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Nature uses a limited set of metabolites to perform all of the biochemical reactions. To increase the metabolic capabilities of biological systems, we have expanded the natural metabolic network, using a nonnatural metabolic engineering approach. The branched-chain amino acid pathways are extended to produce abiotic longer chain keto acids and alcohols by engineering the chain elongation activity of 2-isopropylmalate synthase and altering the substrate specificity of downstream enzymes through rational protein design. When introduced into Escherichia coli, this nonnatural biosynthetic pathway produces various long-chain alcohols with carbon number ranging from 5 to 8. In particular, we demonstrate the feasibility of this approach by optimizing the biosynthesis of the 6-carbon alcohol, (S)-3-methyl-1-pentanol. This work demonstrates an approach to build artificial metabolism beyond the natural metabolic network. Nonnatural metabolites such as long chain alcohols are now included in the metabolite family of living systems.metabolic engineering ͉ protein engineering ͉ chain elongation ͉ long chain alcohols N ature 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 (1-5) is limited by the existing metabolic capabilities. By supplying living cells with chemically synthesized nonnatural amino acids (6, 7) and sugars (8) 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).Aliphatic alcohols with carbon chain length Ͼ5 (C Ͼ 5) are attractive biofuel targets because they have higher energy density, and lower water solubility [1-pentanol 23 g/L, 1-hexanol 6.2 g/L, 1-heptanol 1.2 g/L (9)] that could facilitate postproduction purification from culture medium through an aqueous/organic 2-phase separation process. The only well-characterized mechanism for aliphatic alcohol production is through the Ehrlich pathway (10), which converts branched-chain amino acids into alcohols. The carbon number (up to 5) of the alcohols derived from this type of pathway is limited by the carbon number in the branched chain amino acid pathways (11). To overcome this limitation, existing metabolic networks need to be expanded. This is a daunting task because a metabolic pathway usually involves the collective function of multiple enzymes, which have to be engineered by rational design (12) or directed evolution (13,14) to perform nonnat...

Significance In recent years there has been extensive research toward the development of sustainable polymeric materials. However, environmentally benign, bioderived polymers still represent a woefully small fraction of plastics and elastomers on the market today. To displace the widely useful oil-based polymers that currently dominate the industry, a bioderived synthetic polymer must be both cost and performance competitive. In this paper we address this challenge by combining the efficient bioproduction of β-methyl-δ-valerolactone with controlled polymerization techniques to produce economically viable block polymer materials with mechanical properties akin to commercially available thermoplastics and elastomers.

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Conversion of lignocellulosic biomass into value-added products provides important environmental and economic benefits. Here we report the engineering of an unconventional metabolism for the production of tricarboxylic acid (TCA)-cycle derivatives from D-xylose, L-arabinose and D-galacturonate. We designed a growth-based selection platform to identify several gene clusters functional in Escherichia coli that can perform this nonphosphorylative assimilation of sugars into the TCA cycle in less than six steps. To demonstrate the application of this new metabolic platform, we built artificial biosynthetic pathways to 1,4-butanediol (BDO) with a theoretical molar yield of 100%. By screening and engineering downstream pathway enzymes, 2-ketoacid decarboxylases and alcohol dehydrogenases, we constructed E. coli strains capable of producing BDO from D-xylose, L-arabinose and D-galacturonate. The titers, rates and yields were higher than those previously reported using conventional pathways. This work demonstrates the potential of nonphosphorylative metabolism for biomanufacturing with improved biosynthetic efficiencies.