The Knockout Of Ldh And Ack In K.oxytoca HD79 Improves The Production Of 2,3-butanediol

2,3-Butanediol(2,3-BD)and its derivates are multi-functional platform chemicals that can be widely applied in fields of chemicals,foods,pharmaceuticals,fuels and aerospace.Therefore,the costly,complicated,and pollutional 2,3-BD production by chemical synthesis draw strong interests in microbial production of 2,3-BD production from renewable biomass as an alternative way with soft conditions,safety and green environmental process.As 2,3-BD producer,Klebisella oxytoca was considered as most promising industrialized potential strain due to the advantages of adaptable to circumstances,broad substrates spectrum,metabolic completely,less by-products,higher conversion efficiency and product concentration.However,the accumulation of acidic by-products such as lactate and acetate is limitation to further enhancement of 2,3-BD production for their inhibition to cell growth at higher concentrations,which not only decrease 2,3-BD yield but also increase the difficulty as subsequently isolation and purification of2,3-BD.ldh and ack are the key enzyme genes encoding lactate dehydrogenase and acetate kinase in their metabolic routes.In this paper,?Red homologous DNA technology was used to knock out ldh and ack genes one by one to interrupt the generation of lactate and acetate respectively,which led more carbon flux to 2,3-BD pathway and a high-yielding strain was desirable.First of all,genome DNA of K.oxytoca HD79 preserved in the lab with ability to produce 2,3-BD was served as template to clone two homologous sequence of lactate dehydrogenase that were 489 bp and 400 bp,pGP704-Cm was used as template to clone resistance gene sequence of Chloramphenicol that was 876 bp.The Cm~r gene was inserted internal of ldh gene by splicing operations in vitro through the way of digestion and connection and homologous recombination sequence ldhL-Cm~r-ldhR was constructed.The recombinant strain K.oxytoca HD79-01 successfully screened by?Red homologous DNA technology was verified by PCR detection,qRT-PCR,SDS-PAGE,determination of ldh enzyme activity and 2,3-BD yield.The results indicated that the ldh enzyme activity of K.oxytoca HD79 that was 0.254±0.01 U/mg was 55.9%less than that of K.oxytoca HD79-01 that was 0.112±0.02 U/mg.The yield,convert ratio and production intensity of the recombinant strain were 40.20 g/L,0.38 g/g and 0.48 g/L·h respectively,26.8%,11.8%,45.5%higher than that of parent strain which were 31.71g/L,0.34 g/g,0.33 g/L·h respectively;while the yield of lactate of recombinant strain was 49.3%less than the parent strain from 4.83 g/L to 2.45 g/L.Secondly,genome DNA of the recombinant strain K.oxytoca HD79-01 preserved was served as template to clone two homologous sequence of acetate kinase that were308 bp and 312 bp,pET-28a(+)was used as template to clone resistance gene sequence of Kanamycin that was 813 bp.The Kan~r gene was inserted internal of ack gene by splicing operations in vitro through the way of digestion and connection and homologous recombination sequence ackL-Kan~r-ackR was constructed.The recombinant strain K.oxytoca HD79-02 successfully screened by?Red homologous DNA technology was verified by PCR,qRT-PCR,SDS-PAGE,determination of ack enzyme activity and 2,3-BD yield.The results indicated that the ack enzyme activity of K.oxytoca HD79 that was 0.233±0.003 U/mg was 73.4%less than that of K.oxytoca HD79-02 that was 0.062±0.02 U/mg.The yield,productivity and production intensity of the recombinant strain K.oxytoca HD79-02 were 46.21 g/L,0.47 g/g and 0.64 g/L·h respectively,54.9%,20.5%,106.5%higher than that of parent strain which were 29.83g/L,0.39 g/g,0.31 g/L·h respectively;while the yield of lactate and acetate of recombinant strain were 48.2%and 62.8%less than the parent strain from 4.94 g/L to2.45 g/L and 4.28 g/L to 1.59 g/L respectively.The double knock-out of ldh and ack genes was successfully and the engineering strain of K.oxytoca HD79-02 was constructed.Compared with K.oxytoca HD79,The yield,productivity and production intensity of the recombinant strain were 54.9%,20.5%,106.5%higher than that of parent strain.The study providing guiding significance to construct 2,3-BD high producing strain as well as amplifying bacterial source of industrial production of 2,3-BD.