease by Bacillus subtilis and improvement of staphylolytic activity by protein engineering

Background Most of the proteases classified into the M23 family in the MEROPS database exhibit staphylolytic activity and have potential as antibacterial agents. The M23 family is further classified into two subfamilies, M23A and M23B. Proteases of the M23A subfamily are thought to lack the capacity for self-maturation by auto-processing of a propeptide, which has been a challenge in heterologous production and application research. In this study, we investigated the heterologous expression, in Bacillus subtilis, of the Lysobacter enzymogenes beta-lytic protease (BLP), a member of the M23A subfamily. Results We found that B. subtilis can produce BLP in its active form. Two points were shown to be important for the production of BLP in B. subtilis. The first was that the extracellular proteases produced by the B. subtilis host are essential for BLP maturation. When the host strain was deficient in nine extracellular proteases, pro-BLP accumulated in the supernatant. This observation suggested that BLP lacks the capacity for self-maturation and that some protease from B. subtilis contributes to the cleavage of the propeptide of BLP. The second point was that the thiol-disulfide oxidoreductases BdbDC of the B. subtilis host are required for efficient secretory production of BLP. We infer that intramolecular disulfide bonds play an important role in the formation of the correct BLP conformation during secretion. We also achieved efficient protein engineering of BLP by utilizing the secretory expression system in B. subtilis. Saturation mutagenesis of Gln116 resulted in a Q116H mutant with enhanced staphylolytic activity. The minimum bactericidal concentration (MBC) of the wild-type BLP and the Q116H mutant against Staphylococcus aureus NCTC8325 was 0.75 μg/mL and 0.375 μg/mL, respectively, and the MBC against Staphylococcus aureus ATCC43300 was 6 μg/mL and 3 μg/mL, respectively. Conclusions In this study, we succeeded in the secretory production of BLP in B. subtilis. To our knowledge, this work is the first report of the successful heterologous production of BLP in its active form, which opens up the possibility of industrial use of BLP. In addition, this study proposes a new strategy of using the extracellular proteases of B. subtilis for the maturation of heterologous proteins.

A hemolysin secretion pathway-based novel secretory expression platform for efficient manufacturing of tag peptides and anti-microbial peptides in Escherichia coli

Background Although Escherichia coli has been widely used for the expression of exogenous proteins, the secretory expression in this system is still a big obstacle. As one of the most important secretion pathways, hemolysin A (HlyA) system of E. coli can transport substrates directly from the cytoplasm to extracellular medium without the formation of any periplasmic intermediate, making it an ideal candidate for the development of the secretory production platform for exogenous proteins. Results In this work, we developed a novel production platform, THHly, based on the HlyA secretion system, and explored its applications in the efficient preparation and quick detection of tag peptides and anti-microbial peptides. In this novel platform the signal sequence of HlyA is fused to the C-terminal of target peptide, with Tobacco Etch Virus (TEV) protease cleavage site and 6*His tag between them. Five tag peptides displayed good secretory properties in E. coli BL21 (DE3), among which T7 tag and S tag were obtained by two rounds of purification steps and TEV cleavage, and maintained their intrinsic immunogenicity. Furthermore, Cecropin A and Melittin, two different types of widely explored anti-microbial peptides, were produced likewise and verified to possess anti-microbial/anti-tumor bioactivities. No significant bacterial growth inhibition was observed during the fusion protein expression, indicating that the fusion form not only mediated the secretion but also decreased the toxicity of anti-microbial peptides (AMPs) to the host bacteria. To the best of our knowledge, this is the first report to achieve the secretory expression of these two AMPs in E. coli with considerable potential for manufacturing and industrialization purposes. Conclusions The results demonstrate that the HlyA based novel production platform of E. coli allowed the efficient secretory production and purification of peptides, thus suggesting a promising strategy for the industrialized production of peptide pharmaceuticals or reagents. Graphical Abstract

Microbial Production of Biodegradable Lactate-Based Polymers and Oligomeric Building Blocks From Renewable and Waste Resources

Polyhydroxyalkanoates (PHAs) are naturally occurring biopolymers produced by microorganisms. PHAs have become attractive research biomaterials in the past few decades owing to their extensive potential industrial applications, especially as sustainable alternatives to the fossil fuel feedstock-derived products such as plastics. Among the biopolymers are the bioplastics and oligomers produced from the fermentation of renewable plant biomass. Bioplastics are intracellularly accumulated by microorganisms as carbon and energy reserves. The bioplastics, however, can also be produced through a biochemistry process that combines fermentative secretory production of monomers and/or oligomers and chemical synthesis to generate a repertoire of biopolymers. PHAs are particularly biodegradable and biocompatible, making them a part of today’s commercial polymer industry. Their physicochemical properties that are similar to those of petrochemical-based plastics render them potential renewable plastic replacements. The design of efficient tractable processes using renewable biomass holds key to enhance their usage and adoption. In 2008, a lactate-polymerizing enzyme was developed to create new category of polyester, lactic acid (LA)–based polymer and related polymers. This review aims to introduce different strategies including metabolic and enzyme engineering to produce LA-based biopolymers and related oligomers that can act as precursors for catalytic synthesis of polylactic acid. As the cost of PHA production is prohibitive, the review emphasizes attempts to use the inexpensive plant biomass as substrates for LA-based polymer and oligomer production. Future prospects and challenges in LA-based polymer and oligomer production are also highlighted.

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.