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The efficiency of whole-cell biotransformation is often affected by genetic instability of plasmid-based expression systems, which require selective pressure to maintain the stability of the plasmids. To circumvent this shortcoming, we constructed a chromosome engineering strain for synthesis of phenylpyruvic acid (PPA) from L-phenylalanine. Firstly, L-amino acid deaminase (pmLAAD) from Proteus myxofaciens was incorporated into E. coli BL21 (DE3) chromosome and the copy-numbers of pmLAAD were increased by chemically induced chromosomal evolution (CIChE). 59 copies of pmLAAD was obtained in E. coli BL8. The PPA titer of E. coli BL8 reached 2.22 g/L at 6 h. Furthermore, the deletion of lacI improved PPA production. In the absence of Isopropyl-β-D-thiogalactopyranoside (IPTG), the resulting strain, E. coli BL8△recA△lacI, produced 2.65 g/L PPA at 6 h and yielded a 19.37 % increase in PPA production compared to E. coli BL8△recA. Finally, the engineered E. coli BL8△recA△lacI strain achieved 19.14 g/L PPA at 24 h in 5 L bioreactor. The culture of the strain does not require the addition of antibiotics and inducers. The production level of CIChE strains can catch up with plasmid expression strains. This work extends production methods for whole-cell biotransformation. This article is protected by copyright. All rights reserved.

Phenyllactic acid (PLA) is a novel antimicrobial compound. A novel NADH‐dependent d‐lactate dehydrogenase (d‐LDH), named as LF‐d‐LDH0653, with high phenylpyruvate (PPA) reducing activity was isolated from Lactobacillus fermentumJN248. Its optimum pH and temperature were 8.0 and 50 °C, respectively. The Michaelis–Menten constant (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for NADH were 1.20 mmol/L, 67.39 s−1, and 56.16 (mmol/L)−1s−1, respectively. The (Km), (kcat), and (kcat/Km) for phenylpyruvate were 1.68 mmol/L, 122.66 s−1, and 73.01 (mmol/L)−1s−1, respectively. This enzyme can catalyze phenylpyruvate and the product presented excellent optical purity (enantioselectivity >99%). The results suggest that LF‐d‐LDH0653 is a promising biocatalyst for the efficient synthesis of optically pure d‐PLA. A novel d‐LDH with phenylpyruvate reducing activity has been isolated and identified. It could be used as a reference for improving the production of optically pure d‐PLA. d‐PLA has a potential for application as antimicrobial an agent in dairy industry and baking industry, pharmaceutical agent in medicine and cosmetics.

Isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxypropanoate (IDHP) is an investigational new drug having the capacity for treating ailments in cardiovascular and cerebrovascular system. In this work, a rapid and sensitive method using high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (HPLC-ESI-Q-TOF-MS) was developed to reveal the metabolic profile of IDHP in rats after oral administration. The method involved pretreatment of the samples by formic acid-methanol solution (v:v, 5:95), chromatographic separation by an Agilent Eclipse XDB-C18 column (150 × 4.6 mm i.d., 5 µm) and online identification of the metabolites by Q-TOF-MS equipped with electrospray ionizer. A total of 16 metabolites from IDHP, including 4 phase I metabolites and 12 phase II metabolites, were detected and tentatively identified from rat plasma, urine and feces. Among these metabolites, Danshensu (DSS), a hydrolysis product of IDHP, could be further transformed to 11 metabolites. These results indicated that DSS was the main metabolite of IDHP in rats and the major metabolic pathways of IDHP in vivo were hydrolysis, O-methylation, sulfation, glucuronidation and reduction. The results also demonstrated that renal route was the main pathway of IDHP clearance in rat. The present study provided valuable information for better understanding the efficacy and safety of IDHP. This article is protected by copyright. All rights reserved.

Background and Purpose Cardiac fibrosis is a common feature of advanced coronary heart disease and is characteristic of heart disease. However, currently available drugs against cardiac fibrosis are still very limited. Here, we have assessed the role of isopropyl 3-(3,4-dihydroxyphenyl)-2-hydroxylpropanoate (IDHP), a new metabolite of Danshen Dripping Pills, in cardiac fibrosis mediated by the β-adrenoceptor agonist, isoprenaline, and its underlying mechanisms. Experimental Approach Identification of IDHP was identified by mass spectrometry, and proton and carbon nuclear magnetic resonance spectra. Myocardial collagen was quantitatively assessed with Picrosirius Red staining. Expression of mRNA for collagen was evaluated with real-time PCR. Phosphorylated and total p38 MAPK, NADPH oxidase (NOX) and superoxide dismutase (SOD) were analysed by Western blot. Generation of reactive oxygen species (ROS) generation was evaluated by dihydroethidium (DHE) fluorescent staining. NOX2 was knocked down using specific siRNA. Key Results IDHP attenuated β-adrenoceptor mediated cardiac fibrosis in vivo and inhibited isoprenaline-induced proliferation of neonatal rat cardiac fibroblasts (NRCFs) and collagen I synthesis in vitro. Phosphorylation of p38 MAPK, which is an important mediator in the pathogenesis of isoprenaline-induced cardiac fibrosis, was inhibited by IDHP. This inhibition of phospho-p38 by IDHP was dependent on decreased generation of ROS. These effects of IDHP were abolished in NRCFs treated with siRNA for NOX2. Conclusions and Implications IDHP attenuated the cardiac fibrosis induced by isoprenaline through a NOX2/ROS/p38 pathway. These novel findings suggest that IDHP is a potential pharmacological candidate for the treatment of cardiac fibrosis, induced by β-adrenoceptor agonists. Linked Articles This article is part of a themed section on Chinese Innovation in Cardiovascular Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-23

Simple and inexpensive copper-mediated N-arylation ofheteroarylamines was achieved by using N,N′-dimethylethylenediamine as a ligand and K2CO3 as a base in dioxane heated at 100 °C. In this coupling reaction, the influence of the copper species, ligand, base and solvent was investigated in detail. N-Arylated derivatives of several heteroarylamines were synthesized under optimized reaction conditions, and all the products were isolated in good yields. By controlling the amount of CuI/DMEDA added, heteroarylamines with weak nucleophilic activity were coupled with aryl iodides or aryl bromides. The activity of the copper catalyst for this C–N bond-forming reaction follows the order CuI > Cu0 > CuII. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)