MicroFuelPro: Microbial fuel development framework using synthetic biology for next generation drop-in renewable fuel production

利用细菌作为细胞工厂是可持续大规模生产能量分子的一种有吸引力的手段。生物工程研究在识别和优化参与类燃料碳氢化合物生物合成的微生物代谢途径方面取得了令人印象深刻的进展。这些代谢途径包括：氨基酸途径、甲羟戊酸途径、聚酮途径和脂肪酸途径的衍生物。这些天然代谢途径已经在原生和非原生宿主中被设计和适度实施，使微生物细胞能够将单糖吸收成增值分子。然而，工业生产速度尚未达到，而且，这种生物合成不能被认为是完全可持续的，除非它与单糖作为原料的使用脱钩。MicroFuelPro项目面临的挑战是，通过微生物将废物（如木质纤维素）同化为能量分子，提高代谢通量，实现大规模生物燃料生产。将废物直接转化为类燃料分子的方法尚未得到充分的探索，因此，该项目的目标将在于识别使这种分解代谢和合成代谢过程有效的基因。一个有吸引力的候选宿主生物燃料合成是革兰氏阴性细菌的活动单胞菌，它具有高底物吸收和惊人的乙醇产量。然而，Z. mobilis不能自然降解木质纤维素，因此，为了使其具有这一功能，将在木质纤维素降解微生物中鉴定相关基因并在宿主平台中表达。最有可能的是，单独的异源基因表达将不足以实现有效的底物同化，因此，进一步的生物工程工具（例如CRISPR，基因组编辑，核糖体结合分子等）将被用来产生合成的高性能代谢。同样的方法将应用于其他不同的细菌，以合成脂肪酸、萜类、聚酮和高级醇等化合物。参与这些产物合成的途径要么在原生宿主中进行调整，要么在模型细菌中进行异种工程改造，以显示出高生长速度、对溶剂毒性的高耐受性或代谢替代原料的能力。最终目标将是对一系列工程细菌进行比较，以检测最适合木质纤维素生物降解和转化为增值分子的候选细菌。在生产出燃料分子后，将确定燃料的设计特性，并选择替代燃料托盘。理想情况下，每种调色板化合物将代表目标可再生燃料中发现的一类化合物。替代调色板将包含市场上碳氢燃料中发现的每一个主要碳氢化合物家族的代表：正烷烃、异烷烃、环烷烃、芳烃和萘芳烃。13C（碳）和1H（质子）核磁共振（NMR）波谱和GC-MS将用于量化本研究中每种目标可再生燃料的组成特征。下一步将是确定并进行优化研究，以确定每种调色板化合物应该包含在替代品中，以实现可再生燃料的性能目标。一旦确定了每种替代组合物，将纯调色板化合物混合在一起以产生替代物，并对每种替代物进行测试，以确定是否在其期望的公差范围内实现了属性目标。燃料设计过程的结果将与代谢途径相关。通过这一分析，我们将能够确定并设计出最有效的途径，不仅在产量方面，而且在燃料性能、可持续性以及对传统和未来发电系统的适用性方面。

The employment of bacteria as cell factories is an attractive means for sustainable large production of energy molecules. Bioengineering research have made impressive progresses in identifying and optimizing microbial metabolic pathways involved in the biosynthesis of fuel-like hydrocarbons. Such metabolic routes include: derivations of amino acid pathway, the mevalonate pathway, the polyketide pathway, and the fatty acid pathway. These natural metabolic routes have been engineered and modestly implemented in native and non-native hosts, 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. MicroFuelPro project sets the challenge of enhancing metabolic fluxes to achieve large scale biofuel production through the microbial assimilation of waste material, such as lignocelluloses, into energy molecules. The direct conversion of waste materials into fuel-like molecules has not been fully explored, thus, the objective of the project will lies in the identification of genes that enable this catabolic and anabolic process efficiently. An attractive candidate to host biofuel synthesis is the Gram-negative bacterium Zymomonas mobilis, which has a high substrate uptake and striking ethanol yield. However, Z. mobilis does not naturally degrade lignocellulose, hence, to provide it with this function, relevant genes will be identified in lignocellulolytic microorganisms and expressed in the host platform. Most likely, the solely heterologous expression of genes will not be sufficient to enable an efficient substrate assimilation, thus, further bioengineering tools (e.g. CRISPR, genome editing, ribosome binding molecules etc.) will be used to generate a synthetic high-performance metabolism. The same approach will be exerted in other different bacteria to synthesize compounds such as fatty acids, terpenoids, polyketides and higher alcohols. The pathways involved in these products synthesis will be either tweaked in native host or heterogously engineered in model bacteria that show features like high growth rate, high tolerance to solvent toxicity, or ability of metabolizing alternative feedstocks. The ultimate goal will be the comparison of an array of engineered bacteria to detect the most suitable candidates for lignocellulose biodegradation and conversion into value-added molecules. After the production of fuel molecules, the fuel design properties will be identified, and surrogate fuel pallet will be chosen. Ideally, each palette compound would be representative of a class of compounds found in the target renewable fuel. The surrogate palette will contain representatives from each of the major hydrocarbon families found in market hydrocarbon fuels: n-alkanes, iso-alkanes, cycloalkanes, aromatics, and naphtho-aromatics. 13C (carbon) and 1H (proton) nuclear magnetic resonance (NMR) spectroscopy and GC-MS will be used to quantify the compositional characteristics of each target renewable fuel in this study. The next step will be to identify and run an optimization study to determine how much of each palette compound should be included in the surrogate to achieve the property targets of renewable fuels. Once each surrogate composition is determined, the pure palette compounds will be blended together to produce the surrogates, and each surrogate will be tested to determine whether the property targets are achieved within their desired tolerances. The outcomes of the fuel design process will be correlated with the metabolic pathways. Through this analysis we will be able to identify and design the most efficient pathway not only in terms of yield but also in terms of fuel properties, sustainability and suitability to conventional and future power generation systems.