Co-augmentation of a transport gene mfsT1 in Mycolicibacterium neoaurum with genome engineering to enhance ergothioneine production.

Ergothioneine (EGT) is a rare thiohistidine derivative with exceptional antioxidant properties. The blood level of EGT is considered highly reliable predictors for cardiovascular diseases and mortality, yet animals lack the ability to synthesize this compound. Free plasmids have been previously used to overexpress genes involved in the EGT biosynthetic pathway of Mycolicibacterium neoaurum. Here, we tentatively introduced a putative transporter gene mfsT1 into high-copy plasmids and sharply increased the ratio of extracellular EGT concentration from 18.7% to 44.9%. Subsequently, an additional copy of egtABCDE, hisG, and mfsT1 was inserted into the genome with a site-specific genomic integration tool of M. neoaurum, leading a 2.7 times increase in EGT production. Co-enhancing the S-adenosyl-L-methionine regeneration pathway, or alternatively, the integration of three copies of egtABCDE, hisG and mfsT1 into the genome further increased the total EGT yield by 16.1% (64.6 mg/L) and 21.7% (67.7 mg/L), respectively. After 168-h cultivation, the highest titer reached 85.9 mg/L in the latter strain with three inserted copies. This study provided a solid foundation for genome engineering to increase the production of EGT in M. neoaurum.

Simple and cheap CRISPR/Cas12a biosensor based on plug-and-play of DNA aptamers for the detection of endocrine-disrupting compounds.

Endocrine-disrupting compounds (EDCs) are widely distributed in the environment. Here, we present a CRISPR/Cas12a (CAS) biosensor based on DNA aptamers for point-of-care detection of EDCs. Two typical EDCs, 17ß-estradiol (E2) and bisphenol A (BPA), were selected to be detected by the CAS biosensors via the plug-and-play of their DNA aptamers. The results indicated that the performance of the CAS biosensors can be well regulated by controlling the trans-cleavage activity of Cas12a on a single-stranded DNA reporter and optimizing the sequence and ratio of DNA aptamer and activator DNA. Ultimately, two reliable and specific biosensors were developed, with the linear range and limit of detection of 0.2-25 nM and 0.08 nM for E2 and of 0.1-250 nM and 0.06 nM for BPA, respectively. Compared to the existing detection methods, the CAS biosensors showed higher reliability and sensitivity with simple operation, short detection time, and no costly equipment.

Improvement of the Biocompatibility and Potential Stability of Chronically Implanted Electrodes Incorporating Coating Cell Membranes.

The development of chronically implanted electrodes attracts much attention since these electrodes are much favorable for monitoring changes of neurotransmitters in brain science. The research in this field focused mainly on chemical modification to improve the potential stability and less on the biocompatibility. In this work, for the first time, we proposed the concept of cell-membrane electrodes based on a basic hypothesis using animal's self-cell membrane to reduce animal exclusiveness (hyperacute rejection and chronic rejection). As a proof of concept, we first studied cell-membrane reference electrodes for chronically implanted electrodes. Red cell membrane (RCM) was extracted from rat blood and coated on the chemically modified Ag/AgCl electrodes. It was found that ionic liquid (IL) 1-butyl-2,3-dimethylimidazolium hexafluorophosphate (BDMI) showed good performance rather than Nafion used as coating film for protection of silver chloride on Ag wire and support of the cell membrane. Electrochemical impedance spectra supported that charge-transfer resistance nearly kept constant before and after the electrodes were implanted into the rat's brain tissues for 28 days. Immunohistochemical analysis of the implant sites in the rat's brain tissues indicated that the extent of glial scarring arising from the Ag/AgCl/BDMI/RCM electrodes was smaller than that of both Ag/AgCl/Nafion and Ag/AgCl/Nafion/RCM electrodes after 28 days of implantation. The RCM-coated Ag/AgCl/IL electrodes showed a relatively potential stability compared to RCM-noncoated Ag/AgCl/IL electrodes after 28 days of implantation. Additionally, the current-voltage curve demonstrated that the RCM-coated electrodes can be used as polarized electrodes. This work demonstrated that the RCM, which was coated on the Ag/AgCl/IL electrodes, can significantly improve the biocompatibility and potential stability of the RCM-noncoated Ag/AgCl/IL electrodes implanted in the rat brain. The cell-membrane-coated electrodes will serve as a lighthouse in guiding the design of chronically implanted electrodes for in vivo electrochemical detection.