Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.

doi:10.1021/acssynbio.6b00115

Title: Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

Journal Article · Tue Jul 12 00:00:00 EDT 2016 · ACS Synthetic Biology

DOI:https://doi.org/10.1021/acssynbio.6b00115· OSTI ID:1393096

Brown, Margaret E. ^[1]; Mukhopadhyay, Aindrila ^[1]; Keasling, Jay D. ^[2]

Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States)
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Univ. of California, Berkeley, CA (United States); Technical Univ. of Denmark, Horsholm (Denmark)

In this paper, 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. Finally, 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.

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Research Organization:: Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); Defense Advanced Research Projects Agency (DARPA) (United States)

Grant/Contract Number:: AC02-05CH11231; HR001134783

OSTI ID:: 1393096

Journal Information:: ACS Synthetic Biology, Vol. 5, Issue 12; ISSN 2161-5063

Publisher:: American Chemical Society (ACS)Copyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 4 works

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Title: Engineering Bacteria to Catabolize the Carbonaceous Component of Sarin: Teaching E. coli to Eat Isopropanol

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