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

Directed evolution and secretory expression of xylose isomerase for improved utilisation of xylose in Saccharomyces cerevisiae

Background Xylose contained in lignocellulosic biomass is an attractive carbon substrate for economically viable conversion to bioethanol. Extensive research has been conducted on xylose fermentation using recombinant Saccharomyces cerevisiae expressing xylose isomerase (XI) and xylose reductase/xylitol dehydrogenase (XR/XDH) pathways along with the introduction of a xylose transporter and amplification of the downstream pathway. However, the low utilization of xylose in the presence of glucose, due to the varying preference for cellular uptake, is a lingering challenge. Studies so far have mainly focused on xylose utilization inside the cells, but there have been little trials on the conversion of xylose to xylulose by cell before uptake. We hypothesized that the extracellular conversion of xylose to xylulose before uptake would facilitate better utilization of xylose even in the presence of glucose. To verify this, XI from Piromyces sp. was engineered and hyper-secreted in S. cerevisiae for the extracellular conversion of xylose to xylulose. Results The optimal pH of XI was lowered from 7.0 to 5.0 by directed evolution to ensure its high activity under the acidic conditions used for yeast fermentation, and hyper-secretion of an engineered XI-76 mutant (E56A and I252M) was accomplished by employing target protein-specific translational fusion partners. The purified XI-76 showed twofold higher activity than that of the wild type at pH 5. The secretory expression of XI-76 in the previously developed xylose utilizing yeast strain, SR8 increased xylose consumption and ethanol production by approximately 7–20% and 15–20% in xylose fermentation and glucose and xylose co-fermentation, respectively. Conclusions Isomerisation of xylose to xylulose before uptake using extracellular XI was found to be effective in xylose fermentation or glucose/xylose co-fermentation. This suggested that glucose competed less with xylulose than with xylose for uptake by the cell. Consequently, the engineered XI secretion system constructed in this study can pave the way for simultaneous utilization of C5/C6 sugars from the sustainable lignocellulosic biomass.

Coordinately Express Hemicellulolytic Enzymes in Kluyveromyces Marxianus to Improve the Saccharification and Ethanol Production from Corncobs

BackgroundHemicelluloses act as one factor contributing to the recalcitrance of lignocelluloses that prevent cellulases to degrade the cellulose efficiently even in low quantities. Supplement of hemicellulases can enhance performance of commercial cellulases in the enzymatic hydrolyses of lignocellulose. Kluyveromyce marxianu is an attractive yeast for cellulosic ethanol fermentation, as well as a promising host for heterologous protein production, since it has remarkable thermotolerance, high growth rate, and broad substrate spectrum etc. In this study, we attempted to coordinately express multiple hemicellulases in K. marxianus through a 2A-mediated ribosomes skipping to self-cleave polyproteins, and investigated their capabilities for saccharification and ethanol production from corncobs.ResultsTwo polycistronic genes IMPX and IMPαX were constructed to test the self-cleavage of P2A sequence from Foot and Mouth Disease virus (FMDV) in K. marxianus. The IMPX gene consisted of a β-mannanase gene M330 (without the stop codon), a P2A sequence and a β-xylanase gene Xyn-CDBFV in turn, while in the IMPαX gene there was an additional α-factor signal sequence in frame with the N-terminus of Xyn-CDBFV. The extracellular β-mannanase activities of IMPX and IMPαX strains were 21.34 and 15.50 U/mL repectively. By contrast, the IMPαX strain secreted 136.17 U/mLof the β-xylanase, which was much higher than that of IMPX strain 42.07 U/mL. Based on these, two recombinant strains, the IXαR and IMPαXPαR, were constructed to coordinately and secretorily express two xylantic enzymes a β-D-xylosidase RuXyn1 and Xyn-CDBFV, or three hemicellulolytic enzymes including M330, Xyn-CDBFV and RuXyn1. In fed-batch fermentations, extracellular activities of β-xylanase and β-xylosidase in the IMPαX strain were 1664.2 and 0.90 U/mL, while productions of secretory β-mannanase, β-xylanase, and β-xylosidase in the IMPαXPαR strain were 159.8, 2210.5, and 1.25 U/ml of respectively. Hemicellulolytic enzymes of these two strains enhanced the yields of both glucose and xylose from diluted acid pretreated (DAP) corncobs when acted synergistically with commercial cellulases. In hybrid saccharification and fermentation (HSF) of DAP corncobs, hemicellulases of the IMPαXPαR strain increased the ethanol yields by 8.7% at 144 h. When using aqueous ammonia pretreated (AAP) corncobs as HSF feedstocks, the IMPαXPαR strain increased both ethanol and xylose yields, which were about 12.7% and 18.2% more than that of the control at 120 h. Our results indicated that coordinately expression of hemicellulolytic enzymes in K. marxianus could promote the saccharification and ethanol production from corncobs.ConclusionsThe FMDV P2A sequence showed high efficiency in self-cleavage of polyproteins in K. marxianus, and could be used for secretory expression of multiple enzymes in present of their own signal sequences. The IMPαXPαR strain that coexpressed three hemicellulolytic enzymes improved the saccharification and ethanol production from corncobs, and could be used as a promising strain for ethanol production from lignocelluloses.

Cloning and secretory expression of functional diisopropyl-fluorophosphatase (DFPase) in Bacillus subtilis

Background and aims: Synthetic organophosphates (OPs) inhibit acetylcholinesterase resulting in the accumulation of acetylcholine, failure of organs, and eventually death. Diisopropyl-fluorophosphatase (DFPase) is one of the OPs degrading enzymes that has broad substrate from OPs. In this study, for the first time, the secretory expression of DFPase in Bacillus subtilis was investigated in order to accelerate the biodegradation rate of OPs. Methods: DFPase gene was amplified using polymerase chain reaction (PCR) from the pET28-inaV/N-dfpase plasmid. The PCR product was subcloned in the pWB980 plasmid. Competent B. subtilis WB600 were transformed with recombinant plasmid. SDS PAGE technique was used to study the expression of protein secreted in superrich medium. Results: Appearance of the 946 bp band in agarose gel after digestion of transformed plasmid confirmed the presence of DFPase gene in this construct. Approximately, 35 kDa protein band was shown in culture medium after incubating at 35°C for 72 hours and 150 rpm. Measurement of enzyme’s activity was done by monitoring the release of fluoride from diisopropyl fluorophosphate (DFP), using ion-meter. Results showed that enzyme’s activity was 3333 U/L. Conclusion: Bacillus subtilis is a suitable host for production of secretory and active form of DFPase.