Phosphate toxicity and SERCA2a dysfunction in sudden cardiac arrest.

Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one-third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion-channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end-product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end-product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low-phosphate dietary interventions to reduce the risk of sudden cardiac arrest.

Improvement of succinate production by release of end-product inhibition in Corynebacterium glutamicum.

Succinate is a renewable-based platform chemical that may be used to produce a wide range of chemicals including 1,4-butanediol, tetrahydrofurane, and γ-butyrolactone. However, industrial fermentation of organic acids is often subject to end-product inhibition, which significantly retards cell growth and limits metabolic activities and final productivity. In this study, we report the development of metabolically engineered Corynebacterium glutamicum for high production of succinate by release of end-product inhibition coupled with an increase of key metabolic flux. It was found that the rates of glucose consumption and succinate production were significantly reduced by extracellular succinate in an engineered strain, S003. To understand the mechanism underlying the inhibition by succinate, comparative transcriptome analysis was performed. Among the downregulated genes, overexpression of the NCgl0275 gene was found to suppress the inhibition of glucose consumption and succinate production, resulting in a 37.7% increase in succinate production up to 55.4g/L in fed-batch fermentation. Further improvement was achieved by increasing the metabolic flux from PEP to OAA. The final engineered strain was able to produce 152.2g/L succinate, the highest production reported to date, with a yield of 1.1g/g glucose under anaerobic condition. These results suggest that the release of end-product inhibition coupled with an increase in key metabolic flux is a promising strategy for enhancing production of succinate.

A glutathione responsive rice glyoxalase II, OsGLYII-2, functions in salinity adaptation by maintaining better photosynthesis efficiency and anti-oxidant pool.

Glyoxalase II (GLY II), the second enzyme of glyoxalase pathway that detoxifies cytotoxic metabolite methylglyoxal (MG), belongs to the superfamily of metallo-ß-lactamases. Here, detailed analysis of one of the uncharacterized rice glyoxalase II family members, OsGLYII-2 was conducted in terms of its metal content, enzyme kinetics and stress tolerance potential. Functional complementation of yeast GLY II mutant (∆GLO2) and enzyme kinetics data suggested that OsGLYII-2 possesses characteristic GLY II activity using S-lactoylglutathione (SLG) as the substrate. Further, Inductively Coupled Plasma Atomic Emission spectroscopy and modelled structure revealed that OsGLYII-2 contains a binuclear Zn/Fe centre in its active site and chelation studies indicated that these are essential for its activity. Interestingly, reconstitution of chelated enzyme with Zn(2+), and/or Fe(2+) could not reactivate the enzyme, while addition of Co(2+) was able to do so. End product inhibition study provides insight into the kinetics of GLY II enzyme and assigns hitherto unknown function to reduced glutathione (GSH). Ectopic expression of OsGLYII-2 in Escherichia coli and tobacco provides improved tolerance against salinity and dicarbonyl stress indicating towards its role in abiotic stress tolerance. Maintained levels of MG and GSH as well as better photosynthesis rate and reduced oxidative damage in transgenic plants under stress conditions seems to be the possible mechanism facilitating enhanced stress tolerance.

Cultivation of Mycolicibacterium spp. Mutants in Miniaturized and High-Throughput Format to Characterize Their Growth, Phytosterol Conversion Ability, and Resistance to the Steroid Products.

This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis.

Ameliorating end-product inhibition to improve cadaverine production in engineered Escherichia coli and its application in the synthesis of bio-based diisocyanates.

Cadaverine is an important C5 platform chemical with a wide range of industrial applications. However, the cadaverine inhibition on the fermenting strain limited its industrial efficiency of the strain. In this study, we report an engineered Escherichia coli strain with high cadaverine productivity that was generated by developing a robust host coupled with metabolic engineering to mitigate cadaverine inhibition. First, a lysine producing E. coli was treated with a combination of radiation (ultraviolet and visible spectrum) and ARTP (atmospheric and room temperature plasma) mutagenesis to obtain a robust host with high cadaverine tolerance. Three mutant targets including HokD, PhnI and PuuR are identified for improved cadaverine tolerance. Further transcriptome analysis suggested that cadaverine suppressed the synthesis of ATP and lysine precursor. Accordingly, the related genes involved in glycolysis and lysine precursor, as well as cadaverine exporter was engineered to release the cadaverine inhibition. The final engineered strain was fed-batch cultured and a titer of 58.7 g/L cadaverine was achieved with a yield of 0.396 g/g, both of which were the highest level reported to date in E. coli. The bio-based cadaverine was purified to >99.6% purity, and successfully used for the synthesis of polyurethane precursor 1,5-pentamethylene diisocyanate (PDI) through the approach of carbamate decomposition.

Growth Enhancement of Probiotic Pediococcus acidilactici by Extractive Fermentation of Lactic Acid Exploiting Anion-Exchange Resin.

