title: Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol.
abstract: We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis. Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls and was able to grow on isopropanol as a sole carbon source. In the process, reconstitution of this large ACX complex (370 kDa) in a system naïve to its structural and mechanistic requirements allowed us to study this otherwise cryptic enzyme in more detail than would have been possible in the less genetically tractable native Xanthobacter system.
authors.affiliation: Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: Margaret E
authors.initials: ME
authors.lastname: Brown
authors.affiliation: Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|Joint BioEnergy Institute , Emeryville, California 94608, United States.
authors.forename: Aindrila
authors.initials: A
authors.lastname: Mukhopadhyay
authors.affiliation: Biological Systems and Engineering Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.|Joint BioEnergy Institute , Emeryville, California 94608, United States.|Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kogle Alle, DK2970-Hørsholm, Denmark.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D019840:2-Propanol / Q000378:metabolism*; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D005818:Genetic Engineering / Q000379:methods*; D060847:Metabolic Engineering / Q000379:methods; D009876:Operon; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012524:Sarin / Q000378:metabolism*; D020584:Xanthobacter / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D012524:Sarin; D000426:Alcohol Dehydrogenase; D002262:Carboxy-Lyases; C100963:acetone carboxylase; D003066:Coenzyme A Ligases; C023747:acetoacetyl-CoA synthetase; D019840:2-Propanol
keywords: acetone carboxylase; biodegradation; bioengineering; carbon catabolism pathway; sarin; synthetic biology