Microbial fuel development framework using synthetic biology and fuel design for next generation renewable fuel production

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"EPSRC : Melissa Poma : EP/S023836/1"Biofuel production from organic waste and biomass is a promising source of renewable energy [1]. The employment of bacteria as cell factories is an attractive means for sustainable large-scale production of energy molecules. Bioengineering research has made impressive progress in identifying and optimizing microbial metabolic pathways involved in the biosynthesis of fuel-like hydrocarbons. Such metabolic routes include derivations of the amino acid [2], the mevalonate [3], the polyketide [4], and the fatty acid pathways [5]. These natural metabolic routes have been engineered and modestly implemented in native and non-native hosts [6], enabling the microbial cell to assimilate simple sugars into value-added molecules. However, industrial production rate has not been achieved yet. Moreover, this biosynthesis cannot be considered entirely sustainable, unless it is decoupled from the use of simple sugars as feedstock. The use of lignocellulosic biomass as feedstocks for the production of substrate needed for microbial fuel production has gained attention due to its abundance and low cost [7]. The cellulose and hemicellulose portions of the biomass can be fermented by microbes into useful simple sugars, which will serve as carbon sources for microbial fuel production [8]. This process is accompanied with loss of simple sugars since these are incorporated into the cells during growth. The use of enzymes instead of microbes can circumvent the sugar loss, but enzymes are costly and faced with low biochemical reaction rates or product feedback inhibition, leading to low product formation [9]. At the heart of efficient biofuel production lays the challenge of metabolic engineering [8]. Thanks to recent developments in synthetic biology and genetic engineering, manipulating heterologous enzyme expression to construct biofuel-producing pathways in a microbial host is no longer the roadblock it once was [10]. However, optimal pathway activity is rarely achieved by simple expression (or overexpression) of required enzymes; product formation can be affected by many other factors including consumption of substrates by competing pathways, energetic and redox imbalances caused by engineered pathway activity, and inhibition due to product accumulation. Metabolomic analyses can guide pathway optimization by identifying sources of metabolic inefficiency, revealing strategies to increase activity of engineered pathways.The novelty of my project and its contribution to the existing literature will be proposed through experimental research where I develop a model bacterium Zymomonas mobilis that could serve as host for the engineering of a designer cellulosome apparatus, enabling control over the composition and the position of the cellulases, and the linkers' length. I will develop processes which mimic natural cellulolysis in model industrial bacteria to provide them with a novel function and employ them as biofuel refineries. This model bacterium will be expected to have high growth rate, survive under standard cultivation conditions, be capable for high substrate uptake, have high ethanol tolerance, produce attractive metabolic precursors, and generally recognized as safe, with well-known genetic engineering tool-set.1. Liao JC, Mi L, Pontrelli S, Luo S. Nat Rev Microbiol 2016, 14.2. Akita et al. Appl Microbiol Biotechnol. 2015, 99, 991-9993. Peralta-Yahya, et al. Nature 2012, 488, 320-328.4. Zargar et al. Current Opinion in Biotechnology 2017, 45, 156-1635. Ledesma-Amaro et al. Progress in Lipid Research 2016, 61, 40-506. Schirmer at al. Science 2010, 329, 559-5627. Alfenore S, Molina-Jouve C. Process Biochemistry 2016;51.8. Ruffing AM. Liquid, Gaseous and Solid Biofuels-Conversion Techniques. 2013:263-99.9. Ravindran R, Jaiswal AK. Bioresource technology. 2016;199:92-102.10. Liu R, Bassalo MC, Zeitoun RI, Gill RT. Metab Eng 2015, 32:143-154.