Combinatorial optimization and spatial remodeling of CYPs to control product profile.

Activating inert substrates is a challenge in nature and synthetic chemistry, but essential for creating functionally active molecules. In this work, we used a combinatorial optimization approach to assemble cytochrome P450 monooxygenases (CYPs) and reductases (CPRs) to achieve a target product profile. By creating 110 CYP-CPR pairs and iteratively screening different pairing libraries, we demonstrated a framework for establishing a CYP network that catalyzes six oxidation reactions at three different positions of a chemical scaffold. Target product titer was improved by remodeling endoplasmic reticulum (ER) size and spatially controlling the CYPs' configuration on the ER. Out of 47 potential products that could be synthesized, 86% of the products synthesized by the optimized network was our target compound quillaic acid (QA), the aglycone backbone of many pharmaceutically important saponins, and fermentation achieved QA titer 2.23 g/L.

The formation process and mechanism of gastrointestinal anastomosis after retrieval anchor-assisted endoscopic ultrasound-guided gastroenterostomy: a preclinical study.

BACKGROUND AND AIMS: Endoscopic ultrasound-guided gastroenterostomy (EUS-GE) is a promising method of treating gastric outlet obstruction. However, no study has investigated gastrointestinal anastomosis formation after EUS-GE. We aimed to evaluate the formation of gastrointestinal anastomosis after EUS-GE in a porcine model. METHODS: Retrieval anchor-assisted EUS-GE was performed on 15 Bama mini pigs. Five pigs each were randomly euthanized 3, 7, and 14 days postoperatively to evaluate the formation of gastrointestinal anastomosis and measure the anastomotic distance. The expression of transforming growth factor-ß1(TGF-ß1) and Smad3 in the anastomosis site were examined by immunohistochemistry. RESULTS: EUS-GE was successfully performed in all 15 pigs. The mean procedure time was 29.2 ± 6.0 (range 18-40) minutes. The anastomotic distance was 34.0 ± 3.6 cm in 14 pigs. The site of gastroenterostomy of one pig was at the ileum. For pigs euthanized 3 days postoperatively, the structure was mechanically maintained by a stent. For pigs euthanized 7 or 14 days postoperatively, the stomach and small intestine were anastomosed to form a stable structure. The level of TGF-ß1 and Smad3 in the anastomosis site gradually increased from 3 to 14 days after EUS-GE. TGF-ß1 and Smad3 expression had a significant difference between 3 days, 7 days, and 14 days after EUS-GE (P < 0.05). CONCLUSIONS: For EUS-GE, the stomach and small intestine were initially linked together mechanically and spontaneously anastomosed to form a stable structure 7 days postoperatively. TGF-ß1 and Smad3 play an important role in the formation of a stable structure of gastrointestinal anastomosis.

Efficient Synthesis of Phycocyanobilin by Combinatorial Metabolic Engineering in Escherichia coli.

Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD+ kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO4·7H2O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-ß-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO4·7H2O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.

Double anchor lock fixing method to prevent stent displacement in endoscopic ultrasound-guided gastroenterostomy: a porcine study.

BACKGROUND: Endoscopic ultrasound-guided gastroenterostomy (EUS-GE) is widely used in patients with gastric outlet obstruction (GOO). Recurrence of obstruction symptoms caused by stent migration is the major reason for reintervention in EUS-GE. To solve this problem, we proposed developing a double anchor lock (DAL) fixing stent method for EUS-GE. The safety and efficacy of the DAL fixing stent method were evaluated in this study. METHODS: Sixteen Bama miniature pigs were randomly divided into an experimental group (n = 8) and a control group (n = 8). A gastric outlet obstruction model was established for all the pigs. The experimental group was treated with the DAL fixing stent method for EUS-GE, while the control group was treated with EUS-GE. Three-month stent migration rate, remission rate of GOO, re-occlusion intervention rate, weight change, and incidence of procedure-related complications of EUS-GE were analyzed and estimated in the two groups. RESULTS: EUS-GE was successfully completed in every subject in both groups. Symptoms of digestive tract obstruction can be relieved in all animals. The stent existence rate in the 3 months was higher in the experimental group than in the control group (87.5% vs. 12.5%, P = 0.012). Except for one animal in the experimental group in which there was pneumoperitoneum due to a stent insertion failure, no animal experienced bleeding or perforation. CONCLUSION: The DAL fixing stent method, which can effectively prevent stent migration, is safe and simple. EUS-GE can effectively relieve the symptoms of digestive tract obstruction.