Sensitive electrochemical flow injection analysis of H2O2 released from cells with a pass-through mode.

Conventional plate electrodes were commonly used in electrochemical flow injection analysis and only part of molecules diffused to the plane of electrodes could be detected, which would limit the performance of electrochemical detection. In this study, a low-cost native stainless steel wire mesh (SSWM) electrode was integrated into a 3D-printed device for electrochemical flow injection analysis with a pass-through mode, which is different compared with previous flow-through mode. This strategy was applied for sensitive analysis of hydrogen peroxide (H2O2) released from cells. Under the optimal conditions (the applied potentials, the flow rate and the sample volume), the device exhibits high sensitivity toward H2O2. Linear relationships could be achieved between electrochemical responses and the concentration of H2O2 ranging from 1 nM to 1 mM. The excellent analytical performance of the SSWM-based device could be attributed to the pass-through mode based on the mesh microstructure and intrinsic catalytic properties for H2O2 by stainless steel. This approach could be further successfully extended for screening of H2O2 released from HeLa cells with electrochemical responses linear to the number of cells in a range of 3 - 1.35 × 104 cells with an injection volume of 30 µL. This study revealed the potential of mesh electrodes in electrochemical flow injection analysis for cellular function and pathology and its possible extension in cell counting and on-line analysis.

Redirection of acyl donor metabolic flux for lipopeptide A40926B0 biosynthesis.

The metabolic flux of fatty acyl-CoAs determines lipopeptide biosynthesis efficiency, because acyl donor competition often occurs from polyketide biosynthesis and homologous pathways. We used A40926B0 as a model to investigate this mechanism. The lipopeptide A40926B0 with a fatty acyl group is the active precursor of dalbavancin, which is considered as a new lipoglycopeptide antibiotic. The biosynthetic pathway of fatty acyl-CoAs in the A40926B0 producer Nonomuraea gerenzanensis L70 was efficiently engineered using endogenous replicon CRISPR (erCRISPR). A polyketide pathway and straight-chain fatty acid biosynthesis were identified as major competitors in the malonyl-CoA pool. Therefore, we modified both pathways to concentrate acyl donors for the production of the desired compound. Combined with multiple engineering approaches, including blockage of an acetylation side reaction, overexpression of acetyl-CoA carboxylase, duplication of the dbv gene cluster and optimization of the fermentation parameters, the final strain produced 702.4 mg l-1 of A40926B0, a 2.66-fold increase, and the ratio was increased from 36.2% to 81.5%. Additionally, an efficient erCRISPR-Cas9 editing system based on an endogenous replicon was specifically developed for L70, which increased conjugation efficiency by 660% and gene-editing efficiency was up to 90%. Our strategy of redirecting acyl donor metabolic flux can be widely adopted for the metabolic engineering of lipopeptide biosynthesis.

Molecular mechanism of azoxy bond formation for azoxymycins biosynthesis.

Azoxy bond is an important chemical bond and plays a crucial role in high energy density materials. However, the biosynthetic mechanism of azoxy bond remains enigmatic. Here we report that the azoxy bond biosynthesis of azoxymycins is an enzymatic and non-enzymatic coupling cascade reaction. In the first step, nonheme diiron N-oxygenase AzoC catalyzes the oxidization of amine to its nitroso analogue. Redox coenzyme pairs then facilitate the mutual conversion between nitroso group and hydroxylamine via the radical transient intermediates, which efficiently dimerize to azoxy bond. The deficiency of nucleophilic reactivity in AzoC is proposed to account for the enzyme's non-canonical oxidization of amine to nitroso product. Free nitrogen radicals induced by coenzyme pairs are proposed to be responsible for the efficient non-enzymatic azoxy bond formation. This mechanism study will provide molecular basis for the biosynthesis of azoxy high energy density materials and other valuable azoxy chemicals.