Metabolic Engineering Of Ethanologenic Bacterium Zymomonas Mobilis For Isobutanol Production

Due to the lack of global energy and serious environment problem,advanced and environmentally friendly biofuel attracts a lot of attention.Isobutanol has many advantages and is a promising candidate for the replacement of current fossil fuel.In recent years,many microorganisms were transformed for advanced biofuels production by metabolic engineering and synthetic biology approaches.Zymomonas mobilis,a generally regarded as safe status(GRAS)bacterium,has many advantages for biofuel production,such as high specific glucose uptake rate,high ethanol tolerance,low aeration cost in fermentation process,is an industrial platform for future biorefineries.This work used metabolic engineering methods to transform Z.mobilis for isobutanol production.We first tested the tolerance of Z.mobilis toward isobutanol at different isobutanol concentrations(0%,0.5%,1%,1.5%,2%,2.5%),and discovered Z.mobilis can tolerate up to 1.5% isobutanol.We then introduced a codon-optimized ketonic-acid decarboxylase gene kdc A of Lactococcus Lactis into Z.mobilis,and obtained a recombinant strain for isobutanol production successfully.Furthermore,we integrated the kdc A gene to the genome and a native plasmid of Z.mobilis through homologous recombination to improve the stability of gene expression,and obtained ZMQ1 and ZMQ2.The mutant ZMQ1 can produce 104 mg/L isobutanol with 1 ?g/m L tetracycline induction.We further changed the inducible promoter Ptet of the kdc A gene with a strong constitutive promoter Pgap or Peno to improve isobutanol production.Under the condition of overexpression of endogenous ketone reductoisomerase(ilv C)and dihydroxy acid dehydrating enzyme(ilv D),we compared endogenous acetolactate synthetase Zmals and three heterologous acetolactate synthetases(Ecals from Enterobacter cloacae,Blals from Bacillus licheniformis,Bsals from Bacillus subtilis).Our result indicated that the combination of Bsals,ilv C,ilv D was the best so far,reaching 4 g/L isobutanol.This work explored the possibility to develop Z.mobilis as a microbial cell factory to produce other biochemicals such as isobutanol,which can lay the foundation for future metabolic engineering practices in Z.mobilis.