Fermentation employing lactic acid bacteria (LAB) often suffers end-product inhibition which reduces the cell growth rate and the production of metabolite. The utility of adsorbent resins for in situ lactic acid removal to enhance the cultivation performance of probiotic, Pediococcus acidilactici was studied. Weak base anion-exchange resin, Amberlite IRA 67 gave the highest maximum uptake capacity of lactic acid based on Langmuir adsorption isotherm (0.996 g lactic acid/g wet resin) compared to the other tested anion-exchange resins (Amberlite IRA 410, Amberlite IRA 400, Duolite A7 and Bowex MSA). The application of Amberlite IRA 67 improved the growth of P. acidilactici about 67 times compared to the control fermentation without resin addition. Nevertheless, the in situ addition of dispersed resin in the culture created shear stress by resins collision and caused direct shear force to the cells. The growth of P. acidilactici in the integrated bioreactor-internal column system containing anion-exchange resin was further improved by 1.4 times over that obtained in the bioreactor containing dispersed resin. The improvement of the P. acidilactici growth indicated that extractive fermentation using solid phase is an effective approach for reducing by-product inhibition and increasing product titer.

Strategies for improving production performance of probiotic Pediococcus acidilactici viable cell by overcoming lactic acid inhibition.

Lactic acid bacteria are industrially important microorganisms recognized for fermentative ability mostly in their probiotic benefits as well as lactic acid production for various applications. Fermentation conditions such as concentration of initial glucose in the culture, concentration of lactic acid accumulated in the culture, types of pH control strategy, types of aeration mode and different agitation speed had influenced the cultivation performance of batch fermentation of Pediococcus acidilactici. The maximum viable cell concentration obtained in constant fed-batch fermentation at a feeding rate of 0.015 L/h was 6.1 times higher with 1.6 times reduction in lactic acid accumulation compared to batch fermentation. Anion exchange resin, IRA 67 was found to have the highest selectivity towards lactic acid compared to other components studied. Fed-batch fermentation of P. acidilactici coupled with lactic acid removal system using IRA 67 resin showed 55.5 and 9.1 times of improvement in maximum viable cell concentration compared to fermentation without resin for batch and fed-batch mode respectively. The improvement of the P. acidilactici growth in the constant fed-batch fermentation indicated the use of minimal and simple process control equipment is an effective approach for reducing by-product inhibition. Further improvement in the cultivation performance of P. acidilactici in fed-bath fermentation with in situ addition of anion-exchange resin significantly helped to enhance the growth of P. acidilactici by reducing the inhibitory effect of lactic acid and thus increasing probiotic production.

Increased biohydrogen yields, volatile fatty acid production and substrate utilisation rates via the electrodialysis of a continually fed sucrose fermenter.

Electrodialysis (ED) removed volatile fatty acids (VFAs) from a continually-fed, hydrogen-producing fermenter. Simultaneously, electrochemical removal and adsorption removed gaseous H2 and CO2, respectively. Removing VFAs via ED in this novel process increased H2 yields by a factor of 3.75 from 0.24molH2mol-1hexose to 0.90molH2mol-1hexose. VFA production and substrate utilisation rates were consistent with the hypothesis that end product inhibition arrests H2 production. The methodology facilitated the recovery of 37g of VFAs, and 30L H2 that was more than 99% pure, both of which are valuable, energy dense chemicals. Typically, short hydraulic and solid retention times, and depressed pH levels are used to suppress methanogenesis, but this limits H2 production. To produce H2 from real world, low grade biomass containing complex carbohydrates, longer hydraulic retention times (HRTs) are required. The proposed system increased H2 yields via increased substrate utilisation over longer HRTs.

Increased expression of ß-glucosidase A in Clostridium thermocellum 27405 significantly increases cellulase activity.

ß-glucosidase A (bglA) in Clostridium thermocellum 27405 was increased by expression from shuttle vector pIBglA in attempts to increase cellulase activity and ethanol titer by lowering the end product inhibition of cellulase. Through a modified electrotransformation protocol C. thermocellum transformant (+MCbglA) harbouring pIBglA was produced. The ß-glucosidase activity of +MCbglA was 2.3- and 1.6-fold greater than wild-type (WT) during late log and stationary phases of growth. Similarly, total cellulase activity of +MCbglA was shown to be 1.7-, 2.3- and 1.6-fold greater than WT during, log, late log and stationary phases of growth. However, there was no significant correlation found between increased cellulase activity and increased ethanol titers for +MCbglA compared with the WT. C. thermocellum has industrial potential for consolidated bioprocessing (CBP) to make a more cost effective production of biofuels; however, the hydrolysis rate of the strain is still hindered by end product inhibition. We successfully increased total cellulase activity by increased expression of bglA and thereby increased the productivity of C. thermocellum during the hydrolysis stage in CBP. Our work also lends insights into the complex metabolism of C. thermocellum for future improvement of this strain.