FUEL AND CHEMICAL PRODUCTION

FROM GLYCEROL FERMENTATION

In order to ensure the long-term viability of biorefineries, it is essential to go beyond the carbohydrate-based platform and develop complementing technologies capable of producing fuels and chemicals from a wide array of available materials. Glycerol, a readily available and inexpensive compound, is generated during biodiesel, oleochemical, and bioethanol production processes, making its conversion into value-added products of great interest. The high degree of reduction of carbon atoms in glycerol confers the ability to produce fuels and reduced chemicals at higher yields when compared to the use of carbohydrates. However, this highly reduced nature also results in unique challenges for microorganisms in order to utilize glycerol under fermentative conditions (i.e., absence of external electron acceptors), a metabolic mode essential to fully exploit the high degree of reduction (Clomburg and Gonzalez, 2013).

Building off our discovery that Escherichia coli, an organism previously thought to require external electron acceptors for glycerol utilization, is able utilize glycerol under fermentative conditions (Dharmadi et al., 2006), we have elucidated key pathways and mechanisms critical for the ability of this biotechnology workhorse to ferment glycerol (Gonzalez et al., 2008; Murarka et al., 2008). Central to this glycerol fermentation ability are: (i) the production of 1,2-propanediol (1,2-PDO) providing a means to consume reducing equivalents generated in the synthesis of cell mass, thus facilitating redox balance, and (ii) the conversion of glycerol to ethanol, through a redox-balanced pathway, fulfilling energy requirements by generating ATP via substrate-level phosphorylation. Furthermore, glycerol dissimilation into glycolytic intermediates under fermentative conditions was determined to be mediated by a type II glycerol dehydrogenase (glyDH-II) and a dihydroxyacetone kinase (DHAK), the former of previously unknown physiological role.  The activity of the formate hydrogen-lyase and F0F1-ATPase systems were also found to facilitate the fermentative metabolism of glycerol. Additional key insights into the fermentative metabolism of glycerol by E. coliwere elucidated through a quantitative analysis employing kinetic modeling and metabolic control analysis (MCA). This analysis indicated that glycolytic flux during glycerol fermentation is almost exclusively controlled by the aforementioned glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM), and overexpression of these genes was shown to significantly increase glycerol utilization flux under anaerobic conditions (Cintolesi et al., 2012). Furthermore, we have established a comprehensive understanding of glycerol utilization under microaerobic conditions in which availability of low amounts of oxygen enables redox balance, in the absence of 1,2-PDO synthesis, while preserving the ability to synthesize reduced products (Durnin et al., 2009).

These findings have provided a platform for implementing metabolic engineering strategies aimed at the conversion of glycerol into a range of value added products. Targeted production of specific fuel and chemical compounds has utilized combinatorial engineering of glycerol dissimilation pathways and downstream product synthesis pathways. The overexpression of key glycerol utilization pathways to increase the rate of glycerol dissimilation or the availability of specific intermediate metabolites, integrated with specific product synthesis pathways provides overall metabolic engineering strategies for individual product synthesis uniquely tailored to exploiting glycerol as a carbon source. Using this approach, we have demonstrated the high titer production of ethanol (Cintolesi et al., 2012; Durnin et al., 2009; Yazdani and Gonzalez, 2008), 1,2-PDO (Clomburg and Gonzalez, 2011), succinic acid (Blankschien et al., 2010), D-lactic acid (Mazumdar et al., 2010), and L-lactic acid (Mazumdar et al., 2013) from glycerol. We are also continually expanding our knowledge base on and use of glycerol as a carbon source for fuel and chemical production, and through the development of novel pathways for advanced fuels and chemicals (i.e. longer carbon chain length) we have demonstrated the synthesis of varying chain length fatty acids (Clomburg et al., 2012; Vick et al., 2015; Kim et al., 2015), n-alcohols (Kim et al., 2015), unsaturated carboxylic acids (Kim et al., 2016), w-hydroxyacids (Clomburg et al., 2015; Cheong et al., 2016), and dicarboxylic acids (Clomburg et al., 2015; Cheong et al., 2016) among other compounds using glycerol as a carbon source. These and continuing efforts are key to diversifying the range of products that can be produced from glycerol, capitalizing on this abundant, low-price feedstock.