Engineered Respiro-Fermentative Metabolism for the Production of Biofuels and Biochemicals from Fatty Acid-Rich Feedstocks

Abstract

Although lignocellulosic sugars have been proposed as the primary feedstock for the biological production of renewable fuels and chemicals, the availability of fatty acid (FA)-rich feedstocks and recent progress in the development of oil-accumulating organisms make FAs an attractive alternative. In addition to their abundance, the metabolism of FAs is very efficient and could support product yields significantly higher than those obtained from lignocellulosic sugars. However, FAs are metabolized only under respiratory conditions, a metabolic mode that does not support the synthesis of fermentation products. In the work reported here we engineered several native and heterologous fermentative pathways to function in Escherichia coli under aerobic conditions, thus creating a respiro-fermentative metabolic mode that enables the efficient synthesis of fuels and chemicals from FAs. Representative biofuels (ethanol and butanol) and biochemicals (acetate, acetone, isopropanol, succinate, and propionate) were chosen as target products to illustrate the feasibility of the proposed platform. The yields of ethanol, acetate, and acetone in the engineered strains exceeded those reported in the literature for their production from sugars, and in the cases of ethanol and acetate they also surpassed the maximum theoretical values that can be achieved from lignocellulosic sugars. Butanol was produced at yields and titers that were between 2-and 3-fold higher than those reported for its production from sugars in previously engineered microorganisms. Moreover, our work demonstrates production of propionate, a compound previously thought to be synthesized only by propionibacteria, in E. coli. Finally, the synthesis of isopropanol and succinate was also demonstrated. The work reported here represents the first effort toward engineering microorganisms for the conversion of FAs to the aforementioned products.

Synthesis of customized petroleum-replica fuel molecules by targeted modification of free fatty acid pools in Escherichia coli

“…The size of the challenge facing advanced biofuels can be appreciated when considering the effort necessary to progress the engineered biosynthesis of semisynthetic artemisinin from the laboratory to commercial production: many changes beyond manipulating the endogenous mevalonate pathway have been required, including changes to host cell biology, fermentation conditions, and extraction procedures (34,35). The application of engineering, life-cycle, and economic costing analysis for scale-up procedures combined with the exploitation of alternative, nonfood carbon sources to break the link between food and fuel prices (17)(18)(19)(36)(37)(38)(39) and new approaches to engineering metabolism [exemplified by the development of dynamic sensor-regulator systems for FAderived products (40), the remarkable reversal of the β-oxidation cycle (41), and introduction of molecular scaffolds for improving metabolic efficiency (42,43)] will facilitate this aim. The results presented in this article, although at a very early stage in product development, contribute toward the goals of advanced biofuels by providing metabolic pathways for the production of industrially relevant, petroleum-replica fuel molecules.…”

Microbial Engineering for Aldehyde Synthesis

“…Although microbial FFAs have been produced for decades, recent work has demonstrated the potential for obtaining advanced fuels or valuable chemicals as derivatives of FFAs (50)(51)(52)(53). Based on the broad substrate range and known activities of carboxylic acid reductases, their addition to these pathways can result in production of C 4 to C 18 aliphatic aldehydes (25,26).…”

Deletion of Genes Encoding Cytochrome Oxidases and Quinol Monooxygenase Blocks the Aerobic-Anaerobic Shift in Escherichia coli K-12 MG1655

“…The ECOM4 strain was unable to undergo an aerobic-anaerobic shift and exhibited similar phenotypes under both conditions, making it suitable for use as a platform strain for the implementation and adaptation of strain designs. Moreover, a strain exhibiting fermentative growth under oxic and anoxic conditions has a potential to be used for overproduction of reduced metabolic products, such as ethanol (11,18,24,32), pyruvate (52), lactate (35,43,51,53), and succinate (34), in industrial settings. A comprehensive understanding of the metabolism and physiology of the platform strain is important, as it provides insights for further engineering.…”

“…Cells were grown overnight, harvested, washed twice with water, and used to inoculate 50-ml flasks containing 25 ml medium with 2 g/liter 13 C-labeled D-glucose, with an initial OD 600 of 0.005 to 0.01. Glucose was supplied as 100% [1][2][3][4][5][6][7][8][9][10][11][12][13] C labeled, 100% 6-13 C labeled, or a mixture of 20% uniformly (U-13 C) labeled and 80% natural (which is randomly 1% 13 C). Cells were grown to mid-log phase, corresponding to an OD 600 of 0.6 (WT) or 0.25 (ECOM4LA).…”

“…Corrected mass distributions for amino acid fragments from [U- 13 C]glucoselabeled cells were used to infer the trafficking and reassortment through metabolism of linked chains of carbons derived from glucose, while mass distribution data from [1][2][3][4][5][6][7][8][9][10][11][12][13] C]glucose-or [6][7][8][9][10][11][12][13] C]glucose-labeled cells were used to track the fate of individual carbon atoms. The analysis is summarized here and is described in more detail in the supplemental methods (see the supplemental material).…”