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Bio-based renewable chemical production is becoming an increasingly important process for the production of industrial bulk and specialty chemicals due to concerns over finite nature of petroleum and other fossil-based resources, as well as the potential net greenhouse gas emissions resulting from the use of these feedstocks, which are thought to have an adverse effect on global climate. Potential advantages related to process safety and green chemistry as well as opportunities to develop value added chemicals not readily obtainable via traditional petrochemical processes are also propelling these trends.

Feedstocks for current biocatalytic processes are either C5-C6 sugars, obtained from hydrolytic degradation of starch, cellulose, and hemicellulose, or oils and fats extracted from vegetables and animals. Extensive studies have focused on the development of biochemical pathways to convert sugars into fuels and chemicals. Some products such as hydrogen, ethanol, lactic acid, and succinic acid can be produced directly or be readily synthesized from common compounds within central metabolism. More elaborate pathways such as the fatty acid biosynthesis, isoprenoid, polyketide, and shikimate pathways and more recently β-oxidation reversal (r-BOX) pathway (Dellomonaco et al., 2011) have also been employed to produce various value-added chemicals ranging from antibiotics to jet fuels.

The r-BOX pathway is usually initiated by thiolase-catalyzed non-decarboxylative Claisen condensation between two acetyl-CoAs. One complete cycle of this pathway consists of four core enzymes: (i) a thiolase (THL) that catalyzes the condensation of acetyl-CoA, which serves as the extender unit for carbon chain elongation, with an acyl-CoA, which serves as the primer, yielding a 3-ketoacyl-CoA; (ii) a hydroxyacyl-CoA dehydrogenase that reduces the 3-ketoacyl-CoA to 3-hydroxyacyl-CoA; (iii) an enoyl-CoA dehydratase that converts the 3-hydroxyacyl-CoA to trans-2-enoyl-CoA; (iv) a trans-enoyl-CoA reductase that generates acyl-CoA two carbon longer than the initial acyl-CoA from enoyl-CoA.

This r-BOX platform can utilize fatty acid oxidation pathways to obtain various chemicals. These pathways contain enzymes that allow for the selective functionalization of fatty acids, such as generation of C-C double bonds, additional reactions on the C-C double bond, and functionalization of C-H bond in the alkyl chain leading to microbial synthesis of short-, and medium-chain length aliphatic fatty acids/alcohol, and unsaturated α,β-carboxylic acids (Clomburg et al., 2012) (Cintolesi et al., 2014) (Kim et al., 2015) (Kim et al., 2016).  Further functionalization can be achieved through usage of different ω and  ω-1-functionalized acyl-CoAs as primers  and  α-functionalized acyl-CoAs as extender units instead of acetyl-CoA, or integration of carbon chain functionalization pathway, such as ω-oxidation pathway, after termination of r-BOX cycle, leading to microbial synthesis of dicarboxylic acids, ω-hydroxy acids, phenylakanoic acids and methyl-branched fatty acids (Clomburg et al., 2015) (Cheong et al., 2016).  Those chemicals can be used for the manufacture of products such as fuels, paints, food additives, resins, foams, lubricants, plasticizers, and cosmetics.

Bio-based chemicals, however, suffer from economic competition from much cheaper counterparts derived from conventional fossil-based routes. Nevertheless, economical industrialization of bio-based chemical production that have no petrochemical counterparts, such as alkylpolyglucoside and PLA polymers, highlights the importance of bio-based approaches to developing newly functionalized chemicals. While the r-BOX platform is expected to play a crucial role in this effort, one of the major challenges is the selective production of functionalized chemicals with a specific carbon chain length. The r-BOX uses an iterative mechanism to elongate alkyl chains, which in turn can lead to the production of molecules with the same functional groups but various carbon chain lengths. The synthesis of a single product with a specific chain length (as opposed to a mixture of products with various chain lengths) will require the engineering of enzymes with high chain-length specificity, including thiolases, thioesterases, and acyl-CoA or acyl-ACP reductases. Moreover, the efficient operation of platforms like the β-oxidation reversal will also require the engineering of pathways involved in central metabolism to achieve a balanced supply of primers, extender units, and reducing equivalents from a single carbon source. In addition, engineering of pathways will be also required for further diversification of products of the platform.

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