| Acetaldehyde is a mutagenic and carcinogenic substance that can be oxidized to acetic acid by acetaldehyde dehydrogenase.Acetaldehyde dehydrogenase can be utilized to reduce or eliminate the hazards of acetaldehyde.Alcoholism also harms human health.Alcohol can be converted to relatively non-toxic acetic acid by alcohol dehydrogenase(ADH)and acetaldehyde dehydrogenase(ALDH).Supplement of ADH and ALDH can help the body degrade ethanol,resulting in decrease of blood and liver ethanol amount,and thereby reducing alcoholic liver injury.Therefore,the preparation of hangover products based on this idea has great research and application prospects.Microbial heterologous expression is the most commonly used method for producing enzymes because of its advantage of low cost,wherein B.subtilis is a "super cell factory" that produces proteins in industry and can achieve high levels of heterologous expression.B.subtilis is also a potential probiotic,and has a certain ability to withstand acidic environment.To our knowledge,there are no reports of heterologous expression of ADH and ALDH in B.subtilis.Therefore,recombinant B.subtilis co-expressing ADH and ALDH has important research value for the development of oral hangover preparations.In this work,B.subtilis was used as an expression host to secrete express Issatchenkia terricola acetaldehyde dehydrogenase(istALDH),and the effects of signal peptides,promoters and secrete chaperones on the secretion efficiency of istALDH were studied.Then,an intracellular co-expressing istALDH and Saccharomyces cerevisiae alcohol dehydrogenase(scADH)recombinant B.subtilis was constructed.The ability of co-expressing recombinant strain to degrade ethanol and tolerance to artificially simulated gastrointestinal environment were exaimed,and its protective effect on acute alcoholic liver injury in mice was evaluated by animal experiments.Finally,double-crossover homologous recombination method was used to construct a food-grade co-expressing scADH and istALDH B.subtilis.The in vitro and in vivo experiments were carried out to study the ability of this food-grade recombinant strain to degrade ethanol at low pHs and the effect of alleviating acute alcoholic liver injury.The formulation of the protective agent for preparing the freeze-dried powder of the food-grade co-expressing strain was optimized by orthogonal experiment.The main findings are as follows:1.Secretory expression of istALDH in Bacillus subtilis.Firstly,the recombinant B.subtilis-1 harboring pBE/istALDH was constructed and the highest yield of istALDH in the fermentation supernatant was 113.71±3.62 U/mL at 36h.Then,the signal peptide was optimized and 5 high-efficiency signal peptides were screened.Among them,the yqzG signal peptide had the best effect,and the yield of istALDH was 204.85±3.31 U/mL(increased by 80.15%).Further,the aprE promoter was replaced by Pglv,P43 and P43-Pglv promoter.The yield of istALDH was 254.82±9.79 U/mL(increased by 19.97%),245.76±8.72 U/mL(increased by 12.40%)and 268.26±11.22U/mL(increased by 30.96%),respectively.Secondly,the fermentation conditions of the recombinant B.subtilis were optimized,and the fermentation was carried out at 35?and 2%inoculum.The optimized fermentation medium was 20 g/L peptone,20 g/L yeast extract,20 g/L glucose,20 g/L maltose and 5 g/L(NH4)2SO4.Under this condition,the yield of istALDH was 331.19±21.19 U/mL,which was increased by 191.26%compared with B.subtilis-1.Thirdly,the integrated expression recombinant B.subtilis was constructed and the yield of istALDH in the fermentation supernatant was 38.30±4.57 U/mL.Then,secrete chaperones csaA and prsA was overexpressed in this recombinant B.subtilis,the istALDH activity was 69.45±3.09 U/mL(increased by 81.36%)and 180.10±5.89 U/mL(increased by 370.28%),respectively.2.Co-expression of scADH and istALDH in Bacillus subtilis and its application in ethanol degradation.Firstly,an intracellular expression strategy was used to study the heterologous expression of scADH and istALDH in B.subtilis.After 48 hours of fermentation,the recombinant strain with intracellular expression of scADH had a dehydrogenation yield of 20.48±1.13 U/mL.The recombinant strain with intracellular expression of istALDH had an acetaldehyde dehydrogenase yield of 33.34±1.26 U/mL.Then,the recombinant B.subtilis co-expressing scADH and istALDH was constructed by double promoter strategy.The scADH and istALDH activity