l-Aspartate is an important chemical in the food and pharmaceutical industries. Herein, a dual-enzyme system was constructed to synthesize l-aspartate from maleic anhydride at 50 °C, which can reduce the byproduct production. Maleate transformed from maleic anhydride in the solution was converted into l-aspartate via fumarate catalyzed by maleate isomerase (MaiA) and thermostable aspartase (AspB), respectively. Because MaiA is a rate-limiting enzyme, enzyme activities of various MaiAs were compared, and the efficient and thermostable maleate isomerase AaMaiA from Alicyclobacillus acidoterrestris was chosen. The K cat/K m value of AaMaiA was 264.4 mM–1 min–1. AaMaiA and AspB were coexpressed in E. coli to produce l-aspartate. To improve the l-aspartate production rate, the ribosome binding site (RBS) sequence located upstream of AaMaiA was optimized and the Tat signal peptide was fused with AaMaiA. The conversion rate was 96% within 60 min, and the intermediate was not detected, the possible reason of which is that high temperature inhibits the activity of bacterial endogenous enzymes, but functional enzymes remain active. Cells from fermentation produced 243.6 g/L (1.83 M) of l-aspartate with a 2 M substrate. Our study revealed an effective method to produce l-aspartate without using gene knockout and provided a strategy for l-aspartate production in the industrial field.

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant, Due to the large amount of use, its presence in the environment threatens to human health. However, the molecular mechanism of pretilachlor degradation is unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and found to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, 50 mg/L initial substrate, pH 6.98, and 30.1°C. One novel metabolite was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, comprising a gene cluster (EthRABCDB2), was absent in the mutant strain TB2 which could not degrade pretilachlor. The Eth gene system, encodes an AraC/XylS family transcriptional regulator, a ferredoxin reductase, a cytochrome P-450 monooxygenase, a ferredoxin and a 10-kDa unknown protein. Complementation of EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its activity against chloroacetamide herbicides. The codon of EthABCDB2 was optimized, expressed in Escherichia coli, and purified utilizing Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation activity toward alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation activity toward pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation or N-dealkoxymethylation of pretilachlor and propisochlor. And the results provide microbial resource for the remediation of chloroacetamide herbicides-contaminated sites,

Background: The chloroacetamide herbicides pretilachlor is an emerging pollutant. Due to the large amount of use, its presence in the environment threatens human health. However, the molecular mechanism of pretilachlor degradation remains unknown. Results: Now, Rhodococcus sp. B2 was isolated from rice field and shown to degrade pretilachlor. The maximum pretilachlor degradation efficiency (86.1%) was observed at a culture time of 5 d, an initial substrate concentration 50 mg/L, pH 6.98, and 30.1°C. One novel metabolite N-hydroxyethyl-2-chloro-N-(2, 6-diethyl-phenyl)-acetamide was identified by gas chromatography-mass spectrometry (GC-MS). Draft genome comparison demonstrated that a 32,147-bp DNA fragment, harboring gene cluster (EthRABCDB2), was absent from the mutant strain TB2 which could not degrade pretilachlor. The Eth gene cluster, encodes an AraC/XylS family transcriptional regulator (EthRB2), a ferredoxin reductase (EthAB2), a cytochrome P450 monooxygenase (EthBB2), a ferredoxin (EthCB2) and a 10-kDa protein of unknown function (EthDB2). Complementation with EthABCDB2 and EthABDB2, but not EthABCB2 in strain TB2 restored its ability to degrade chloroacetamide herbicides. Subsequently, codon optimization of EthABCDB2 was performed, after which the optimized components were separately expressed in Escherichia coli, and purified using Ni-affinity chromatography. A mixture of EthABCDB2 or EthABDB2 but not EthABCB2 catalyzed the N-dealkoxymethylation of alachlor, acetochlor, butachlor, and propisochlor and O-dealkylation of pretilachlor, revealing that EthD acted as a ferredoxin in strain B2. EthABDB2 displayed maximal activity at 30 °C and pH 7.5. Conclusions: This is the first report of a P450 family oxygenase catalyzing the O-dealkylation and N-dealkoxymethylation of pretilachlor and propisochlor, respectively. And the results of the present study provide a microbial resource for the remediation of chloroacetamide herbicides-contaminated sites.

Background: To establish an animal model of the minipigs of ileal ureter-bladder anastomosis and ureteral-ureteral anastomosis using the Yang-Monti technique, and compare the advantages and disadvantages. Methods: 14 minipigs were randomly divided into two groups. Group A received the left Yang-Monti ileal ureter-ureteral anastomosis ; group B received the left Yang-Monti ileal ureter-bladder anastomosis. The length of the incision and the operation time of the two groups were compared. Changes in serum creatinine and urea nitrogen were observed. IVU,cystography,Urinary culture examination and histological examination were performed. Results: the remaining 11 miniature pigs had no urinary fistula or intestinal fistula.The operation time and incision length of group A were longer than group B. All minipigs had normal renal function before and after surgery. There was no stenosis obstruction in the venous pyelography. The narrowest diameter of group A was larger than that of group B; no contrast agent was returned to the upper urinary tract in the two groups. Postoperative urine culture examinations were colonized with Escherichia coli. the obstruction and stenosis were found in group A; the ileal ureteral diameter of group A was larger than that of group B. Histological examination showed that the transitional epithelium and columnar epithelial mucosa in group A were not close together; the intestinal villi are slightly atrophied and shortened. Conclusions: Compared with the Yang-Monti ileal ureteral anastomosis, the Yang-Monti ileal ureter-bladder anastomosis is simpler, more reliable, and less complication.

Background: To establish an animal model of the minipigs of ileal ureter-bladder anastomosis and ureteral-ureteral anastomosis using the Yang-Monti technique, and compare the advantages and disadvantages. Methods: 14 minipigs were randomly divided into two groups. Group A received the left Yang-Monti ileal ureter-ureteral anastomosis ; group B received the left Yang-Monti ileal ureter-bladder anastomosis. The length of the incision and the operation time of the two groups were compared. Changes in serum creatinine and urea nitrogen were observed. IVU,cystography,Urinary culture examination and histological examination were performed. Results: the remaining 11 miniature pigs had no urinary fistula or intestinal fistula.The operation time and incision length of group A were longer than group B. All minipigs had normal renal function before and after surgery. There was no stenosis obstruction in the venous pyelography. The narrowest diameter of group A was larger than that of group B; no contrast agent was returned to the upper urinary tract in the two groups. Postoperative urine culture examinations were colonized with Escherichia coli. the obstruction and stenosis were found in group A; the ileal ureteral diameter of group A was larger than that of group B. Histological examination showed that the transitional epithelium and columnar epithelial mucosa in group A were not close together; the intestinal villi are slightly atrophied and shortened. Conclusions: Compared with the Yang-Monti ileal ureteral anastomosis, the Yang-Monti ileal ureter-bladder anastomosis is simpler, more reliable, and less complication.