Engineering Zymomonas mobilis for the production of biofuels and other value-added products

Zymomonas mobilis is a promising organism for lignocellulosic biofuel production as it can efficiently produce ethanol from simple sugars using unique metabolic pathways. Z. mobilis displays what is known as the “uncoupled growth” phenomenon, meaning cells will rapidly convert sugars to ethanol regardless of their energy requirements for growth. This makes Z. mobilis attractive not only for ethanol production, but for alternative product synthesis as well. One limitation of Z. mobilis for cellulosic ethanol production is that this organism cannot natively ferment the pentose sugars, like xylose and arabinose, which are present in lignocellulosic hydrolysates. While it has been engineered to do so, the fermentation rates of these sugars are still extremely low. In this work, we have investigated sugar transport as a possible bottleneck in the fermentation of xylose by Z. mobilis. We showed that transport limits xylose fermentations in this organism, but only when the starting sugar concentration is high. To discern additional bottlenecks in pentose fermentations by Z. mobilis, we then used adaptation and high-throughput sequencing to pinpoint genetic mutations responsible for improved growth phenotypes on these sugars. We found that the transport of both xylose and arabinose through the native sugar transporter, Glf, limits pentose fermentations in Z. mobilis, thereby confirming our previous results. We also found that mutations in the AddB protein increase plasmid stability and can reduce cellular aggregation in these strains. Consistent with previous research, we found that reduced xylitol production improves xylose fermentations in Z. mobilis. We also found that increased transketolase activity and reduced glyceraldehyde-3-phosphate dehydrogenase activity improve arabinose fermentations in Z. mobilis. In order for Z. mobilis to prosper as an industrial host for alternative product synthesis, the genetic techniques utilized in this organism must be improved. Toward this goal, we have adapted the λ Red recombinase system for use in Z. mobilis. We have shown that this system increases the frequency of double crossover events for the purpose of constructing gene knockouts in the strain. We have also constructed an expression system for the type II CRISPR/CRISPR-associated (Cas) bacterial adaptive immunity system in Z. mobilis. We have shown that the Cas9 nuclease can be directed by small RNAs to target the Z. mobilis genome for the purpose of genome editing, and that this system can also likely be used to facilitate gene knockouts in this organism. Finally, toward improving heterologous protein production in Z. mobilis, we have constructed a constitutive promoter library that leads to a range of gene expression levels in the strain. Collectively, our results provide the framework for the development of an industrial production process utilizing Z. mobilis as the microbial host.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemical Engineering

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Dunn, Kori Lynn

Contributors dc:contributor

• Rao, Christopher V.
• Jin, Yong-Su
• Kong, Hyun Joon
• Kraft, Mary

• cellulosic ethanol
• pentose transport
• adaptive evolution
• recombineering
• DNA endonuclease
• metabolic engineering

Statement dc:rights

• Copyright 2015 Kori Lynn Dunn

Language dc:language

en

Handle dc:identifier

http://hdl.handle.net/2142/88278

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/88278