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

“EPSRC：Melissa Poma：EP/S023836/1”利用有机废物和生物质生产生物燃料是一种很有前途的可再生能源[1]。利用细菌作为细胞工厂是一种有吸引力的可持续大规模生产能量分子的方法。生物工程研究在确定和优化生物合成类燃料碳氢化合物的微生物代谢途径方面取得了令人印象深刻的进展。这种代谢途径包括氨基酸[2]、甲氧戊酸[3]、聚酮[4]和脂肪酸途径[5]的衍生物。这些自然代谢途径已经在原生和非原生宿主中设计并适度实施[6]，使微生物细胞能够将单糖同化为增值分子。然而，工业生产率还没有达到。此外，这种生物合成不能被认为是完全可持续的，除非它与以单糖为原料的使用脱钩。利用木质纤维生物质作为原料生产微生物燃料生产所需的底物，由于其丰富和低成本而受到关注[7]。生物质中的纤维素和半纤维素部分可以被微生物发酵成有用的单糖，作为微生物燃料生产的碳源[8]。这个过程伴随着单糖的损失，因为这些单糖在生长过程中被合并到细胞中。使用酶而不是微生物可以避免糖的损失，但酶的成本很高，而且面临着低生化反应速率或产物反馈抑制，导致产物形成低[9]。高效生物燃料生产的核心是代谢工程的挑战[8]。由于合成生物学和基因工程的最新发展，操纵异源酶表达在微生物宿主中构建生物燃料生产途径不再是曾经的障碍[10]。然而，最优的途径活性很少通过简单地表达(或过度表达)所需的酶来实现；产物的形成可以受到许多其他因素的影响，包括竞争途径对底物的消耗，工程途径活性引起的能量和氧化还原失衡，以及产物积累造成的抑制。代谢组学分析可以通过识别代谢低效的来源，揭示增加工程途径活性的策略来指导途径优化。我的项目的新颖性及其对现有文献的贡献将通过实验研究提出，我开发了一种模式细菌运动发酵单胞菌，它可以作为设计纤维素体装置的宿主，能够控制纤维素酶的组成和位置，以及连接物的长度。我将开发在模拟工业细菌中模拟自然纤维素分解的过程，为它们提供新的功能，并将它们用作生物燃料精炼厂。这种模式菌将有望具有高生长速度，在标准培养条件下存活，能够高底物吸收，具有高乙醇耐受性，产生诱人的代谢前体，并普遍被认为是安全的，拥有众所周知的基因工程工具1。廖锦昌，米L，庞特雷利S，罗世南，微生物学评论，2016，14.Akita et al.应用微生物生物技术。2015，99,991-9993。Peralta-Yahya等人。《自然》杂志，2012年，488,320-328.4。Zargar等人。生物技术的当前意见2017，45,156-1635。Ledesma-Amaro等人。脂类研究进展2016，61，40-506。Schirmer at al.科学，2010年，329,559-5627。S，《过程生化》；51.8.发怒的AM液体、气体和固体生物燃料.转化技术2013年：263-99.9。Ravindran R，Jaiswal AK.生物资源技术。2016年；199年：92-102.10。刘R，Bassalo MC，Zeitoun RI，Gill RT.Metab Eng 2015，32：143-154。

"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.