Format

Send to

Choose Destination
Sci Rep. 2015 Jun 8;5:11128. doi: 10.1038/srep11128.

Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli.

Author information

1
1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.
2
1] Joint BioEnergy Institute, Emeryville, CA 94608, USA [2] Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA [3] Department of Bioengineering, University of California, Berkeley, CA 94720, USA [4] Department of Chemical &Biomolecular Engineering, University of California, Berkeley, CA 94720, USA.

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. Finally, 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.

PMID:
26052683
PMCID:
PMC4459108
DOI:
10.1038/srep11128
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for Nature Publishing Group Icon for PubMed Central
Loading ...
Support Center