Identification, characterization, and application of a highly sensitive lactam biosensor from Pseudomonas putida

ABSTRACTCaprolactam is an important polymer precursor to nylon traditionally derived from petroleum and produced on a scale of 5 million tons per year. Current biological pathways for the production of caprolactam are inefficient with titers not exceeding 2 mg/L, necessitating novel pathways for its production. As development of novel metabolic routes often require thousands of designs and result in low product titers, a highly sensitive biosensor for the final product has the potential to rapidly speed up development times. Here we report a highly sensitive biosensor for valerolactam and caprolactam from Pseudomonas putida KT2440 which is >1000x more sensitive to exogenous ligand than previously reported sensors. Manipulating the expression of the sensor oplR (PP_3516) substantially altered the sensing parameters, with various vectors showing Kd values ranging from 700 nM (79.1 μg/L) to 1.2 mM (135.6 mg/L). Our most sensitive construct was able to detect in vivo production of caprolactam above background at ~6 μg/L. The high sensitivity and range of OplR is a powerful tool towards the development of novel routes to the biological synthesis of caprolactam.

Copper (ii) dimers stabilized by bis(phenol) amine ligands: theoretical and experimental insights

Comparative structural and magnetic studies on the binuclear Cu complexes of 1L(2−)/2L(2−) have been done and their electronic structures and observed electronic transitions (DFT and TD-DFT calculations) rationalized. The dimer complex can be converted into the corresponding monomeric Cu(ii) complex, [1L2CuII2(X)] (X = py), by adding an exogenous ligand such as pyridine (py).

Role of the membrane cholesterol-glycosphingolipid complex as a ‘transistor’ to regulate GSL receptor function and signaling of both lipids

Cholesterol and glycosphingolipids (GSL) are the major species that accumulate in plasma membrane lipid rafts. These complexes imbue the membrane with increased order, which in turn, plays a central role in the transmembrane signaling foci lipid rafts provide. In addition, both GSL and cholesterol binding can mediate (separate) signal pathways. We have shown that cholesterol and GSLs however, form a complex in which the GSL sugar is reoriented from a membrane perpendicular to parallel format, becoming largely unavailable for exogenous ligand binding. Similarly, the steroid hydroxyl is masked, restricting access of cholesterol ligands. This was observed in model and cell membranes and in human tumour frozen tissue sections. We now show the order of exogenous ligand binding plays a significant role to determine the extent of GSL or cholesterol receptor activity. Ligand binding to cholesterol enhances subsequent GSL recognition and vice versa, suggesting that ligand binding to “free” receptor (membrane perpendicular GSL carbohydrate, nonmasked cholesterol) can result in partial dissociation of the GSL/cholesterol complex to allow additional GSL ligand and cholesterol ligand binding. Since many GSLs can complex with membrane cholesterol, the binding of a single cholesterol ligand may unmask cholesterol-complexed GSL for increased binding of both a single or multiple GSL-specific ligands. We show that multiple cholesterol-masked GSLs can be coincident in tissues. This provides a mechanism for GSL-dependent signal amplification and diversification, representing a biological ‘transistor’, regulating amplitude and potentially, diversity of GSL signaling. The process represents a new mechanism of ‘cross-talk’ between GSL and cholesterol signaling. This is of clinical importance since we have found cholesterol/GSL masking applies to monoclonal anti GSL antibodies in development and in current use as antineoplastic therapeutics.