Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

Heparin is an anionic polysaccharide that is widely used as a clinical anticoagulant. This glycosaminoglycan is prepared from animal tissues in metric ton quantities. Animal‐sourced heparin is also widely used in the preparation of low molecular weight heparins that are gaining in popularity as a result of their improved pharmacological properties. The recent contamination of pharmaceutical heparin together with concerns about increasing demand for this life saving drug and the fragility of the heparin supply chain has led the scientific community to consider other potential sources for heparin. This review examines progress toward the preparation of engineered heparins through chemical synthesis, chemoenzymatic synthesis, and metabolic engineering.

Importance: Particulate respirators such as N95 masks are an essential component of personal protective equipment (PPE) for front-line workers. This study describes a rapid and effective UVC irradiation system that would facilitate the safe re-use of N95 respirators and provides supporting information for deploying UVC for decontamination of SARS-CoV-2 during the COVID19 pandemic. Objective: To assess the inactivation potential of the proposed UVC germicidal device as a function of time by using 3M 8211 - N95 particulate respirators inoculated with SARS-CoV-2. Design: A germicidal UVC device to deliver tailored UVC dose was developed and snippets (2.5cm2) of the 3M-N95 respirator were inoculated with 106 plaque-forming units (PFU) of SARS-CoV-2 and were UV irradiated. Different exposure times were tested (0-164 seconds) by fixing the distance between the lamp (10 cm) and the mask while providing an exposure of at least 5.43 mWcm-2. Setting: The current work is broadly applicable for healthcare-settings, particularly during a pandemic such as COVID-19. Participants: Not applicable. Main Outcome(s) and Measure(s): Primary measure of outcome was titration of infectious virus recovered from virus-inoculated respirator pieces after UVC exposure. Other measures included the method validation of the irradiation protocol, using lentiviruses (biosafety level-2 agent) and establishment of the germicidal UVC exposure protocol. Results: An average of 4.38x103 PFUml-1(SD 772.68) was recovered from untreated masks while 4.44x102 PFUml-1(SD 203.67), 4.00x102 PFUml-1(SD 115.47), 1.56x102 PFUml-1(SD 76.98) and 4.44x101 PFUml-1(SD 76.98) was recovered in exposures 2s,6s,18s and 54 seconds per side respectively. The germicidal device output and positioning was monitored and a minimum output of 5.43 mWcm-2 was maintained. Infectious SARS-CoV-2 was not detected by plaque assays (minimal level of detection is 67 PFUml-1) on N95 respirator snippets when irradiated for 120s per side or longer suggesting 3.5 log reduction in 240 seconds of irradiation. Conclusions and Relevance: A scalable germicidal UVC device to deliver tailored UVC dose for rapid decontamination of SARS-CoV-2 was developed. UVC germicidal irradiation of N95 snippets inoculated with SARS-CoV-2 for 120s per side resulted in 100% (3.5 log in total) reduction of virus. These data support the reuse of N95 particle-filtrate apparatus upon irradiation with UVC and supports use of UVC-based decontamination of SARS-CoV-2 virus during the COVID19 pandemic.

Isoflavonoids are a class of plant natural products gaining attention due to their pharmaceutical properties. These natural compounds constitute a subclass of flavonoids, which belong to a broader class of plant products known as phenylpropanoids.

No abstract