Degree Type

Dissertation

Date of Award

2011

Degree Name

Doctor of Philosophy

Department

Biochemistry, Biophysics and Molecular Biology

First Advisor

Thomas A. Bobik

Abstract

Biomass is widely accepted as a good alternative to petroleum due to concerns about global warming and energy security. Microbial conversion of renewable resources into fuel molecules and important chemicals has been achieved through efforts in genetic engineering of the biocatalysts. In this work, we demonstrated efficient production of an important bulk chemical in glucose fermentation by recombinant Escherichia coli strains. Escherichia coli K12 strain MG1655 was engineered to co-produce acetaldehyde and hydrogen during glucose fermentation using an exogenous acetyl-CoA reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 y 1.63 ymol min-1 mg-1) and the desired acetyl-CoA reductase activity (49.23 y 2.88 ymol min-1 mg-1). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, ΔfrdC). The engineered strain (ZH88) produced of 4.91 y 0.29 mM acetaldehyde while consuming 11.05 mM glucose, but also produced 6.44 y 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated and all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g/L yeast extract and lowering the pH to 6.0 further increased the co-production of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 y 0.20 g·h-1·g-1 dry cell weight, and in 86% of theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation based on the co-production of acetaldehyde and hydrogen is discussed.

Copyright Owner

Huilin Zhu

Language

en

Date Available

2012-04-06

File Format

application/pdf

File Size

110 pages

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