Acetaldehyde is an important carbonyl compound commonly detected in wines. A high concentration of acetaldehyde can affect the flavor of wines and result in adverse effects on human health. Alcohol dehydrogenase I (ADH1) in Saccharomyces cerevisiae catalyzes the reduction reaction of acetaldehyde into ethanol in the presence of cofactors, showing the potential to reduce the content of acetaldehyde in wines. In this study, ADH1 was successfully expressed in Pichia pastoris GS115 based on codon optimization. Then, the expression level of ADH1 was enhanced by replacing its promoter with optimized promoters and increasing the copy number of the expression cassette, with ADH1 being purified using nickel column affinity chromatography. The enzymatic activity of purified ADH1 reached 605.44 ± 44.30 U/mg. The results of the effect of ADH1 on the content of acetaldehyde in wine revealed that the acetaldehyde content of wine samples was reduced from 168.05 ± 0.55 to 113.17 ± 6.08 mg/L with the addition of 5 mM NADH and the catalysis of ADH1, and from 135.53 ± 4.08 to 52.89 ± 2.20 mg/L through cofactor regeneration. Our study provides a novel approach to reducing the content of acetaldehyde in wines through enzymatic catalysis.

Recently, a putative alcohol dehydrogenase 3, termed EhADH3B of the Entamoeba histolytica isolate HM-1:IMSS was identified, which is expressed at higher levels in non-pathogenic than in pathogenic amoebae and whose overexpression reduces the virulence of pathogenic amoebae. In an in silico analysis performed in this study, we assigned EhADH3B to a four-member ADH3 family, with ehadh3b present as a duplicate (ehadh3ba/ehadh3bb). In long-term laboratory cultures a mutation was identified at position 496 of ehadh3ba, which codes for a stop codon, which was not the case for amoebae isolated from human stool samples. When using transfectants that overexpress or silence ehadh3bb, we found no or little effect on growth, size, erythrophagocytosis, motility, hemolytic or cysteine peptidase activity. Biochemical characterization of the recombinant EhADH3Bb revealed that this protein forms a dimer containing Ni2+ or Zn2+ as a co-factor and that the enzyme converts acetaldehyde and formaldehyde in the presence of NADPH. A catalytic activity based on alcohols as substrates was not detected. Based on the results, we postulate that EhADH3Bb can reduce free acetaldehyde released by hydrolysis from bifunctional acetaldehyde/alcohol dehydrogenase-bound thiohemiacetal and that it is involved in detoxification of toxic aldehydes produced by the host or the gut microbiota.

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

Alcoholic liver fatty disease (ALFD) is caused by excessive and chronic alcohol consumption. Alcohol consumption causes an imbalance in the intestinal microflora, leading to liver disease induced by the excessive release of endotoxins into the hepatic portal vein. Therefore, research on the intestinal microflora to identify treatments for ALFD is increasing. In this study, the protective effects of lactic acid bacteria (LAB) strains, including Levilactobacillus brevis, Limosilactobacillus reuteri, and Limosilactobacillus fermentum, were evaluated in ethanol-induced HepG2 cells. Among the evaluated LAB, nine strains increased aldehyde dehydrogenase (ALDH) levels and downregulated lipid peroxidation and liver transferase in the ethanol-induced HepG2 cells. Moreover, L. brevis MG5280 and MG5311, L. reuteri MG5458, and L. fermentum MG4237 and MG4294 protected against ethanol-induced HepG2 cell damage by regulating CYP2E1, antioxidant enzymes (SOD, CAT, and GPX), lipid synthesis factors (SREBP1C and FAS), and lipid oxidation factors (PPARα, ACO, and CPT-1). Moreover, five LAB were confirmed to be safe probiotics based on antibiotic susceptibility and hemolysis assays; their stability and adhesion ability in the gastrointestinal tract were also established. In conclusion, L. brevis MG5280 and MG5311, L. reuteri MG5458, and L. fermentum MG4237 and MG4294 may be useful as new probiotic candidates for ALFD prevention.

Thermoanaerobacter species have recently been observed to reduce carboxylic acids to their corresponding alcohols. The present investigation shows that Thermoanaerobacter pseudoethanolicus converts C2-C6 short-chain fatty acids (SCFAs) to their corresponding alcohols in the presence of glucose. The conversion yields varied from 21% of 3-methyl-1-butyrate to 57.9% of 1-pentanoate being converted to their corresponding alcohols. Slightly acidic culture conditions (pH 6.5) was optimal for the reduction. By increasing the initial glucose concentration, an increase in the conversion of SCFAs reduced to their corresponding alcohols was observed. Inhibitory experiments on C2-C8 alcohols showed that C4 and higher alcohols are inhibitory to T. pseudoethanolicus suggesting that other culture modes may be necessary to improve the amount of fatty acids reduced to the analogous alcohol. The reduction of SCFAs to their corresponding alcohols was further demonstrated using 13C-labelled fatty acids and the conversion was followed kinetically. Finally, increased activity of alcohol dehydrogenase (ADH) and aldehyde oxidation activity was observed in cultures of T. pseudoethanolicus grown on glucose as compared to glucose supplemented with either 3-methyl-1-butyrate or pentanoate, using both NADH and NADPH as cofactors, although the presence of the latter showed higher ADH and aldehyde oxidoreductase (ALDH) activity.

