Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli
Abstract
Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Lastly, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.
- Authors:
-
- 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)
- Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); Univ. of California, Berkeley, CA (United States)
- Publication Date:
- Research Org.:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1256054
- Grant/Contract Number:
- AC02-05CH11231
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Scientific Reports
- Additional Journal Information:
- Journal Volume: 5; Journal ID: ISSN 2045-2322
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES
Citation Formats
George, Kevin W., Thompson, Mitchell G., Kang, Aram, Baidoo, Edward, Wang, George, Chan, Leanne Jade G., Adams, Paul D., Petzold, Christopher J., Keasling, Jay D., and Soon Lee, Taek. Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli. United States: N. p., 2015.
Web. doi:10.1038/srep11128.
George, Kevin W., Thompson, Mitchell G., Kang, Aram, Baidoo, Edward, Wang, George, Chan, Leanne Jade G., Adams, Paul D., Petzold, Christopher J., Keasling, Jay D., & Soon Lee, Taek. Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli. United States. https://doi.org/10.1038/srep11128
George, Kevin W., Thompson, Mitchell G., Kang, Aram, Baidoo, Edward, Wang, George, Chan, Leanne Jade G., Adams, Paul D., Petzold, Christopher J., Keasling, Jay D., and Soon Lee, Taek. Mon .
"Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli". United States. https://doi.org/10.1038/srep11128. https://www.osti.gov/servlets/purl/1256054.
@article{osti_1256054,
title = {Metabolic engineering for the high-yield production of isoprenoid-based C5 alcohols in E. coli},
author = {George, Kevin W. and Thompson, Mitchell G. and Kang, Aram and Baidoo, Edward and Wang, George and Chan, Leanne Jade G. and Adams, Paul D. and Petzold, Christopher J. and Keasling, Jay D. and Soon Lee, Taek},
abstractNote = {Branched five carbon (C5) alcohols are attractive targets for microbial production due to their desirable fuel properties and importance as platform chemicals. In this study, we engineered a heterologous isoprenoid pathway in E. coli for the high-yield production of 3-methyl-3-buten-1-ol, 3-methyl-2-buten-1-ol, and 3-methyl-1-butanol, three C5 alcohols that serve as potential biofuels. We first constructed a pathway for 3-methyl-3-buten-1-ol, where metabolite profiling identified NudB, a promiscuous phosphatase, as a likely pathway bottleneck. We achieved a 60% increase in the yield of 3-methyl-3-buten-1-ol by engineering the Shine-Dalgarno sequence of nudB, which increased protein levels by 9-fold and reduced isopentenyl diphosphate (IPP) accumulation by 4-fold. To further optimize the pathway, we adjusted mevalonate kinase (MK) expression and investigated MK enzymes from alternative microbes such as Methanosarcina mazei. Next, we expressed a fusion protein of IPP isomerase and the phosphatase (Idi1~NudB) along with a reductase (NemA) to diversify production to 3-methyl-2-buten-1-ol and 3-methyl-1-butanol. Lastly, we used an oleyl alcohol overlay to improve alcohol recovery, achieving final titers of 2.23 g/L of 3-methyl-3-buten-1-ol (~70% of pathway-dependent theoretical yield), 150 mg/L of 3-methyl-2-buten-1-ol, and 300 mg/L of 3-methyl-1-butanol.},
doi = {10.1038/srep11128},
journal = {Scientific Reports},
number = ,
volume = 5,
place = {United States},
year = {Mon Jun 08 00:00:00 EDT 2015},
month = {Mon Jun 08 00:00:00 EDT 2015}
}
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