Improved cryptic plasmids in probiotic Escherichia coli Nissle 1917 for antibiotic-free pathway engineering.

The engineered probiotic Escherichia coli Nissle 1917 (EcN) is expected to be employed in the diagnosis and treatment of various diseases. However, the introduced plasmids typically require antibiotics to maintain genetic stability, and the cryptic plasmids in EcN are usually eliminated to avoid plasmid incompatibility which may change the inherent probiotic characteristics. Here, we provided a simple design to minimize the genetic change of probiotics by eliminating native plasmids and reintroducing the recombinants carrying functional genes. Specific insertion sites in the vectors showed significant differences in the expression of fluorescence proteins. Selected integration sites were applied in the de novo synthesis of salicylic acid, leading to a titer of 142.0 ± 6.0 mg/L in a shake flask with good production stability. Additionally, the design successfully realized the biosynthesis of ergothioneine (45 mg/L) by one-step construction. This work expands the application scope of native cryptic plasmids to the easy construction of functional pathways. KEY POINTS: • Cryptic plasmids of EcN were designed to express exogenous genes • Insertion sites with different expression intensities in cryptic plasmids were provided • Target products were stably produced by engineering cryptic plasmids.

[Effect of electroacupuncture on expression of hippocampal Caspase-3 in rats with cerebral ischemia/reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the expression level of Caspase-3, so as to explore its mechanism in inhibiting apoptosis after cerebral ischemia reperfusion. METHODS: SD male rats were randomly divided into sham-operation, model, EA and Caspase-3 inhibitor groups (n=20 rats in each group). The focal cerebral ischemia/reperfusion injury rat model was established by occlusion of the middle cerebral artery. Rats of the EA group received EA at "Hegu" (LI4), "Chize" (LU5), "Zusanli" (ST36) and "Sanyinjiao" (SP6) on the affected side for 20 min. Rats of the inhibitor group were given intracerebroventricular injection of inhibitor Z-DEVD-FMK 5 µg before modeling. The neurological deficit scores (NDS) were assessed by using Longa's method, the infarct size of the brain assessed after staining with 2% triphenyltetrazolium chloride. The apoptosis index of nerve cells were observed by TUNEL staining, PCR and Western blot were used to detect the mRNA and protein expressions of Caspase-3 in the hippocampus, separately. RESULTS: After modeling, the NDS, infarct volume, the apoptosis index of hippocampus CA1 area, and Caspase-3 mRNA and protein expression levels were significantly increased in the model group compared with the sham-operation group (P<0.01). After intervention, the NDS, infarct volume, the apoptosis index, Caspase-3 mRNA and protein expression levels were all significantly decreased in the EA and Caspase-3 inhibitor groups re-levant to the model group (P<0.05). CONCLUSION: EA can improve the neurological function in cerebral ischemia/reperfusion rats, which may be related to its effect in inhibiting of Caspase-3 expression.

Mechanism of Electroacupuncture Against Cerebral Ischemia-Reperfusion Injury: Reducing Inflammatory Response and Cell Pyroptosis by Inhibiting NLRP3 and Caspase-1.

Cell pyroptosis is one of the main forms of neuronal injury after cerebral ischemia-reperfusion. It is accompanied by an inflammatory reaction and regulated by the caspase gene family. Electroacupuncture (EA) can reduce neuronal injury caused by cerebral ischemia-reperfusion, and we speculated that EA can prevent neuronal pyroptosis after cerebral ischemia-reperfusion by regulating the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/caspase-1 pathway. The cerebral ischemia-reperfusion injury model of C57 and caspase-1 gene knockout (Cas-1 ko) mice was established by Longa's method. EA was conducted at acupoints Chize (LU5), Hegu (LI4), Sanyinjiao (SP6), and Zusanli (ST36) for 1.5 h after cerebral ischemia-reperfusion injury for 20 min, and observation was carried out after 24 h. Neurological deficit scores evaluated the neurological function, cerebral infarction volume was observed by triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, TUNEL and caspase-1 double-labeled fluorescence staining, and NLRP3 and caspase-1 double-labeled immunofluorescence staining that were used to observe the morphology of neurons in hippocampus, and the protein expression of NLRP3, pro-caspase-1, cleaved caspase-1 p20, pro-interleukin-1ß (IL-1ß), cleaved IL-1ß, and GSDMD was detected by Western blot assay. Results showed that EA could reduce the score of neurological deficit, reduce the volume of cerebral infarction and improve the degree of nerve cell injury, and inhibit NLRP3, pro-caspase-1, cleaved caspase-1 p20, pro-IL-1ß, cleaved IL-1ß, and GSDMD protein expression. In summary, EA plays a neuroprotective role by reducing the pyroptotic neurons that were caspase 1-mediated and inflammatory response after cerebral ischemia-reperfusion.

Improving the biotransformation efficiency of soybean phytosterols in Mycolicibacterium neoaurum by the combined deletion of fbpC3 and embC in cell envelope synthesis.

Biotransformation of soybean phytosterols into 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) by mycobacteria is the core step in the synthesis of adrenocortical hormone. However, the low permeability of the dense cell envelope largely inhibits the overall conversion efficiency of phytosterols. The antigen 85 (Ag85) complex encoded by fbpA, fbpB, and fbpC was proposed as the key factor in the combined catalysis of mycoloyl for producing mycolyl-arabinogalactan (m-AG) and trehalose dimycolate (TDM) in mycobacterial cell envelope. Herein, we confirmed that fbpC3 was essential for the biotransformation of trehalose monomycolate (TMM) to TDM in Mycolicibacterium neoaurum. The deficiency of this gene raised the cell permeability, thereby enhancing the steroid uptake and utilization. The 9-OHAD yield in the fbpC3-deficient 9-OHAD-producing strain was increased by 21.3%. Moreover, the combined deletion of fbpC3 and embC further increased the 9-OHAD yield compared to the single deletion of fbpC3. Finally, after 96 h of bioconversion in industrial resting cells, the 9-OHAD yield of 11.2 g/L was achieved from 20 g/L phytosterols and the productivity reached 0.116 g/L/h. In summary, this study suggested the critical role of the fbpC3 gene in the synthesis of TDM in M. neoaurum and verified the feasibility of improving the bioconversion efficiency of phytosterols through the cell envelope engineering strategy.