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Title:An RNA-interference-based platform for genome analysis and engineering in Saccharomyces cerevisiae
Author(s):Si, Tong
Advisor(s):Zhao, Huimin
Department / Program:Chemical & Biomolecular Engr
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):RNA interference
high-throughput screening
directed evolution
Genome Engineering
Saccharomyces cerevisiae
suppressor analysis
acetic acid tolerance
Abstract:Efficient microbial production of fuels and chemicals from lignocellulosic feedstock requires intensive reprogramming of cellular machinery. Given our limited knowledge of the complex biological systems, such tasks prove to be better fulfilled with directed evolution than with rational design, by performing iterative cycles of mutagenesis and selection. However, the success of directed evolution is mostly confined to individual proteins, due to the lack of efficient tools to introduce mutations globally and iteratively on a genome scale. Thanks to its simplicity and effectiveness, the pooled RNA interference (RNAi) screening should satisfy the requirement of directed genome evolution. The RNAi pathway is absent in Saccharomyces cerevisiae, also known as the baker’s yeast. Recently, functional RNAi machinery has been reconstituted in this eukaryotic model, upon the introduction of Dicer and Argonaute proteins from a related species Saccharomyces castellii. We first explored the possibility of applying RNAi screening in S. cerevisiae. The design of convergent promoters was adapted to drive the in vivo synthesis of double strand RNAs (dsRNA), which were further processed as small interference RNAs (siRNA) to mediate the knockdown of homologous genes. A plasmid-based dsRNA library was constructed from enzymatically digested genomic DNA based on convergent-promoter design. The library was confirmed to achieve a nearly complete (>99.75%) coverage of yeast genome, and was employed to perform a suppressor analysis of the yku70 knockout. A colony-size based screening method was used to identify two known and three novel knockdown modifications that can alleviate the growth arrest of the Δyku70 strain at higher temperature. Further analysis confirmed that these genes are indeed the modulators of the Δyku70 phenotype. Encouraged by these results, we further developed an efficient, genome-scale and generally applicable method for eukaryotic reprogramming, RNAi-Assisted Genome Evolution (RAGE). In each round of RAGE, one knockdown modification conferring desired trait was identified by RNAi screening. The best knockdown cassette was integrated into the chromosome to create a new strain, serving as the parent strain for the next round of RAGE. Repeated cycles of RAGE accumulated the beneficial genetic modifications in the host genome, and thus continuously improve the target properties. As proof-of-concept, RAGE was applied to improve acetic acid (HAc) tolerance of S. cerevisiae. After three rounds, the 100% growth inhibition concentration of HAc was elevated from 0.7% to 0.9% (v/v). The best engineered strain exhibited a significantly improved fermentation performance than the wild type strain under HAc stress. By combining the powerful toolboxes of RNAi and directed evolution, RAGE may greatly accelerate the design and evolution of organisms with desired traits and provide new insights on genome structure, function, and evolution.
Issue Date:2013-05-28
Rights Information:Copyright 2013 Tong Si
Date Available in IDEALS:2013-05-28
Date Deposited:2013-05

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