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

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 ; Lee, Taek Soon

Scientific reports, 2015-06, Vol.5 (1), p.11128-11128, Article 11128 [Periódico revisado por pares]

England: Nature Publishing Group

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Título:
Metabolic engineering for the high-yield production of isoprenoid-based C₅ alcohols in E. coli
Autor: 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 ; Lee, Taek Soon
Assuntos: Alcohol ; Alcohols ; Alcohols - metabolism ; BASIC BIOLOGICAL SCIENCES ; Biofuels ; Biosynthetic Pathways - genetics ; Biosynthetic Pathways - physiology ; Butanol ; E coli ; Enzymes ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Escherichia coli - metabolism ; Escherichia coli Proteins - biosynthesis ; Escherichia coli Proteins - genetics ; Fusion protein ; IPP isomerase ; Isopentenyl diphosphate ; Isopentyl alcohol ; Kinases ; Metabolic engineering ; Metabolic Engineering - methods ; Mevalonate kinase ; Mevalonic acid ; Pentanols - metabolism ; Phosphatase ; Protein Engineering - methods ; Protein folding ; Pyrophosphatases - biosynthesis ; Pyrophosphatases - genetics ; Recombinant Fusion Proteins - genetics ; Recombinant Fusion Proteins - metabolism ; Reductase ; Terpenes - metabolism
É parte de: Scientific reports, 2015-06, Vol.5 (1), p.11128-11128, Article 11128
Notas: ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
AC02-05CH11231
USDOE Office of Science (SC), Biological and Environmental Research (BER)
Descrição: 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.
Editor: England: Nature Publishing Group
Idioma: Inglês

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

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 ; Lee, Taek Soon

England: Nature Publishing Group

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