Construction And H₂S Formation As Well As Ethanol Production Of Mutant Strains Of Zymomonas Mobilis Deficient In Sulfate Reduction Pathway

Assimilatory sulfate reduction pathway is one of the ways to produce H₂S formicroorganisms. Though this pathway in Zymomonas mobilis has been shown byresults of bioinformatic research, and the relating genes have been predicted, however,research about the pathway including verification of function of the genes andregulation of the metabolic approach on experiment level has not been reported yet.At the same time, we found that Z. mobilis ZM4could generate the irritant odour gasin the process of ethanol production from sweet sorghum juice and it proved tocontain H₂S as the main component. Therefore, constructing the gene-inactivatedmutant strains of Z. mobilis deficient in sulfate reduction pathway and investigatingtheir H₂S production, would be of greatly scientific significance to verify theassimilatory sulfate reduction pathway and function of the relating genes, and furtherto recognize the whole sulfur metabolism in Z. mobilis.With a method of homologous recombination and FLP-FRT based site-specificrecombination in this study, we firstly constructed a series of mutant strains of Z.mobilis deficient in the assimilatory sulfate reduction pathway. And they wereATP-sulfurylase （coded by cysN and cysD） inactivated cysND-cat and cysND, andsulfite reductase （coded by cysI and cysJ） inactivated cysIJ-cat and cysIJ, as wellas the two enzyme co-inactivated cysND-cat cysIJ and cysND cysIJ-cat.This research tested the H₂S production of all the mutant strains in differentcarbon source-containing medium （glucose, sucrose, fructose and sweet sorghumjuice）. And results showed that all the mutant strains did not produce H₂S while thecontrol ZM4could generate H₂S in all the medium except the fructose-containingmedium at the condition tested. While SO₃^2-was added, only mutant strainscysND-cat and cysND could restore H₂S production but it was not the case for theother mutant strains, indicating that cysND and cysIJ did have relations to H₂Sproduction, and that CysND catalyze a reaction ahead of the intermediate SO₃^2-in theassimilatory sulfate reduction pathway and CysIJ behind SO₃^2-.The research also investigated the growth and ethanol production of the mutantstrains cysND, cysIJ and cysND-cat cysIJ in RM medium containing10%glucose and15%sucrose, respectively, as well as in the10%sweet sorghum juice inthe shaking flask. And results showed that: firstly, while SO₃^2-was not added into themedium, the mutant strains showed no apparent differences in growth pattern which was also near to the ZM4as a control （the maximum OD600was all about5）. Themaximum ethanol concentration of the mutant strains in RM medium containing10%glucose were47.4g/L,48.3g/L and47.3g/L, respectively, which were all higher thanthe result46.7g/L obtained by ZM4. The maximum ethanol concentration of themutant strains in RM medium containing15%sucrose were46.4g/L,46.2g/L and46.3g/L, respectively, which were all lower than the result47.7g/L obtained by ZM4,and the corresponding data for the mutant strains in10%sweet sorghum juice were35.0g/L,29.1g/L and34.7g/L, respectively, which were all lower than the result46.2g/L obtained by ZM4as well. Secondly, while SO₃^2-was added, in RM mediumcontaining10%glucose, cysND showed the lag phase of growth for24h （the lagphase for ZM4was32h）, and the maximum OD600was about5（the maximum OD600for ZM4was also about5） and the maximum ethanol concentration was44.9g/L（thecorresponding result obtained by ZM4was43.3g/L）. The growth of cysIJ andcysND-cat cysIJ was completely retarded in the whole cultivation process, and theethanol could therefore not be detected; in RM medium containing15%sucrose,cysND also showed the lag phase of growth for24h （the lag phase for ZM4was also32h）, but the maximum OD600was about5.5which was also near to the ZM4, and themaximum ethanol concentration was48.8g/L （the corresponding result abtained byZM4was51.2g/L）. The lag phase of growth of cysIJ and cysND-cat cysIJ wereall about40h, and the maximum OD600were about3³.5and the maximum ethanolconcentration were40.6g/L and48.6g/L, respectively. All the varying resultsindicated that the effect of cysND-and cysIJ-inactivation on the growth and ethanolproduction of the mutant strains would be dependent greatly on the substrate, i.e., thetypes of the carbon sources.Besides, the ethanol concentration and yield in the fermentation of10%sweetsorghum juice by the mutant strains of Z. mobilis in the fermenters were all lower thanthe results obtained by ZM4, which was in good agreement with the results showed inthe shaking flask as mentioned above. And the ethanol concentration and yieldobtained by ZM4at the same conditions were47.41g/L and0.42. The result wasshown to well match the data reported in the ethanol production from sweet sorghumjuice by other Z. mobilis strains, and this was also the first report of ZM4strain usedin the ethanol fermentation of sweet sorghum juice.