Alcoholic liver disease (ALD), caused by excessive alcohol consumption, leads to high mortality. We investigated the hepatoprotective effect of Levilactobacillus brevis MG5311 in C57BL/6 mice with liver injuries induced by chronic ethanol plus binge feeding. L. brevis MG5311 was administered orally at a dose of 1 × 109 CFU/mouse once daily for 32 days. L. brevis MG5311 administration significantly reduced serum ALT, AST, and triglyceride (TG) levels in ethanol-fed mice. L. brevis MG5311 also decreased malondialdehyde levels and increased glutathione peroxidase (GPx) activity in liver tissues. In addition, hepatic TG content and histopathological scores were significantly reduced. L. brevis MG5311 increased the protein expression of SIRT1, PPARα, SOD1, CAT, and GPx 1/2 in liver tissue, while inhibiting CYP2E1 and SREBP-1c. These results indicated that L. brevis MG5311 alleviated ethanol-induced liver injury by inhibiting hepatic oxidative stress and promoting lipid metabolism. Therefore, L. brevis MG5311 may be a useful probiotic candidate for ameliorating or preventing ALD.

Cytosolic pyruvate is an essential metabolite in lactic acid production during microbial fermentation. However, under aerobiosis, pyruvate is transported to the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) and oxidized in cell respiration. Previous reports using Saccharomyces cerevisiae or Aspergillus oryzae have shown that the production of pyruvate-derived chemicals is improved by deleting the MPC1 gene. A previous lactate-producing K. phaffii strain engineered by our group was used as a host for the deletion of the MPC1 gene. In addition, the expression of a bacterial hemoglobin gene under the alcohol dehydrogenase 2 promoter from Scheffersomyces stipitis, known to work as a hypoxia sensor, was used to evaluate whether aeration would supply enough oxygen to meet the metabolic needs during lactic acid production. However, unlike S. cerevisiae and A. oryzae, the deletion of Mpc1 had no significant impact on lactic acid production but negatively affected cell growth in K. phaffii strains. Furthermore, the relative quantification of the VHb gene revealed that the expression of hemoglobin was detected even in aerobic cultivation, which indicates that the demand for oxygen in the bioreactor could result in functional hypoxia. Overall, the results add to our previously published ones and show that blocking cell respiration using hypoxia is more suitable than deleting Mpc for producing lactic acid in K. phaffii.

The property differences between bacteria produced from solid-state and liquid-state fermentations have always been the focus of attention. This study analyzed the stress tolerance and transcriptomic differences of the probiotic Lacticaseibacillus casei Zhang produced from solid-state and liquid-state fermentations under no direct stress. The total biomass of L. casei Zhang generated from liquid-state fermentation with MRS medium (LSF-MRS) was 2.24 times as much as that from solid-state fermentation with soybean meal-wheat bran (SSF-SW) medium. Interestingly, NaCl, H2O2, and ethanol stress tolerances and the survival rate after L. casei Zhang agent preparation from SSF-SW fermentation were significantly higher than those from LSF-MRS fermentation. The global transcriptomic analysis revealed that in L. casei Zhang produced from SSF-SW fermentation, carbohydrate transport, gluconeogenesis, inositol phosphate metabolism were promoted, that pentose phosphate pathway was up-regulated to produce more NADPH, that citrate transport and fermentation was extremely significantly promoted to produce pyruvate and ATP, and that pyruvate metabolism was widely up-regulated to form lactate, acetate, ethanol, and succinate from pyruvate and acetyl-CoA, whereas glycolysis was suppressed, and fatty acid biosynthesis was suppressed. Moreover, in response to adverse stresses, some genes encoding aquaporins (GlpF), superoxide dismutase (SOD), nitroreductase, iron homeostasis-related proteins, trehalose operon repressor TreR, alcohol dehydrogenase (ADH), and TetR/AcrR family transcriptional regulators were up-regulated in L. casei Zhang produced from SSF-SW fermentation. Our findings provide novel insight into the differences in growth performance, carbon and lipid metabolisms, and stress tolerance between L. casei Zhang from solid-state and liquid-state fermentations.