PubMed · 8041259
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S L MacDonald, D S Pepper. 1994. Hemoglobin polymerization.. https://doi.org/10.1016/0076-6879(94)31021-1
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We have addressed critical challenges in probiotic design to develop a commercially viable bacterial strain capable of removing the intestinal toxin, acetaldehyde. In this study, we report the engineering of the hag locus, a σD-dependent flagellin expression site, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter, following a deletion of the gene encoding a post-translational regulator of σD, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate activity at the hag locus after germination, with a steady increase in heterologous expression throughout outgrowth and vegetative growth. To evaluate the chassis as a spore-based probiotic solution, we identified the physiologically relevant ethanol metabolic pathway and the subsequent accumulation of gut-derived acetaldehyde following alcohol consumption. We integrated a Cupriavidus necator aldehyde dehydrogenase gene (acoD) into the hag locus under the control of the flagellin promoter and observed a rapid reduction in acetaldehyde levels in gut-simulated conditions post-germination. This work demonstrates a promising approach for the development of genetically engineered spore-based probiotics.
Erythrose inhibited the growth of a sodA sodB strain of Escherichia coli under aerobiosis; but did not inhibit anaerobic growth of the sodA sodB strain, or the aerobic growth of the superoxide dismutase (SOD)-competent parental strain. A SOD mimic protected the sodA sodB strain against the toxicity of erythrose as did the carbonyl-blocking reagents hydrazine and aminoguanidine. Three carbon sugars, such as glyceraldehyde and dihydroxy acetone, and the two carbon sugar glycolaldehyde, were similarly toxic in an O-2-dependent manner. An unidentified dialyzable component in E. coli extract augmented the oxidation of short chain sugars, and this was partially inhibitable by SOD. The toxicity of the short chain sugars appears to be because of an O-2-dependent oxidation to alpha, beta-dicarbonyl compounds. In keeping with this view was the O-2-independent toxicity of methylglyoxal.
Biotransformation of ethanol by liver nuclei was studied. The formation of acetaldehyde was determined by GC/FID. The 1-hydroxyethyl (1HEt) formation was established by spin trapping of the radical with N-t-butyl-alpha-phenylnitrone (PBN) followed by GC/MS. Liver nuclei, free of endoplasmic reticulum, cytosol or mitochondria, were able to biotransform ethanol to acetaldehyde in the presence of NADPH under air. Only 22% activity was observed in the absence of the cofactor. Twenty-six percent of the NADPH-dependent activity and 47% of the NADPH-independent activity were observable under nitrogen. Aerobic biotransformation was inhibited by CO, SKF 525A, 4-methylpyrazole and by diethyldithiocarbamate. This suggests that CYP2E1 is involved in the process. However, the formation of acetaldehyde was able to proceed under a pure CO atmosphere. The lack of inhibitory effects of 2-mercapto-1-methylimidazol and thiobenzamide excludes the potential participation of the NADPH flavin monooxigenase system. The formation of hydroxyl radicals in the process is suggested by the partial inhibitory effect of 5 mM mannitol and 5 mM sodium benzoate and by the fact that the 1HEt was detected. The NADPH-dependent anaerobic ethanol biotransformation pathway was stimulated by FAD and inhibited to some extent by iron chelators. The relevance of a liver nuclear ethanol biotransformation, generating reactive metabolites, such as acetaldehyde and free radicals, nearby DNA, nuclear proteins and lipids is discussed.