Co-expression Of Alcohol Dehydrogenase And Aldehyde Dehydrogenase In Lactococcus Latctis And The Protective Effects Of Recombinant Cells On Acute Alcoholic Liver Injury In Mice

Alcohol dehydrogenase(ADH)and aldehyde dehydrogenase(ALDH)play important roles in catalyzing the oxdization of ethanol into non-toxic acetate with NAD+or NADP+working as co-enzyme.However the inactive ALDH2*2 ellele and the highly actice ADHIB*2 in East Asian population resulted in the alcohol use disorders,which can further lead to alcoholic deseases.An effective way to attenuate the damage caused by ehanol is the exogenous supply of these two enzymes in stomach as they might accelerate the oxidation of ethanol into nontoxic acetate.However the two en2ymes might lose activity at low pH value and expressing them in E.coli cells is not allowed in food industry.Lactic acid bacteria is one of the probiotics that are widely used in foods and it can survive the gastric intestinal tracts which implys that it is a perfect organism for the delivery of ADH and ALDH.Moreover there was no report on expression of ADH and ALDH in LAB at present.In a previous study in this lab,IstALDH was cloned from Issatchenkia terricola and expressed in E.coli.In the current study,IstALDH was expressed in Lactococcu lactis NZ3900 and used as whole-cell catalysts in catalyzing detoxification of acetaldehyde at low pH value.Moreover ScADH and IstALDH were co-expressed in NZ3900 via the NICE expression system and used as treatments towards acute alcoholic liver injury in mice.A new food grade selection marker based on the suriactin immune gene was appiled in the expression of IstALDH.The main findings are as follows:1.Expressing of IstALDH in L.lactis NZ3900 and catalyzing detoxification of acetaldehyde with L lactis cells expressing IstALDH as whole cell catalysts.A recombinant L.lactis NZ3900/pNZ8148-istALDH was constructed and the nisin induction conditions were optimized.An ALDH activity of 36.4 U/mL was got when induced with 50 ng/mL nisin for 2 h at 20? as the OD600 reached 0.5.A remaining ALDH activity of 15%was detected in L.lactis expressing IstALDH when kept in a pH 3.0 to 5.0 enviroment while neither the crude enzyme nor the E.coli expressing IstALDH could have any ALDH activity.The resting cells of L.lactis expressing IstALDH showed a 2.5 fold higher activity and better stability in catalyzing oxidation of acetaldehyde at pH 2.0 compared with that of E.coli via a GC analysis method.2.Cloning,expression and purification of ScADH in NZ3900 and characterization of ScADH.A recombinant L.lactis NZ3900/pNZ8148-istALDH was constructed and an ADH activity of 75.09 U/mL was got when induced with nisin at 50 ng/mL.The ScADH was purified by 19.2 fold throuth Blue Sepharose 6FF infinity chromatograph followed by a QFF ion-exchange chromatograph and yield of 14.3%was got.The ScADH was found to have the highest activity at 40? and be stable from 25 to 37?.The optimial pH of ScADH was 8.5.These parameters are quite similar to that of IstALDH.3.Co-expression of ScADH and IstALDH in L.lactis under two separate nisA promoter,and preparation of freeze-dried powders of recombinant L.lactis expressing ScADH and IstALDH.The co-expression of ScADH and IstALDH was carried out via three methods,the infusion of ScADH and IstALDH,under one nisA promoter with a shine-dalgano sequence between the two genes and under two separate nisA promoter.Activity of ScADH and IstALDH was got only with the method of co-expression under two separate promoter.A recombinant NZ3900/pNZ8148-GBD was constructed and an ADH activity of 6.6 U/mL plus an ALDH activity of 1.4 U/mL was got when induced with nisin.A cryoprotectant consists 10%skim milk,4%glycerol and 4%soudium glutamate was applied in the freeze-drying of L.lactis recombinant SeADH-IstALDH.Cells were diluted in cryoprotectant at 10 A 8 CFU/mL and freezed in liquid nitrogen followed by a 16 h storage in-80?.After freeze-drying process,a cell survival rate of 90.8%was got and 82.4%of ADH activity was remained while the remaining activity of ALDH turned to be 81.3%.4.A dose dependent effect of freeze-dried powders of L.lactis expressing ScADH and IstALDH on attenuating acute alcoholic liver damage in ethanol administered mice.Intragastric ethanol administration was carried out at 5.6 g/kg body weight per day in mice for 15 consecutive days and different doses of recombinant L.lactis treatment were administrated together with ethanol.A high dose of L.lactis recombinant ADH-ALDH treatment(ADH activity of 2000 U/kg BW and ALDH activity of 1000 U/kg BW)reduced the serun alanine aminotransferase,aspartate aminotransferase and alkaline phosphatase levels by 38.1%,54.8%and 23.2%in ethanol-treated mice.Moreover,it also helped maintaining serum lipid levels and liver oxidative stress parameters against ethanol administration.5.Gene swrC,a surfactin immunite gene,working as a food grade selection marker in LAB,and expression of IstALDH with plasmid based on swrC selection.The swrC gene was cloned from the genome DNA of Bacillus subtillus 168 and expressed in NZ3900 with plasmid pMG36e.The transformant with swrC gene developed resistance towards surfactin and found to survi've a surfactin concentration of 100 ?g/mL while the tranformant with plasmid pMG36e only did not.A pepN promoter was amplified form pNZ7021 to replace the P32 promoter in pMG36e and a plasmid pFMB4191 with PpepN was got.The swrC gene was expressed with pFMB4191 under PpepN in Lactobacillus plantarum 163 and was also found to be effective in L.plantarum 163.An IstALDH expression plasmid pFMB4197 was constructed which was selective under either erythrocin or surfactin and NZ3900 was transformed with pFMB4197.The segregational stability of pFMB4197 under surfactin of different concentrations was measured and a remaining of 95%plasmid was found after growing with 100 ?g/mL surfactin for 18 h.The IstALDH activity of the NZ3900 harboring pFMB4197 cultured in GM17 broth with surfaction concentration of 100 ?g/mL surfactin for 18 h was measured and an activity of 2.6 U/mL was got.