Interest 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.