was 16.98±0.39 U/mL and 7.97±0.12 U/mL,respectively.Secondly,The whole cell catalyzed ethanol degradation of recombinant B.subtilis was studied by gas chromatography.The degradation rate of ethanol in 30 min was 46.47%at pH 8.0 and 14.67%at pH 2.0.The recombinant B.subtilis died in large amounts at pH 2.0,while was relatively stable in gastric juice at pH 3.0-5.0 and intestinal juice at pH 8.0.This might be the reason why the low degradation rate of ethanol in co-expressing recombinant B.subtilis at pH 2.0.Thirdly,the fermentation medium for this recombinant B.subtilis was optimized.The optimized fermentation medium was 50 ?g/mL kanamycin,60 g/L maltose syrup,30 g/L glucose,20 g/L corn syrup,4 g/L K2HPO4,5 g/L urea,0.7 g/L MgSO4 and 0.1 g/L MnSO4.The yields of scADH and istALDH was 188.83±18.65 and 160.89.97±4.07 U/mL,respectively,which was 3.35 and 20.18 times of the original yield,respectively.3.Protective effect of Bacillus subtilis co-expressing scADH and istALDH on acute alcoholic liver injury in mice.A model of acute alcoholic liver injury in mice was successfully established by continuous gavage for 15 days at a alcohol dose of 5.6 g/kg body weight/day alcohol and the effects of different doses of B.subtilis co-expressing scADH and istALDH on acute alcohol-induced liver injury were investigated.Recombinant B.subtilis significantly alleviated alcohol-induced increase in the liver index,blood alcohol content,and serum alanine aminotransferase,aspartate aminotransferase,and alkaline phosphatase activities(in BSM group:reduced by 9.36%,46.00%,41.89%,49.10%and 27.17%,respectively).Furthermore,the administration of ADH/ALDH-expressing B.subtilis inhibited lipid peroxidation and oxidative stress in the liver of alcohol-treated mice as evidenced by significant reduction of MDA and induction of T-AOC,SOD and GSH levels(in BSM group:MDA level reduced by 43.22%,T-AOC,SOD and GSH level increased by 114.37%,186.90%and 39.96%level).The recombinant strain also restored the expression of oxidative stress(CYP2E1),lipid metabolism(SREBP-1c,FAS and PPARa)and inflammation-related genes(TLR4 and TLR5)in liver to normal levels.Caecal microbiota diversity analysis indicated that administration of recombinant B.sublilis reversed alcohol-induced decrease in Firmicutes and increase in Proteobacteria,in particular pathogenic Helicobacter hepaticus,restoring the microbial composition in alcohol-treated mice(the relative abundance of H.hepaticus in AL group was1.12%,while in BSM group was 0.46%).In conclusion,recombinant B.subtilis expressing scADH and istALDH could act as a novel candidate for prevention of alcohol-induced acute liver injury.4.Construction of food-grade Bacillus subtilis co-expressing scADH and istALDH and preparation of active freeze-dried Bacillus subtilis powder.A food-grade recombinant B.subtilis co-expressing scADH and istALDH was successfully constructed by the double-crossover homologous recombination method.When cultured at 37? for 48 h,the activities of scADH and istALDH were 57.56±7.44 and 81.41±8.26 U/mL,respectively.In vitro experiments showed that recombinant B.subtilis cells co-expressing scADH and istALDH could be used as whole-cell biocatalysts for biodegradation of alcohols at low pHs.Under the condition of pH 2.0,1%ethanol and 109 cfu/mL recombinant B.subtilis,the 2-hour ethanol degradation rate could reach 50%.Animal experiments indicated that recombinant B.subtilis significantly alleviated alcohol-induced increases in mouse liver index,blood alcohol content,and serum alanine aminotransferase,aspartate aminotransferase,and alkaline phosphatase activities(reduced by 8.25%,45.00%,51.56%,22.86%and 36.55%,respectively).Furthermore,recombinant B.subtilis significantly reduced liver MDA level(26.86%)and increased T-AOC(14.55%)and SOD levels(20.89%)in mouse liver.Overall,our findings suggested that food-grade B.subtilis co-expressing scADH and istALDH could be used as a potential probiotic for alcohol detoxification and alleviation of alcoholic liver injury.The formulation of the protective agent for the preparation of active freeze-dried B.subtilis powder was optimized by orthogonal experiment.The optimal protective agent formula was 1%mannitol,6%sodium glutamate,6%trehalose,6%sorbitol and 6%sucrose,and the activities of scADH and istALDH were 1763.34 and 3071.35 U/g lyophilized powder,respectively. |
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