Microbial production of oleochemicals from simple sugars, such as free fatty acids (FFA) and fatty alcohols (FAOH), has been a subject of research interest in recent years since it could provide with new routes to these important molecules. Oleochemicals, which are currently obtained from oily plants such as coconut and canola, are in high demand and has created tensions between food and fuel industries as well as concerns about the high degree of deforestation. One of the challenges in microbial production of FFA and FAOH is the lack of enzymes that are both highly active and specific towards a particular carbon chain length. As a result, research in this area is often categorized between strains that are either able to produce high titers of FFA and FAOH with mixed chain lengths or strains that have low titers with high chain length specificity. A key step that controls this specificity is the thioesterase, which catalyze the last step in microbial FFA production strategies by hydrolyzing the thioester bond in cellular fatty acyl-ACP producing a FFA. Since this step commits the fatty acyl chain length to its current size it is critical to have a thioesterase that is both highly active and specific towards the desired chain length to drive its production. We developed a screen that relies on high production of a particular chain length of FFA for cell survival. The screen was used to improve thioesterase activity on a library created through random mutagenesis in a low activity plant thioesterase. With this approach we were able to achieve &gt;1 g/L FFA titers with high specificity for the desired chain length. This was then used to assembly the pathway for FAOH production from simple sugars and we were able to obtain &gt;1 g/L of FAOH and high specificity for our desired chain length. These results highlight the potential of fatty acid biosynthesis pathway as a route for the production of FFA and FAOH.

Tailoring chain-length specificity for oleochemical production in bacteria

255th National Meeting (2018)

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