The current research aims to develop a new analytical method applying a dispersive liquid-liquid microextraction (DLLME) assisted by vortex and using an environmentally friendly extractant for the preconcentration of organochlorine and organophosphorus pesticides followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis. The extractant (i.e., natural deep eutectic solvent (NADES)) is safe, cheap, biodegradable and can be prepared by simply mixing DL-menthol and decanoic acid (molar ratio 2:1). The main experimental factors affecting the extraction of all analytes evaluated (19 organochlorine and organophosphorus pesticides) have been optimised using a multivariate analysis consisting in two steps: a Plackett-Burman design followed by a central composite design (CCD). Seven experimental factors have been evaluated: (i) sample volume; (ii) NADES volume; (iii) sample pH; (iv) extraction time; (v) centrifugation time; (vi) centrifugation speed; and (vii) ionic strength (NaCl %, w v^-1). For the significant variables, the optimum values were 10 mL sample and 45 μL NADES. No pH adjustment as well as addition of NaCl were needed. The other variables were set at 3 min extraction time, 5 min centrifugation time and 900×g centrifugation speed, respectively. Under the optimised extraction conditions, the limit of quantification (LOQ) values ranged between 0.2 and 78 ng L^-1 for all analysed pesticides. Furthermore, the proposed analytical method has been successfully applied to drinking water (bottled spring water). The recovery study (n = 3) has been evaluated at 0.1, 1.0 and 5.0 μg L^-1 spiking levels, obtaining relative recovery values within the range of 70 % and 117 % and RSD values between 1 % and 20 % for all the analytes studied, except for p,p-DDT (56-77 % in high conductivity water samples).

2024-11-15·ACS Synthetic Biology

Reconfiguring the Escherichia coli Electron Transport Chain to Enhance trans-2-Decenoic Acid Production

Article

作者: Su, Jing ; Su, ChunLi ; Wang, Junqing ; Li, Piwu ; ShangGuan, SiFan ; Zhang, YiSang ; Wang, HaoYang ; Wang, Ruiming ; Liu, Ben ; Nie, ShiHao

trans-2-Decenoic acid is a pivotal α,β-medium-chain unsaturated fatty acid that serves as an essential intermediary in the synthesis of 10-hydroxy-2-decenoic acid and various pharmaceutical compounds. Biosynthesis yield of trans-2-decenoic acid by decanoic acid has significantly improved in recent years; however, the oxidative stress of Escherichia coli at high fatty acid concentrations restricts the conversion rate. Here, we introduced a combination of rational design and metabolic rewiring of the E. coli electron transport chain (ETC) to improve trans-2-decenoic acid production. Overexpressing ubiquinone (UbQ) biosynthesis genes enhanced the expression of ETC complex III: UbQ to reduce reactive oxygen species (ROS) accumulation. Furthermore, applying rotenone to inhibit ETC complex I improved the electron transfer efficiency of complex II. The integration of Vitamin B₅ and B₂ into the fermentation process increased the activities of fatty acyl-CoA synthetase (^MaMACS) and fatty acyl-CoA dehydrogenase (^PpfadE). Finally, the constructed E. coli BL21(DE3)(ΔfadBJR/pCDFDuet-1-^PpfadE-^MaMACS/pRSFDuet-1-sumo-^CtydiI-ubiI) strain exhibited a 51.50% decrease in ROS and a 93.33% enhancement in trans-2-decenoic acid yield, reaching 1.45 g/L after 66 h, which is the highest yield reported for flask fermentation. This study reports the feasibility of rewiring the ETC regulation and energy metabolism to improve α,β-UCA biosynthesis efficiency.

2024-11-06·Journal of the American Chemical Society

Lipidation Engineering in Daptomycin Biosynthesis

Article

作者: Kang, Hahk-Soo ; Park, Sehong ; Je, Hyun-Woo ; Ji, Chang-Hun ; Lee, Kunwoo

Lipopeptides are an important family of natural products, some of which are clinically used as antibiotics to treat multidrug-resistant pathogens. Although the lipid moieties play a crucial role in balancing antibacterial activity and hemolytic toxicity, modifying the lipid moieties has been challenging due to the complexity of the lipidation process in lipopeptide biosynthesis. Here, we show that the lipid profile can be altered by engineering both secondary and primary metabolisms, using daptomycin as an example. First, swapping the fatty acyl AMP ligase (FAAL) gene dptF with foreign FAAL homologs improved the fatty acyl specificity of the lipidation process for decanoic acid. Then, the introduction of Mycobacterium type I fatty acid synthase operon (MvFAS-Ib/MvAcpS) and Cryptosporidium thioesterase (CpTEII) enriched the fatty acid pool with decanoic acid in Streptomyces roseosporus. The engineered fatty acid metabolism eliminates the need for external decanoic acid supplementation by enabling S. roseosporus to biosynthesize decanoic acid. By complete engineering of the lipidation process, we achieved, for the first time, high-purity, natural production of daptomycin. The lipidation engineering approach we demonstrate here lays the foundation for the lipidation control in lipopeptide biosynthesis.

100 项与碘番酸相关的药物交易

登录后查看更多信息