High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

Kang, Aram; Meadows, Corey W.; Canu, Nicolas; Keasling, Jay D.; Lee, Taek Soon

doi:10.1016/j.ymben.2017.03.010

Title: High-throughput enzyme screening platform for the IPP-bypass mevalonate pathway for isopentenol production

Journal Article · Wed Apr 05 00:00:00 EDT 2017 · Metabolic Engineering

DOI:https://doi.org/10.1016/j.ymben.2017.03.010· OSTI ID:1379821

Kang, Aram ^[1]; Meadows, Corey W. ^[1]; Canu, Nicolas ^[2]; Keasling, Jay D. ^[3]; Lee, Taek Soon ^[1]

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

Isopentenol (or isoprenol, 3-methyl-3-buten-1-ol) is a drop-in biofuel and a precursor for commodity chemicals such as isoprene. Biological production of isopentenol via the mevalonate pathway has been optimized extensively in Escherichia coli, yielding 70% of its theoretical maximum. However, high ATP requirements and isopentenyl diphosphate (IPP) toxicity pose immediate challenges for engineering bacterial strains to overproduce commodities utilizing IPP as an intermediate. To overcome these limitations, we developed an “IPP-bypass” isopentenol pathway using the promiscuous activity of a mevalonate diphosphate decarboxylase (PMD) and demonstrated improved performance under aeration-limited conditions. However, relatively low activity of PMD toward the non-native substrate (mevalonate monophosphate, MVAP) was shown to limit flux through this new pathway. By inhibiting all IPP production from the endogenous non-mevalonate pathway, we developed a high-throughput screening platform that correlated promiscuous PMD activity toward MVAP with cellular growth. Successful identification of mutants that altered PMD activity demonstrated the sensitivity and specificity of the screening platform. Strains with evolved PMD mutants and the novel IPP-bypass pathway increased titers up to 2.4-fold. Further enzymatic characterization of the evolved PMD variants suggested that higher isopentenol titers could be achieved either by altering residues directly interacting with substrate and cofactor or by altering residues on nearby α-helices. These altered residues could facilitate the production of isopentenol by tuning either k_cat or K_i of PMD for the non-native substrate. The synergistic modification made on PMD for the IPP-bypass mevalonate pathway is expected to significantly facilitate the industrial scale production of isopentenol.

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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)

Grant/Contract Number:: AC02-05CH11231

OSTI ID:: 1379821

Alternate ID(s):: OSTI ID: 1417504

Journal Information:: Metabolic Engineering, Vol. 41, Issue C; ISSN 1096-7176

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 32 works

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Metabolic engineering of Escherichia coli for production of mixed isoprenoid alcohols and their derivatives Zada, Bakht; Wang, Chonglong; Park, Ji-Bin Biotechnology for Biofuels, Vol. 11, Issue 1 https://doi.org/10.1186/s13068-018-1210-0	journal	July 2018
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