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Activating inert substrates is a challenge in nature and synthetic chemistry, but essential for creating functionally active molecules. In this work, we used a combinatorial optimization approach to assemble cytochrome P450 monooxygenases (CYPs) and reductases (CPRs) to achieve a target product profile. By creating 110 CYP-CPR pairs and iteratively screening different pairing libraries, we demonstrated a framework for establishing a CYP network that catalyzes six oxidation reactions at three different positions of a chemical scaffold. Target product titer was improved by remodeling endoplasmic reticulum (ER) size and spatially controlling the CYPs configuration on the ER. Out of 47 potential products that could be synthesized, 86% of the products synthesized by the optimized network was our target compound quillaic acid (QA), the aglycone backbone of many pharmaceutically important saponins, and fermentation achieved QA titer 2.23 g/L.

Valerolactam is a monomer used to manufacture high-value nylon-5 and nylon-6,5. However, the biological production of valerolactam has been limited by the inadequate efficiency of enzymes to cyclize 5-aminovaleric acid to produce valerolactam. In this study, we engineered Corynebacterium glutamicum with a valerolactam biosynthetic pathway consisting of DavAB from Pseudomonas putida to convert L-lysine to 5-aminovaleric acid and β-alanine CoA transferase (Act) from Clostridium propionicum to produce valerolactam from 5-aminovaleric acid. Most of the L-lysine was converted into 5-aminovaleric acid, but promoter optimization and increasing the copy number of Act were insufficient to significantly improve the titer of valerolactam. To eliminate the bottleneck at Act, we designed a dynamic upregulation system (a positive feedback loop based on the valerolactam biosensor ChnR/Pb). We used laboratory evolution to engineer ChnR/Pb to have higher sensitivity and a higher dynamic output range, and the engineered ChnR-B1/Pb-E1 system was used to overexpress the rate-limiting enzymes (Act/ORF26/CaiC) that cyclize 5-aminovaleric acid into valerolactam. In glucose fed-batch culture, we obtained 12.33 g/L valerolactam from the dynamic upregulation of Act, 11.88 g/L using ORF26, and 12.15 g/L using CaiC. Our engineered biosensor (ChnR-B1/Pb-E1 system) was also sensitive to 0.01-100 mM caprolactam, which suggests that this dynamic upregulation system can be used to enhance caprolactam biosynthesis in the future.

The directed evolution of AraC to build an arabinose-inducible system, which is more compatible with (isopropyl-[beta]-D-1-thiogalactopyranoside) induction of a lactose-inducible system is presented.

To study in vivo modification of the SAM equivalent circuit when a highly ordered SAM is used as a bioelectrode, dodecanethiolate SAM–Au intramuscular electrodes were studied in living rat heart in a challenging in situ perfused rat model by impedance spectroscopy, cyclic voltammetry, and neutron activation analysis (NAA). The SAM layer experienced disintegration in vivo biological system, as NAA detected the presence of Au atoms that had leached into the surrounding living tissue. Therefore, the underlying Au surface became exposed during biological implant. Study by impedance spectroscopy, however, revealed perfect capacitive behavior for the SAM, similar to in vitro behavior. Electrodes showed a pure capacitive Nyquist plot with 86.1–89.4° near-vertical line segments as the equivalent circuit locus, as for a parallel plate capacitor. Impedance magnitude varied linearly with 1/ω excluding diffusionally limited ionic charge transport. There was no diffusional conductive element ZW∞ or spatially confined Warburg impedance ZD. The effect of in vivo exposure of a highly ordered SAM is a ‘sealing over’ effect of new defects by the binding of proteinaceous or lipid species in the biological milieu, a fact of significance for SAM electrodes used either as pacemaker electrodes or as a platform for in vivo biosensors. [Copyright &y& Elsevier]