Synergy of protein and genome engineering for fuels and chemicals production

Nair, Nikhil U.

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Description

Title

Synergy of protein and genome engineering for fuels and chemicals production

Author(s)

Nair, Nikhil U.

Issue Date

2010-05-14T20:51:41Z

Director of Research (if dissertation) or Advisor (if thesis)

Zhao, Huimin

Doctoral Committee Chair(s)

Zhao, Huimin

Committee Member(s)

• Masel, Richard I.
• Rao, Christopher V.
• Kuzminov, Andrei

Department of Study

Chemical & Biomolecular Engr

Discipline

Chemical Engineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Protein Engineering
• Directed Evolution
• Metabolic Engineering
• Genome Engineering
• Recombination
• Xylitol
• Butanol
• Biocatalysis
• Biofuel
• Biorefinery
• Mutagenesis

Abstract

Recent emphasis on using biomass as raw materials for the production of fuels and chemicals instead of non-renewable sources such as petroleum has accelerated the development of catalysts able to convert inedible plant and crop materials to value added compounds. Biocatalysis would seem to be an excellent choice for the process since organisms have evolved to convert sugars and other biomass materials to a variety of different compounds. Therefore, technologies that use biocatalysts have emerged as a promising foundation to build a green, sustainable bioeconomy. However, to derive value-added compounds from biomass, significant engineering work has to be performed to optimize naturally occurring organisms with better catalytic properties. The rapid advancement in understanding of molecular mechanisms of biological functions as well as development of novel molecular biological tools has now enabled this type of engineering work. Recently, two distinct fields have emerged to develop these self-renewing catalysts – protein engineering and genome/metabolic engineering – with the focus at the protein and organism level, respectively. While both techniques have had great success in developing biocatalysts, there has been little work in combining the two strategies to develop superior biocatalysts. Therefore, the main thrust of this work has been to combine protein and genome engineering efforts in order to exploit possible synergies between the two techniques. The first section describes the development of a selective biocatalyst for the production of the sugar substitute, xylitol, from a mixture of hemicellulosic hydrolysate sugars. First, a xylose-specific xylose reductase enzyme was engineered for higher substrate specificity. However, after significant loss in enzymatic activity, genome engineering was targeted as an orthogonal strategy to increase substrate specificity further. The combined effort yielded a biocatalyst able to produce xylitol to ~100% purity. Next, this strategy of combined synergy was explored for the production of n-butanol, a promising biofuel, using recombinant yeast. Using directed evolution, one of the six butanol biosynthetic enzymes was engineered for soluble, functional expression in yeast. This enabled the production of butanol from galactose, but not glucose. In order to divert carbon flux from glucose to butanol, genome engineering was performed, which finally allowed production of butanol from glucose. These two demonstrate that protein and genome engineering strategies can indeed be synergistic and that they can be used to develop properties in biocatalysts that can be beyond the capability of either one. Finally, two tools were developed to engineer the genomes of yeast, and E. coli. In yeast, MIRAGE (mutagenic inverted repeat assisted genome engineering) consists of a mutagenesis cassette comprising inverted repeat of selection markers. After introduction into the chromosome along with the desired mutations, the inverted repeat is excised due to its inherent instability in the chromosome, leaving behind no unnecessary elements in the chromosome. In E. coli, TRIIAGE (terminal repeat initiated illegitimate recombination assisted genome engineering) was been shown to be a powerful tool for creating precise mutations in the chromosome. The method relies on the ability of 16 bp direct repeats at the termini of linear DNA to enable circularization, and allow plasmid type pop-in/pop-out mutagenesis.

Graduation Semester

2010-5

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Copyright 2010 Nikhil Unni Nair

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