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

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

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohols / metabolism*
  • Biosynthetic Pathways / genetics
  • Biosynthetic Pathways / physiology*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / biosynthesis
  • Escherichia coli Proteins / genetics
  • Metabolic Engineering / methods*
  • Pentanols / metabolism
  • Protein Engineering / methods*
  • Pyrophosphatases / biosynthesis
  • Pyrophosphatases / genetics
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Terpenes / metabolism

Substances

  • Alcohols
  • Escherichia coli Proteins
  • Pentanols
  • Recombinant Fusion Proteins
  • Terpenes
  • isopentyl alcohol
  • NudB protein, E coli
  • Pyrophosphatases