This project aims to develop novel multi-library-based approaches for generating complex phenotypes that would be useful for developing industrially important strains for many bioprocessing applications, such as those currently employed or anticipated in the bioprocessing for the utilization or renewable resources (green technologies) or in bioremediation. In addition, the knowledge generated from this project will make a significant contribution to the basic biology of ethanol tolerance.
Broader Impact:
This project?s strategies can be applied to any desirable organism and bioprocessing application whereby a selectable complex phenotype is desirable, and especially processes for the utilization of renewable resources. The importance of efficient and economically viable technologies for production of chemicals & biofuels from renewable resources has become an urgent national and international priority over the last few months. This was driven by several important facts. First, the depletion of non-renewable energy sources, such as oil, coupled with increased energy demand driven by fast developing economies and world population has led to high oil and energy prices. And a second important fact is the realization that non-renewable fossil fuels lead to significant climatic changes that may lead to catastrophic weather-pattern changes. In contrast, biomass is a carbon-neutral renewable resource for production of biofuels and chemicals. Finally, this project provides unique opportunities for student education and training in experimental genomics, and associated high-throughput technologies.
Goals: Production of chemicals and biofuels through bioprocessing and utilizing renewable resources is one of the highest technological priorities for the US and the world. In bioprocessing, in addition to maximizing the flux for a desirable product, the robustness and prolonged productivity of the biocatalyst (the cells) under realistic bioprocessing conditions is an equally important issue in order to develop economically competitive and efficient bioprocesses. Thus, the ability of cells to withstand "stressful" bioprocessing conditions (such as toxic substrates, accumulation of toxic products, and low pH, as encountered in applications for the production of chemicals and biofuels as well as in bioremediation) without loss of productivity is a most significant goal. The difficulty is that the desirable phenotypic trait is determined by a group of genes. Complex phenotypes are also encountered when one desires to develop a new and novel capability or pathway in a particular cell type. In this project, our aim was to develop tools and strategies which will facilitate the development of complex phenotypes in microbial cells. Complex phenotypes arise from multiple interacting genes and loci. Thus, our aim was to develop genomic approached that can identify advantageous interactions between distantly located genetic elements. Findings: An important approach for identification and development of desirable microbial traits is based on genomic libraries. Genomic libraries can be constructed by cutting a cell’s chromosome (genome) into smaller pieces (DNA fragments) of desirable size and linking those pieces into a genetic vector (a plasmid or fosmid). The collection of such vectors makes up the library. Such libraries can be introduced into a cell so that the genes on the library fragments can be expressed aiming to select those that help develop or improve a desirable trait. Library-insert carrying cells are exposed to selective pressure necessary for the identification of the desirable trait, with the assumption that some gene(s) represented in the library will allow enrichment under pressure. Current, i.e., single genomic libraries cannot capture interactions among distantly located loci on a chromosome. This is because each library cell contains one type of library vector and thus one DNA fragment. This inability to have multiple DNA fragments (library inserts) in a single cell (clone) combined with the DNA-fragment size limitation constrains the combinatorial genomic space that can be sampled and hinders the identification of beneficial interactions among distantly-located genetic loci. To overcome these limitations, we developed and demonstrated the use of Coexisting Genomic Libraries (CoGeLs). CoGeLs enable two or three genomic libraries to coexist in one cell thus allowing to screen for gene interactions in the development or improvement of a screenable trait. The stable maintenance and interactions among two CoGeLs was demonstrated first in a proof-of-concept study in a well-defined genetic background. Four sets of two genes of the L-lysine biosynthesis pathway distantly located on the E. coli chromosome were knocked out to generate auxotrophs. Upon transformation of these auxotrophs with CoGeLs, cells growing without supplementation were found to harbor library inserts containing the knocked-out genes. To demonstrate that the CoGeL technology can be applied to generate a useful complex phenotype, we searched for and identified genetic loci that impart tolerance to low pH, which is a very important bioprocessing trait. We thus generated cells that are acid tolerant and can be used as platform strains for bioprocessing under acid-stress conditions. Intellectual Merit: The concept of coexisting genomic libraries for generating complex phenotypes is novel and of practical and fundamental significance. As demonstrated, it can be applied for developing industrially important strains for bioprocessing applications, such as those currently employed or anticipated for the utilization or renewable resources (green technologies) or in bioremediation. In addition, the knowledge generated from this project will make a significant contribution to the basic biology of developing tolerance to important stressors, incl. solvents, various organic toxic chemicals and low pH. Broader Impacts: The technology of coexisting genomic libraries we developed can be applied to any organism and bioprocessing application whereby a selectable complex phenotype is desirable, and especially processes for the utilization of renewable resources. The importance of efficient and economically viable technologies for production of chemicals, biofuels from renewable resources has become an urgent national and international priority. The depletion of non-renewable energy sources, such as oil, coupled with increased energy demand driven by fast developing economies and world population has led to high oil and energy prices. Moreover, non-renewable fossil fuels lead to significant climatic changes. In contrast, biomass is a carbon-neutral renewable resource for production of biofuels and chemicals.
