A native pathway for the production of n-butanol in Escherichia coli: A new paradigm for synthetic biology

对使用先进生物燃料的兴趣迅速发展，如正丁醇和其他高链线性醇，因为它们与乙醇相比具有几个优点，包括更少的吸湿性和挥发性，更高的能量密度，以及与现有的存储，分销和使用基础设施的兼容性。在目前被认为是先进生物燃料的线性醇中，正丁醇是唯一一种在自然界中发现的主要发酵产物。合成正丁醇的能力被认为是梭菌特有的特征。梭状芽孢杆菌是孢子形成者，专性厌氧菌，生长速度慢，有复杂的营养需求，生产正丁醇以及其他产品的混合物，包括丙酮、乙醇、丁酸盐和醋酸盐。缺乏有效的遗传工具来操纵梭菌及其复杂的代谢，阻碍了代谢工程的努力，这些努力可能导致正丁醇产量、滴度和生产力的提高。为了克服上述问题，在梭状芽胞杆菌等本土生产者中能够合成正丁醇的基因已被引入大肠杆菌、酿酒酵母菌、恶臭假单胞菌、枯草芽孢杆菌、乳酸乳球菌和乳杆菌等遗传和代谢容易处理的工业生物中。迄今为止，所有的努力都是基于我们在本提案中提到的异源代谢工程（HeME）：即将（主要是）源自梭状芽孢杆菌的基因/途径移植到无法产生丁醇的宿主（如大肠杆菌、酿酒杆菌）。过去，基于血红素的方法已被用于设计生物燃料生产，目前，当宿主生物不具备所需的代谢功能时，血红素被视为一种选择策略。然而，在正丁醇和其他线性正醇的情况下，HeME方法面临着重大障碍。例如，经过几年的菌株开发和优化，用于生产正丁醇的生物体以低通量合成这种醇，并且仍然需要补充富含营养的培养基。研究人员假设，使用异源代谢工程方法代表了上述研究有限成功的主要问题，因为它依赖于转移可能与宿主不兼容的异源途径，从而损害其功能。本文提出的工作的智力价值涉及通过开发一种替代策略来解决上述局限性，该策略侧重于鉴定和利用天然大肠杆菌酶/途径，这些酶/途径可以作为外源正丁醇合成途径的替代品，从而在缺乏外源基因的情况下介导非天然产物的合成。由于没有外源基因被招募来建立其他外源途径，研究人员将这种方法称为同源代谢工程（HoME）。本提案的总体目标是确定，表征和利用大肠杆菌中高效生产正丁醇的天然生物合成途径，从而为合成生物学在先进生物燃料生产中的应用建立新的范例。该工作的具体目标是：i)鉴定天然大肠杆菌基因编码的酶，可以催化包括梭状芽胞杆菌丁醇途径的反应步骤；ii)大肠杆菌中天然丁醇途径的体内组装和功能表征；iii)提高天然正丁醇途径的效率；iv)野生型和工程菌株的全系统特性。这一提议的广泛影响是多方面的。HoME作为代谢工程和合成生物学的新范式的建立，将通过功能分化工程来开发天然宿主的多能性。通过同源途径生产正丁醇，这一提议将有助于创造根本的新方法，使许多工业生物能够有效地生产第二代生物燃料。在这些进步的基础上，可以发展高效和经济上可行的化学和生物燃料工业，这将使能源独立和气候保护成为可能。这一建议还将教育我们的社会，在通往可持续能源未来的道路上，科学和工程方面的挑战和机遇。研究人员将利用我们与休斯顿和谐科学学院的合作，在替代能源领域培训初高中学生。这所学校主要为少数民族提供服务，因此研究人员将解决增加他们参与科学和工程的国家需求和挑战。

1067565GonzalezInterest in the use of advanced biofuels, such as n-butanol and other higher-chain linear alcohols, has rapidly developed because they offer several advantages compared to ethanol, including less hygroscopicity and volatility, higher energy density and compatibility with current infrastructure for storage, distribution and usage. Among linear alcohols currently considered as advanced biofuels, n-butanol is the only one found in nature as a major fermentation product. The ability to synthesize n-butanol is considered to be an exclusive feature of clostridial species. Clostridia are spore formers, obligate anaerobes that grow at slow rates, have complex nutritional requirements and produce n-butanol along with a mixture of other products including acetone, ethanol, butyrate, and acetate. The lack of efficient genetic tools to manipulate clostridia, along with their complex metabolism, hinders metabolic engineering efforts that could lead to the improvement of n-butanol yield, titer, and productivity. In an effort to overcome the aforementioned issues, the genes that enable the synthesis of n-butanol in native producers like clostridia have been imported into industrial organisms that are genetically and metabolically tractable such as E. coli, Saccharomyces cerevisiae, Pseudomonas putida, Bacillus subtilis, Lactococcus lactis, and Lactobacillus species. All efforts to date have been based on what we refer to in this proposal as heterologous metabolic engineering (HeME): that is, transplanting genes/pathways of (primarily) clostridial origin to hosts otherwise not able to produce butanol (e.g. E. coli, S. cerevisiae). HeME-based approaches have been used to engineer biofuel production in the past and are currently viewed as the strategy of choice when the host organism does not possess the desired metabolic function. However, in the case of n-butanol and other linear n-alcohols, HeME approaches have faced significant hurdles. For example, after several years of strain development and optimization, organisms engineered for the production of n-butanol synthesize this alcohol at low flux and still require the supplementation of the medium with rich nutrients. The investigators hypothesize that the use of a heterologous metabolic engineering approach represents the main issue accounting for the limited success of the aforementioned studies, as it relies on transferring a heterologous pathway that might not be compatible with the host, thus compromising its functionality.The Intellectual Merit of the work proposed here relates to addressing the aforementioned limitations by developing an alternative strategy that focuses on the identification and harnessing of native E. coli enzymes/pathways that could act as surrogates of the heterologous n-butanol-synthesis pathway and hence mediate the synthesis of a non-native product in the absence of foreign genes. Since no exogenous gene is recruited to establish the otherwise foreign pathway, the investigators have termed this approach homologous metabolic engineering (HoME). The overall goal of this proposal is to identify, characterize and harness native biosynthetic pathways for the efficient production of n-butanol in E. coli, thus establishing a new paradigm for the application of synthetic biology to the production of advanced biofuels. The specific objectives of the proposed work are: i) Identify native E. coli genes encoding enzymes that can catalyze the reaction steps comprising the clostridial butanol pathway; ii) In vivo assembly and functional characterization of a native butanol pathway in E. coli; iii) Improve the efficiency of the native n-butanol pathway; iv) System-wide characterization of wild-type and engineered strains.The Broader Impacts of this proposal are numerous. The establishment of HoME as a new paradigm for metabolic engineering and synthetic biology would lead to exploiting the multi-potent capabilities of native hosts via engineering of functional differentiation. By enabling the production of n-butanol through a homologous pathway, this proposal will contribute to the creation of fundamentally new approaches that could enable efficient production of second-generation biofuels in many industrial organisms. Based on these advances, efficient and economically viable chemical and biofuel industries can be developed that will make possible energy independence and climate protection. This proposal will also educate our society in the scientific and engineering challenges and opportunities on the road to a sustainable energy future. The investigators will capitalize on our collaborations with the Houston Harmony Science Academy to train middle and high schools students in the field of alternative energy. This school serves predominantly minority populations, and thus the investigators will address the national need, and challenge, of increasing their participation in science and engineering.