Engineering Escherichia coli for Terminal (ω) - Functionalized Long Chain Dicarboxylic Acids Production from Renewable Fatty Acids and Plant Oils

Chandran, S. P., Ulsan National Institute of Science and Technology (UNIST)

Dicarboxylic acids particularly, long-chain- ω - functionalized dicarboxylic acids (LDCAs, ≥ C12) are widely used as a raw material for preparing various commodities and polymers, such as nylon, polyamides, polyesters, perfumes, adhesives, and high quality lubricants. Industrial production of LDCAs relies mainly on chemical conversion processes from petrochemical sources that have a number of disadvantages and limitations including limitations in the range of products produced, use of multi-step conversion processes, dependence on non-renewable petrochemical feedstock, and generation of unwanted and hazardous byproducts. Biotechnology offers an innovative solution for overcoming these limitations. With increasing attention towards on reducing petroleum footprint, recent researches have been focused on the sustainable production of industrial important bulk chemicals from low-cost and renewable resources. Fatty acids (FAs) are considered as one of the most abundant renewable resources found in nature. Several studies have previously proposed the production of renewable fuels and industrial chemicals from fatty acids by Escherichia coli and suggesting that fatty acids could become a sustainable feedstock for industrial production.

The production of α,ω-dicarboxylic acids involves the following three steps: (i) terminal (ω) oxidation of fatty acids by a hydroxylase complex composed of cytochrome P450 monooxygenase (CYP450) and an NADPH:cytochrome P450 oxidoreductase (NCP) to form ω-hydroxy fatty acids; (ii) subsequent oxidation of hydroxy fatty acids into oxo fatty acids or fatty acid aldehydes by alcohol dehydrogenase and (iii) finally, the fatty aldehyde is oxidized by aldehyde dehydrogenase to the corresponding dicarboxylic acid (DCA). Hydroxylation of fatty acids by CYP450 and NCP is the rate limiting step and is therefore a critical stage in the production of α,ω-DCA, as the CYP enzymes show broad substrate specificity as well as high regioselectivity.

E. coli is a highly versatile organism that has been widely used for heterologous protein production and for genetic recombination studies because of its rapid growth and potential for high-density cultivation on inexpensive substrates. Additionally, its well characterized genetics and the availability of an increasingly large number of cloning vectors and mutant host strains make E. coli an attractive model for genetic studies. In the present study, an E. coli strain was engineered to produce α,ω-dicarboxylic acids (C12 and C14) directly from fatty acid substrates, through heterologously expressed ω-oxidation pathway. By whole cell biotransformation, the resulting engineered E. coli produced a maximum of 41 mg/L and 163 mg/L of C12 DCA and C14 DCA, respectively, from respective fatty acid substrates. In addition, the production of DCAs was increased (159 mg/L of C12 DCA and 410 mg/L of C14 DCA) by the addition of a heme precursor and the hydroxyl radical scavenger in a shorter culture duration than that of the corresponding controls. The constructed biocatalytic system was used to synthesize DCAs of various chain lengths from coconut oil hydrolysate. Therefore, our system could effectively be used for the production of DCAs from any plant oils high in fatty acids, including the non-edible vegetable oils. This novel synthetic biocatalytic system could be served as an efficient platform for the industrial production of α,ω-dicarboxylic acids.