Inadequate blood supply to the intestine can lead to acute mesenteric ischemia (AMI), with a mortality rate ranging from 60% to 90%. This high mortality rate is partially due to late detection and the lack of efficient early diagnostic tests. There is an urgent need for a point-of-care tool for immediate bedside diagnosis. Here we present for the first time a rapid and non-invasive electrochemical biosensor device based on nonfaradic impedance spectroscopy to detect intestinal fatty-acid binding protein (I-FABP) as an indication of AMI. The electrochemical biosensors consist of gold interdigitated electrodes that were fabricated using photolithographic techniques on top of silicon dioxide substrates. The electrode surfaces were functionalized with an I-FABP capture antibody (CAnB) to entice the target protein, while gold nanoparticles (GNPs) functionalized with detection antibodies (DAnB-GNPs) were utilized as a novel mechanism to enhance the detection signal. Quantification of the I-FABP concentration in the medium depended on its attachment to CAnB and DAnB-GNPs in a sandwich manner, where the latter boosts the impedance signal through its binding to the I-FABP. This non-invasive non-faradic electric biosensor device demonstrates the potential for bench-to-bedside translation with the goal of decreasing morbidity and mortality from AMI.
Background: NADPH-dependent enzymes play important roles in many anabolic reactions and the availability of redox cofactors can influence metabolic flux ultimately influencing titers of bioproducts produced by engineered microbial cells. This may be especially true of oleochemical production when carbon flux through the highly NADPH-dependent fatty acid biosynthesis pathway is increased. While pathway specific approaches are often applied to counter redox imbalance, a study evaluating generalized approaches to improved NADPH availability is lacking in Saccharomyces cerevisiae . Results: Here, we have created four unique synthetic Pyruvate-Oxaloacetate-Malate “POM” cycles consisting of either of the endogenous isoforms of pyruvate carboxylase ( PYC1 or PYC2 ), a modified version of malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and a truncated cytosolic form of the endogenous malic enzyme ( sMAE1 ). Only the POM cycle that combined expression of PYC1 , ‘MDH2 , and sMAE1 increased the titer of fatty alcohols produced; however, it did so in two unique fatty alcohol producing strains. In a FAS1 overexpression background, expression of this synthetic POM cycle increased fatty alcohol titers by 40% from 49.0 ± 2.2 mg/L to 68.6 ± 3.3 mg/L and showed similar results in a zwf1 deletion strain. The effect of overexpression of the endogenous NAD+ kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. We found that expression of POS5c resulted in an ~35% increase in fatty alcohol titer, while the overexpression of the UTR1 or YEF1 did not significantly influence titers. In these minimally engineered cells, combined overexpression of PYC1 , ‘ MDH2 , sMAE1 and POS5c did not further increase titers Conclusions: Overexpression of PYC1 in conjunction with ‘MDH2 and sMAE1 results in a synthetic POM cycle which can be utilized to improve fatty alcohol production in engineered strains of S. cerevisiae . Additionally, overexpression of a truncated version of POS5 ( POS5c ) results in similar increases in fatty alcohol production. These findings may serve to provide a generalized mechanism to increase NADPH production in engineered cells, resulting in increased bioproduct titers.
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