We demonstrate that short single-stranded DNA generated by Pyrococcus furiosus Argonaute (PfAgo) can initiate a second round of cleavage. Based on this principle, we established a molecular diagnostic method, termed PfAgo-mediated Nucleic acid Detection (PAND). This method could detect DNA at attomolar sensitivities, distinguish single-nucleotide mutants and accomplish multiplexed detection.

The large-scale preparation of Polyehylene terephthalate (PET) hydrolysing enzymes in low-cost is critical for the biodegradation of PET in industry. In the present study, we demonstrate that the post-translational glycosylation ofPichia pastorismakes it a remarkable host for the heterologous expression of PETase fromIdeonella sakaiensis201-F6 (IsPETase). Taking advantage of the abundant N- and O-linked glycosylation sites inIsPETase and the efficient post-translational modification in endoplasmic reticulum,IsPETase is heavily glycosylated during secretory expression withP. pastoris, which improves the specific activity and thermostability of the enzyme dramatically. Moreover, the specific activity ofIsPETase increased further after the bulky N-linked polysaccharide chains were eliminated by Endo-β-N-acetylglucosaminidase H (Endo H). Importantly, the partially deglycosylatedIsPETase still maintained high thermostability because of the remaining mono- and oligo-saccharide residues on the protein molecules. Consequently, the partially deglycosylatedIsPETase was able to be applied at 50 °C and depolymerized raw, untreated PET flakes completely in 2 to 3 days. This platform was also applied for the preparation of a famous variant ofIsPETase, Fast-PETase, and the same result was achieved. Partially deglycosylated Fast-PETase demonstrates elevated efficiency in degrading postconsumer-PET trays under 55 °C than 50 °C, the reported optimal temperature of Fast-PETase. The present study provides a strategy to modulate thermostableIsPETase through glycosylation engineering and paves the way for promoting PET biodegradation from laboratories to factories.

Background: Nattokinase is a fibrinolytic enzyme that has huge market value as a nutritional supplement for health promotion. In order to increase nattokinase yields, fermentation conditions, strains, cultivation media, and feeding strategies have been optimized. Nattokinase has been expressed using several heterologous expression systems. Pichia pastoris heterologous expression system was the alternative. Results: This report aimed to express high levels of nattokinase from B. subtilis natto (NK-Bs) using a Pichia pastoris heterologous expression system and assess its fibrinolytic activity in vivo. Multicopy expression strains bearing 1–7 copies of the aprN gene were constructed. The expression level of the target protein reached a maximum at five copies of the target gene. However, multicopy expression strains were not stable in shake-flask or high-density fermentation, causing significant differences in the yield of the target protein among batches. Therefore, P. pastoris bearing a single copy of aprN was used in shake-flask and high-density fermentation. Target protein yield was 320 mg/L in shake-flask fermentation and approximately 9.5 g/L in high-density fermentation. The recombinant nattokinase showed high thermo- and pH-stability. The present study also demonstrated that recombinant NK-Bs had obvious thrombolytic activity. Conclusions: This study suggests that the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of nattokinase.

The hygromycin phosphotransferase (HPT) gene as a selective marker is normally used in screening tests as a first step in detecting and quantifying genetically modified organisms (GMOs) in seeds, food, and feed materials. Nevertheless, if researchers only focus on the HPT gene, it is difficult to distinguish genetically modified (GM) crops from microbial infection, leading to miscalculation of the rate of GM materials in a given sample set. Here, we cloned the 7259 bp sequence carrying the HPT gene from soybean sprouts using the genome walking strategy. BLAST analysis revealed that this sequence was derived from plasmids naturally occurring in microorganisms, such as Escherichia coli, Klebsiella pneumoniae or Salmonella sp. Using the reconstructed plasmid pFP-hpt, qualitative PCR and quantitative real-time PCR (qPCR) methods were established, and 261 bp and 156 bp products were produced. The specificity of these assays was assessed against related pFP-hpt plasmids, plant species with important agronomic traits, and GM crops containing the HPT gene. No unexpected results were observed between samples using these qualitative PCR and qPCR methods. The sensitivity of this qualitative PCR assay was determined at 20 copies, while the limit of detection (LOD) and limit of quantification (LOQ) of qPCR were both 5 copies per reaction. Our in-house validation indicated that the amplification efficiency, linearity, and repeatability of this qPCR assay were in line with performance requirements. Furthermore, a qualitative and quantitative duplex PCR showed high reliability for the simultaneous detection of the HPT gene in a plant sample and environmental micro-organisms harboring the HPT gene in one PCR reaction. These qualitative PCR and qPCR assays were able to differentiate between plants infected with E. coli harboring the HPT gene from GM plants, indicating that these two methods are broadly applicable for routine GMO testing.

Background: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. Results: The sfGFP₍₋₁₅₎ mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn’t improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. Conclusion: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.