The use of microbial systems for the production of valued compounds and fuels provide a potentially greener and sustainable route for chemical production. However, one of the challenges in microbial biofuel and biorenewables production is the low tolerance of the microbial hosts to toxic compounds in the feedstock and products. Unfortunately, the engineering of microorganisms for increased robustness is currently restricted by the lack of knowledge of the molecular mechanisms involved. Our long-term goal is to globally identify the mechanisms involved in microbial adaptation to growth inhibitors present in the feedstock and products for the metabolic engineering of microbial systems. A member of the lactic acid bacteria, Lactobacillus brevis, was recently identified to exhibit tolerance to several growth inhibitors present in cellulosic biomass feedstock and to the second-generation biofuel, butanol. However, L. brevis lacks many biosynthetic pathways and has high nutritional requirements, and thus may not be an ideal production platform. The proposed research focuses on identifying gene(s) and/or sets of genes(s) from L. brevis, when heterologously expressed, will confer butanol tolerance to Escherichia coli, the non-fastidious workhorse of biotechnology. Specifically, the research plan proposes to generate libraries of E. coli single integrants that express L. brevis genes. Genome shuffling between the E. coli single integrants will be used to generate libraries of E. coli double integrants expressing combinations of L. brevis genes at two genomic loci. Serial enrichment strategies will be developed to select for integrants containing L. brevis genes that confer butanol tolerance to E. coli from the libraries. The results from the proposed work will be used as preliminary results for subsequent proposals to further develop and expand the application of this combinatorial engineering approach for desirable complex phenotypes in microbial production hosts. The educational outreach plan aims to broaden the participation and enhance the retention of women and minorities in science and engineering fields. First, we hope to encourage female and/or minority high school students to pursue engineering degrees in college by establishing a summer internship program in the lab; this will be accomplished through the collaboration with local high school science teachers. Second, we will tackle the problem of low retention and decrease in proportion of women and minorities in achieving advanced degrees in engineering disciplines by providing research opportunities and mentorships to science and engineering undergraduates. In addition, an undergraduate/graduate course in metabolic engineering will be established to generate excitement and interest in pursuing advanced degrees in engineering; this will be accomplished by focusing on the integration of engineering and science to solve current societal challenges.
Broader impact The broader impact of the proposed work is three fold. First, L. brevis genes that confer butanol tolerance to E. coli will be identified and used to further engineer more robust biobutanol producers (e.g. E. coli, and C. acetobutylicum). Two, the combinatorial engineering approach developed will be broadly applicable to a variety of desirable complex phenotypes. Third, the educational outreach plans will stimulate interest and increase participation of females and minorities in engineering fields. The successful engineering of microbial hosts with high tolerance to products and inhibitors present in sustainable feedstock will significantly improve the economical viability of industrial biotechnology. Thus, the proposed activities will have significant benefit on the initiation of the PI's independent career and on society.
The use of biocatalysts offers a potentially more environmentally friendly and sustainable route for production of fuels and chemicals. However, product and feedstock toxicity often poses a challenge for the economical viability of bio-based production. Model organisms, while amenable to strain engineering, do not always exhibit the necessary robustness in production environments. Non-model organisms, on the other hand, may possess inherently high tolerance to industrially relevant inhibitors, but may not have tools available for genetic manipulation. In this work, we focused on the n-butanol tolerance in Lactobacillus brevis due to recent work demonstrating its relatively high tolerance to this promising second-generation biofuel. Using genomic technologies, we identified distinct transcriptional regulatory patterns in Lactobacillus brevis compared with E. coli under n-butanol challenge. We have also developed libraries of E. coli expressing fragmented genomic DNA from Lactobacillus brevis that can be used to identify genetic determinants in Lactobacillus brevis that can contribute to industrially relevant complex phenotypes in E. coli. The fundamental knowledge gained from this work and products generated from this work can help facilitate future strain engineering efforts for more robust biocatalysts. This work involved the participation of graduate, undergraduate, and high school students, the majority of whom are female or from underrepresented groups, which contributed to their professional training and growth as scientists and engineers. Results from this work were broadly disseminated via peer-reviewed journal publications and presentations at conferences.
