Engineering Synthetic Non-Fermentative Pathways for Production of Branched-Chain Higher Alcohols as Biofuels
Atsumi, S.; T. Hanai and J.C. Liao (2008) Non-Fermentative Pathways for Synthesis of Branched-Chain Higher Alcohols as Biofuels, Nature, 451:86-89
Compared
to the commercially available biofuel, ethanol, higher alcohols offer advantages
as gasoline substitutes because of their higher energy density and
hydrophobicity. In addition, branched-chain alcohols have higher octane numbers
compared to their straight-chain counterparts. However, these alcohols cannot
be synthesized economically using native organisms. Here we present a synthetic
approach using Escherichia coli to produce higher alcohols including
isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and
2-phenylethanol from a renewable carbon source, glucose. This strategy
leverages the host’s highly active amino acid biosynthetic pathway and diverts
its 2-keto acid intermediates
for alcohol synthesis. In particular, we
have achieved high yield, high specificity production of isobutanol from
glucose. The strategy enables the exploration of biofuels beyond those naturally
accumulated to high quantities in microbial fermentation.
2-Propanol production by E. coli
Hanai, T.; S. Atsumi; J. C. Liao (2007) Engineered synthetic pathway for isopropanol production in Escherichia coli, Appl. Env. Microbiol. 73:7814–7818
We engineered a synthetic pathway into Escherichia coli to produce isopropanol by heterologous expression of the Clostridial metabolic pathway which utilizes the metabolite acetyl-CoA. The pathway was constructed using various combinations of genes from Clostridium acetobutylicum ATCC824, E. coli K-12 MG1655, Clostridium beijerinckii NRRL B593 and Thermoanaerobacter brockii HTD4. For isopropanol production, the strain with the combination of genes thl (acetyl-CoA acetyltransferase from C. acetobutylicum), atoAD (acetoacetyl-CoA transferase from E. coli), adc (acetoacetate decarboxylase from C. acetobutylicum) and adh (secondary alcohol dehydrogenase from C. beijerinckii) achieved the highest titer. This strain produced 81.6 mM isopropanol in a glucose fed batch shake flask experiment with a yield of 43.5% (mol/ mol) in the production phase. To our knowledge, this work is the first to produce isopropanol in E. coli, and the amount produced exceeded that from native producers. When the alcohol dehydrogenase was not used, the strain produced acetone up to 148.3 mM and achieved 73.5% (mol/mol) of conversion in the production phase.
1-Butanol production by E. coli
Atsumi, S.; A. F. Cann; M. Connor; C.R. Shen; K. M. Smith; M.P. Brynildsen; K.J.Y. Chou; T. Hanai, and J. C. Liao (2008) Metabolic engineering of Escherichia coli for 1-butanol Production, Metabolic Eng. In print
Compared to ethanol, butanol offers many advantages as a substitute for gasoline because of higher energy content and higher hydrophobicity. Typically, 1-butanol is produced by Clostridium in a mixed-product fermentation. To facilitate strain improvement for specificity and productivity, we engineered a synthetic pathway in Escherichia coli and demonstrated the production of 1-butanol from this non-native user-friendly host. Alternative genes and competing pathway deletions were evaluated for 1-butanol production. Results show promise for using E. coli for 1-butanol production.