|Schematic representation of the biosynthetic pathway of the 6-carbon alcohol 3-methyl-1-pentanol. The engineered nonnatural metabolic pathway is shaded in lavender. Click to enlarge. Credit: PNAS|
Researchers at UCLA have developed a nonnatural biosynthetic pathway enabling the bacteria Escherichia coli to produce various long-chain alcohols with carbon numbers ranging from 5 to 8. Higher carbon alcohols are attractive biofuel targets because they have higher energy density and lower water solubility. By way of comparison, ethanol has two carbons; butanol has four.
To demonstrate the feasibility of their approach, they optimized the biosynthesis of a 6-carbon alcohol: 3-methyl-1-pentanol. A paper on the work by Dr. James Liao and colleagues was published online 8 December in the Proceedings of the National Academy of Sciences.
In January 2008, Liao reported the genetic modification of E. coli for the efficient production of several higher-chain alcohols including isobutanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol and 2-phenylethanol. (Earlier post.) Instead of relying on fermentation for the production of the alcohols, Liao leveraged E. coli’s highly active amino acid biosynthetic pathway by shifting part of it (its 2-keto acid intermediates) to alcohol synthesis. Gevo, a biofuels startup, licensed that technology, and Liao joined Gevo’s scientific advisory board.
Nature uses a limited set of metabolites such as organic acids, amino acids, nucleotides, lipids and sugars as building blocks for biosynthesis. These chemicals support the biological functions of all organisms. So far, construction of artificial biological systems is limited by the existing metabolic capabilities. By supplying living cells with chemically synthesized nonnatural amino acids and sugars as new building blocks, it is possible to introduce novel physical and chemical properties into biological entities.
These efforts raise an interesting question: Can we rewire metabolism in a bottom-up fashion to produce nonnatural metabolites from simple carbon source? If so, such engineered artificial metabolism should be able to expand the chemical repertoire that living systems can use and produce. To begin to address this question, we developed a strategy to produce 7-(C7) to 9-carbon (C9) 2-keto acids, which can lead to useful nonnatural alcohols (C6–C8).—Zhang et al. 2008
In this work, Liao’ team first used the existing metabolic capability of E. coli to synthesize 2-keto-3-methylvalerate, the 2-keto acid precursor of amino acid Lisoleucine. The chemical structure of 2-keto-3-methylvalerate is similar to that of 2-ketoisovalerate, which is converted to a butanol precursor via chain elongation (increasing its carbon content) by a set of enzymes. The researchers reasoned the enzymes (LeuA, LeuB, LeuC and LeuD) might be promiscuous enough to take 2-keto-3-methylvalerate through the same elongation cycle and produce a novel compound: 2-keto-4-methylhexanoate.
They then speculated that the 2-keto-4-methylhexanoate could be converted to the corresponding aldehyde and then to the 6-carbon alcohol, (S)-3- methyl-1-pentanol, by an enzyme from the bacterium Lactococcus lactis (2-ketoisovalerate decarboxylase, KIVD) and another from the yeast Saccharomyces cerevisiae (alcohol dehydrogenase VI, ADH6).
They engineered three synthetic operons comprising 14 genes to overexpress all the enzymes. While the new pathway produced the 6-carbon sugar, the yield was low: one strain produced 6.5±1.1 mg/L of the 6-carbon alcohol from 20 g/L of glucose. Another strain produced 40.8±5.5 mg/L.
To improve yield, Liao and his team re-engineered two of the enzymes involved in the process. To reduce the formation of byproducts and drive the carbon flux toward the target C6 alcohol, they engineered KIVD with higher selectivity toward 2-keto-4-methylhexanoate. This resulted in production from one strain of 384.3±30.3 mg/L. They then modified LeuA, the other key enzyme determining the carbon flux toward 3-methyl-1-pentanol. This, combined with the KIVD mutant, pushed yield from one of the strains up to 793.5±46.5 mg/L.
For practical applications, further metabolic engineering and enzyme engineering will be needed to increase the production yield and rate of these compounds.—Zhang et al. 2008
The work was supported in part by the UCLA Department of Energy Institute for Genomics and Proteomics.
Kechun Zhang, Michael R. Saway, David S. Eisenberg, and James C. Liao (2008) Expanding metabolism for biosynthesis of nonnatural alcohols. PNAS doi: 10.1073/pnas.0807157106