Coenzyme I

An analytical platform was developed using agarose gel to overcome the issues of device miniaturization and interference effects caused by foreign proteins, which are challenges for field analysis-oriented measurement technology. The lab-on-gel (LoG) device integrates sample preparation, reaction, and detection on a chip, with an agarose gel for separation that preferentially allows only small molecules to pass through due to the size exclusion effect, and an agarose gel for sensing that contains glucose dehydrogenase and substrates. Measurements can be performed simply by dropping in a diluted serum sample and then placing the device in a custom-made compact Raspberry Pi-based fluorescence imager. This analytical approach was demonstrated by glucose measurement. The detection limit for glucose was estimated to be 21 μM (3σ). The method was also able to quantify glucose in serum at a lower dilution than the conventional method using well plates. This suggests that agarose gel works effectively for the pretreatment and sensing of biosensors. The developed method is expected to be useful for rapid and labor-saving medical diagnostics in town clinics and at the bedside.

9α-Hydroxyandrost-4-ene-3,17-dione (9-OHAD) is a representative precursor in steroid drug synthesis.However, during the 9-OHAD production from phytosterol in Mycolicibacterium neoaurum (M. neoaurum), the current manufacturing processes suffer from low productivity and purity due to the generation of a variety of byproducts.In this study, we genetically modified the phytosterol catabolism in M. neoaurum and achieved 13.4 g/L and 91.1 % molar yield of 9-OHAD with no detectable byproducts at a high phytosterol concentration of 20 g/L in shake flask.It was achieved by inactivating Opccr, SalA, TeB, and overexpressing KshA1 and ChsH1-2 to eliminate the production of 20-hydroxymethyl-9,21-dihydroxy-20-methyl-pregna-4-en-3-one (9-OH-4-HBC), 4-androstene-3,17-dione (4-AD) and 3-oxo-4-pregnene-9-OH-20-carboxylic Me ester (9-OH-3-OPCM) as well as overcoming the production limitations of 9-OHAD.Notably, when scaled up in a 5 L bioreactor with 45 g/L phytosterol concentration, the modified M. neoaurum achieved 24.5 g/L and 74.5 % molar conversion of 9-OHAD by using soybean oil and (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) as a co-solvent and antifoam agent.Therefore, our findings demonstrate a method to improve the efficiency and purity of 9-OHAD biosynthesis in M. neoaurum.