Efficient synthesis of hydrocarbons using an engineered reversal of the B-oxidation cycle: A new paradigm for the production of advanced biofuels

与酒精生物燃料相比，基于长链碳氢化合物的先进液体生物燃料具有几个优势，包括吸湿性和挥发性更小，能量密度更高，并且与现有的储存、分配和使用基础设施兼容。许多微生物具有生产烯烃和烷烃的生物合成途径。对于烯烃，头对头？酰基辅酶a硫酯的缩合被认为是主要途径，而烷烃的合成则通过两步途径，包括酰基辅酶a硫酯转化为脂肪醛，然后将脂肪醛脱碳成烷烃。迄今为止，脂肪酸生物合成途径已被用作生成碳氢化合物合成所需的酰基辅酶a硫酯的唯一途径。然而，该途径的操作效率不高，因为它在合成丙二酰acp时消耗ATP，而丙二酰acp是链延伸的二碳单元的供体。因此，通过脂肪酸合成途径生产碳氢化合物的ATP产率非常低。这反过来又极大地限制了细胞的生长和碳氢化合物的产生。提出的研究的总体目标是代谢工程β -氧化循环的功能逆转，作为大肠杆菌宿主菌株高效生物合成长链碳氢化合物的新代谢平台。与脂肪酸生物合成途径不同，β -氧化循环的逆转与辅酶- a （CoA）硫酯中间体一起操作，并直接使用乙酰辅酶a进行酰基链延伸，而不是首先需要atp依赖的丙二酰辅酶a激活。这些特性使产品合成在最大的碳和能源效率。为实现这一目标，提出了四个目标：1)用最少的酶设计β -氧化循环的功能逆转，2)设计β -氧化循环功能逆转中产生的酰基辅酶a中间体合成烷烃和烯烃的途径，3)提高合成碳氢化合物过程中β -氧化循环的工程逆转效率，4)对野生型和工程菌株进行系统范围的表征。更广泛的影响拟议的研究超出了先进生物燃料应用的范围，因为β -氧化酶的普遍特性有可能使工业生物中各种非天然产物的组合合成具有最少数量的外源基因，这种方法有可能导致工程途径的有效运作。拟议的研究将培养博士后研究人员在这些途径的代谢工程。拟议的推广和教育活动将侧重于可持续能源主题，并与主要为西班牙裔人口服务的学区合作。特别是，与休斯顿和谐科学学院的合作努力将使中学生和高中生了解可再生能源概念以及该领域相关的职业机会。

PI: GonzalezProposal Number: 1134541Intellectual MeritAdvanced liquid biofuels based on long-chain hydrocarbons offer several advantages compared to alcohol biofuels, including less hygroscopicity and volatility, higher energy density, and compatibility with current infrastructure for storage, distribution and usage. Many microorganisms possess biosynthetic pathways for production of alkene and alkane hydrocarbons. For alkenes, the ?head-to-head? condensation of acyl-CoA thioesters has been proposed as the primary pathway, whereas alkanes are synthesized through a two-step pathway that involves conversion of acyl-CoA thioesters to fatty aldehydes, which are then decarbonylated to alkanes. To date, the fatty acid biosynthesis pathway has been used as the exclusive means to generate the acyl-CoA thioesters required for the synthesis of hydrocarbons. However, the operation of this pathway is not efficient because it consumes ATP in the synthesis of malonyl-ACP, which is the donor of two-carbon units for chain elongation. As a consequence, the ATP yield associated with the production of hydrocarbon through the fatty acid synthesis pathway is very low. This, in turn, greatly limits cell growth and hydrocarbon production.The overall goal of the proposed research is to metabolically engineer a functional reversal of the beta-oxidation cycle as a new metabolic platform for the efficient biosynthesis of long-chain hydrocarbons in E. coli host strains. Unlike the fatty acid biosynthesis pathway, the reversal of the beta-oxidation cycle operates with coenzyme-A (CoA) thioester intermediates and uses acetyl-CoA directly for acyl-chain elongation, rather than first requiring ATP-dependent activation to malonyl-CoA. These characteristics enable product synthesis at maximum carbon and energy efficiency. To achieve this goal, four objectives are proposed: 1) engineer a functional reversal of the beta-oxidation cycle with a minimal set of enzymes, 2) engineer pathways for the synthesis of alkanes and alkenes from acyl-CoA intermediates generated in the functional reversal of the beta-oxidation cycle, 3) improve the efficiency of the engineered reversal of the beta-oxidation cycle during the synthesis of hydrocarbons, and 4) perform system-wide characterization of wild-type and engineered strains.Broader ImpactsThe proposed research extends beyond the confines of advanced biofuel applications, as the ubiquitous nature of beta-oxidation enzymes has the potential to enable the combinatorial synthesis of a variety of non-native products in industrial organisms with a minimum number of foreign genes, an approach that has the potential to lead to efficient functioning of the engineered pathways. The proposed research will train post-doctoral research associates in metabolic engineering of these pathways.The proposed outreach and education activities will focus on sustainable energy topics, and work with school districts serving predominantly Hispanic populations. In particular, collaborative efforts with the Houston Harmony Science Academy will expose middle and high schools students to renewable energy concepts and associated career opportunities in this field.