Metabolic Engineering Of Clostridium Aceticum For Ethanol Production From CO₂

Bioethanol,as an available and clean fuel for engines,has became an increasingly common alternative to gasoline.Clostridium aceticum is an obligately anaerobic acetogen which can grow autotrophically with H₂+CO₂ or cheterotrophically with fructose,pyruvate or fumarate.C.aceticum can convert H₂+CO₂ to acetyl-CoA via the Wood-Ljungdahl pathway,and then form the acetic acid.Aldehyde Ferredoxin Oxidoreductase（AOR）can reduce acetate to acetaldehyde and under the strict anaerobic condition.Acetaldehyde can be reduced to ethanol by Alcohol dehydrogenase（AdhA）.Previous studies showed that the AOR enzymes from Pyrococcus furiosus and Thermococcus strain ES-1could catalyze the reduction of acetate to aldehyde.This thesis reports metabolic engineering of C.aceticum by overexpressing the aor gene for ethanol production from CO₂.The main results of this thesis provides a new way to constructing industrial microorganism which can produce ethanol from CO₂.（1）Bioinformatics analysis of C.aceticum AOR: Four AOR enzymes coding sequence were searched from the genome sequence of C.aceticum,the phynogenetic tree was constructed based on amino acid sequence of AOR enzyme after multiple sequence alignment.Two AOR enzymes which are numbered as WP₄4825795.1 and WP₄4824935.1 were selected on basis of the evolutionary relationship.Positioning analysis of the sequences annotated three structure domains of the C.aceticum AOR enzyme,including dhV,AFOR_N and AFOR_C.It is verified that the selected two enzyme share same catalytic activity with Thermococcus sp.EP1 and P.furiosus AOR enzymes.（2）Structure prediction of C.aceticum AOR: The secondary structure prediction of AOR enzyme protein showed that the protein numbered as WP₀44825795.1 had more similarity with P.furiosus AOR enzyme,and the enzyme structure differences of different proteins came from the Helix（H）and folding（E）.Tertiary structure of the target protein was predicted by homologymodeling,the results showed that the differences of the tertiary structure between target protein and template protein were concentrated on two terminals of the conservative region,and the highly consistent structures were concentrated on the interior of conservative region.（3）Upstream and downstream sequences of AOR enzyme conding gene:The results of visualizing C.aceticum genome showed that the reading frame direction of AOR coding sequence was against to the direction of its upstream and downstream genes.According to the results of searching and analyzing the upstream and downstream sequences of AOR enzyme conding gene,the ribosome binding site “AGGAGG” was within the range of-10 to-15,the“Pribnow” box was within the range of-17 to-26.（4）Metabolic prediction of AOR overexpressing C.aceticum:Reconstructing the metabolic pathway of C.aceticum on basis of genome-wide enzyme protein,the results showed that the genome-wide enzyme proteins of C.aceticum mainly act on biological metabolism,genetic information processing,signal processing,cell processes,organic system environment and the pathogenic system.Mapping the metabolic network and forecasting the metabolic change of C.aceticum after overexpressing AOR enzyme,the results showed that the recombinant strain would convert acetic acid to ethanol.Besides,it was predicted that the AOR overexpressing C.aceticum would also produce L-lactic acid,formic acid and intermediate in the tricarboxylic acid cycle.（5）Constructing AOR expressing C.aceticum: Three pairs of primers were designed,including aor-prim,aor-prim2 and aor-prim3.The target sequences were obtained after PCR reaction under the optimized annealing temperature.The recombinant plasmids pMTL82151_thl_aor,aor2 and aor3 were obtained after ligation,transformation screening and verification.The recombinant plasmids were then transfered into the wildtype C.aceticum from the recombinant E.coli CA434 through conjugation.Recombinant C.aceticum strains which overexpressed aor,aor2 and aor2 genes were obtained after screening and verification.（6）Analyzing the metaboiltes of wild type and recombinant C.aceticum:The fermentation results of C.aceticum showed that the exclusive metaboilte of wild type C.aceticum and pMTL82151_thl_aor containing C.aceticum wasacetic acid.Besides,metaboiltes of recombinant C.aceticum strains which contained plasmid p MTL82151_thl_aor2 and pMTL82151_thl_aor3 included succinic acid,lactic acid,formic acid,acetic acid and ethanol.