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

大肠杆菌中生产正丁醇的天然途径：合成生物学的新范例

• 批准号: 1067565
• 负责人: James Clomburg
• 金额: $ 36.36万
• 依托单位: William Marsh Rice University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1067565&HistoricalAwards=false
• 关键词: native; pathway; production; butanol; Escherichia

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.

对使用先进生物燃料如正丁醇和其他高链直链醇的兴趣已经迅速发展，因为与乙醇相比，它们提供了几个优点，包括更低的吸湿性和挥发性，更高的能量密度以及与当前存储，分配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直链醇中，正丁醇是自然界中唯一发现的主要发酵产物。合成正丁醇的能力被认为是梭菌物种的独有特征。梭菌是孢子形成菌，专性厌氧菌，生长速度缓慢，具有复杂的营养需求，并产生正丁醇沿着其他产物的混合物，包括丙酮、乙醇、丁酸盐和乙酸盐。缺乏有效的遗传工具来操纵梭菌，沿着它们复杂的代谢，阻碍了可能导致正丁醇产率、滴度和生产率提高的代谢工程努力。为了克服上述问题，已经将能够在天然生产者如梭菌中合成正丁醇的基因输入到遗传和代谢上易于处理的工业生物中，如E.大肠杆菌、酿酒酵母、恶臭假单胞菌、枯草芽孢杆菌、乳酸乳球菌和乳杆菌。迄今为止的所有努力都基于我们在本提案中所称的异源代谢工程（HeME）：即将（主要）梭菌起源的基因/途径移植到原本无法产生丁醇的宿主（例如大肠杆菌）。coli、S. cerevisiae）。过去，基于HeME的方法已被用于工程生物燃料生产，目前被视为宿主生物体不具备所需代谢功能时的选择策略。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，并且仍然需要补充具有丰富营养素的培养基。研究人员假设，异源代谢工程方法的使用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相容的异源途径，本文提出的工作的知识价值涉及通过开发一种替代策略来解决上述限制，该策略侧重于天然E.大肠杆菌酶/途径，其可以作为异源正丁醇合成途径的替代物，并因此在不存在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否则的外源途径，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，表征和利用天然生物合成途径，以有效地生产正丁醇在E。大肠杆菌，从而建立了一个新的范例，应用合成生物学生产先进的生物燃料。本研究的具体目标是：i）鉴定天然E.编码可催化包括梭菌丁醇途径的反应步骤的酶的大肠杆菌基因; ii）天然丁醇途径在大肠杆菌中的体内组装和功能表征。大肠杆菌; 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表征。HoME作为代谢工程和合成生物学的新范式的建立将导致通过功能分化的工程来开发天然宿主的多潜能能力。通过使正丁醇的生产通过同源途径，这一建议将有助于创造一种全新的方法，使第二代生物燃料的有效生产在许多工业有机体。在这些进展的基础上，可以发展高效和经济上可行的化学和生物燃料工业，使能源独立和气候保护成为可能。该提案还将教育我们的社会在通往可持续能源未来的道路上面临的科学和工程挑战和机遇。研究人员将利用我们与休斯顿和谐科学院的合作，对初中和高中学生进行替代能源领域的培训。这所学校主要为少数民族人口服务，因此调查人员将解决国家的需要和挑战，增加他们对科学和工程的参与。