The Construction And Evaluation Of Zymomonas Mobilis ZM4Engineering Strains With Byproducts Producing-related Gene Unmarked Insertional Inactivation

With the situation of global fossil energy depletion and environmental pollutionworsening, it is more and more important to substitute non-renewable fossil energywith biological fuel ethanol. The starch raw material, sugariness raw material andcellulose fuel have become the main raw materials to produce ethanol. However, thefermentation strains still face a great challenge. As a bacteria with the strongestalcohol-producing ability, Zymomonas mobilis still have some shortcomings. Forexample, the ethanol yield is low, the byproducts is much, lignocellulosic materialscan not be utilized as substrate, and the resistance is poor.In order to identify genes related to the byproducts such as lactose, sorbitol andgluconic acid, etc., in the ethanol fermentation, to decrease the byproducts production,and to investigate the suitable sites for intergration of exogenous genes to broaden thecarbon source for Z.mobilis in this study, we firstly constructed lactoseproducing-related genes ldhA and D-ldhA-inactivated mutant strains ZM4(ldhA::FRT)and ZM4(D-ldhA::FRT), respectively, as well as sorbitol and gluconic acidproducing-related gene gfo-inactivated mutant strain ZM4(gfo::FRT). Secondly, withrecyclable use of the selectable marker gene cml, we further constructed the mutantstrains ZM4(ldhA::FRT)(D-ldh::FRT), ZM4(ldhA::FRT)(gfo::FRT) andZM4(ldhA::FRT)(D-ldh::FRT)(gfo::FRT), and thus established the stable andrepeatable method for constructing multiple genes-inactivated and antibioticselectable maker free mutant strains of Z. mobilis.Finally, the engineering strains were evaluate with byproducts, ethanol, growth asthe main indicator. The results of evaluation are as follows.1) ZM4(ldhA::FRT), ZM4(D-ldh::FRT) and ZM4(ldhA::FRT)(D-ldh::FRT): theLDH (lactate dehydrogenase) enzyme activity of three strains were0.609,0.566,0.594U/mg protein, which was lower than the strain ZM4(0.835U/mg protein);There was no big difference between the mutant strains and the wild-type strain in theproduction of lactic acid, but the mutant strains still had a certain reduction and thestrain of ZM4(ldhA::FRT)(D-ldh::FRT) produced the lowest lactic acid. The yield oflactic acid was lower in the MM medium than in the RM medium. TheZM4(ldhA::FRT)(D-ldh::FRT) strain produced more ethanol than the other three strains. Compared to0.900g/100g of ZM4, the yield of ethanol ofZM4(ldhA::FRT)(D-ldh::FRT) was0.945g/100g at12h in the medium of2%RM.The growth of the mutant strains was similar to the wild-type strain ZM4whether theRM medium or the MM medium.2) ZM4(gfo::FRT): ZM4(gfo::FRT) and ZM4strains were cultivated in richmedia containing different concentrations of sucrose, glucose, fructose or mixtures ofglucose and fructose to evaluate and analysis the effect of gfo inactivation on thegrowth and ethanol production. The results indicated that gfo inactivation preventedsorbitol production and thus, as expected, the ethanol yield had an obvious increasedin the medium containing sucrose or mixtures of glucose and fructose. Especially forthe high concentration of sucrose, the ethanol yield were85.06%and83.87%in20%and36%sucrose respectively, by comparison, the wild-type were66.27%and60.56%.. However, the production of ethanol had a little decrease with theconcentration of the glucose increasing when the gfo-inactivated mutant strain wascultivated in the media containing different concentrations of glucose. The ethanolyield of the engineering strain decreased22.26%when growing on the20%glucose.There was no big difference between the mutant strains and the wild-type strain in theproduction of ethanol when growing in the medium of fructose. Growth had a greatrelationship with the yield of ethanol, so better growth would be found in the mediumcontaining sucrose or mixtures of glucose and fructose. The OD600of the engineeringstrain could reach about8, whereas the wild-type was only5. ZM4(gfo::FRT)exhibited a long lag period in20%glucose, when grew normal in the fructose media.When sorbitol was added to the medium, the growth and fermentation ofZM4(gfo::FRT) would only be improved slightly in36%sucrose. But sorbitol couldrestored ZM4(gfo::FRT) to the level of ZM4in20%glucose medium.3)ZM4(ldhA::FRT)(gfo::FRT) and ZM4(ldhA::FRT)(D-ldh::FRT)(gfo::FRT): twostrains and the wild-type strain were cultivated in rich media containing36%sucrose,20%glucose or20%fructose, and then carried out a comparative evaluation.The results of the engineering strains showed that sorbitol was not produce and theyield of lactic acid was lower than ZM4. Meanwhile, the yield of ethanol wasincreased apparently and they were93.22and91.46g/L(ZM4was53.09g/L) in thestable period. Although there was certain delay of growth, the yield of lactic acid wasstill less than ZM4, and the final ethanol yield were77.35and88.33g/L respectively,which was higher than ZM4which was74.23g/L. The growth and fermentation of three strains in the fructose were similar. The gene inactivation led to anapproximately24h lag in sucrose digestion, but the final OD600beyond the wild typestrains. In the medium of20%glucose, the double knocking out strain began to growafter32h and the three knocking out strain began to grow after48h.ZM4, ZM4(gfo::FRT), ZM4(ldhA::FRT)(gfo::FRT) andZM4(ldhA::FRT)(D-ldh::FRT)(gfo::FRT) was cultivated in the fermentor in36%sucrose. The results showed that, the mutant strains grew slower than ZM4, but themaximum of OD600was higher than ZM4. The maximum ethanol concentration of thefour strains were58.78,93.48,108.17and107.52g/L respectively. The overall trendof fermentor was consist with the result of shake flask.