A wide variety of industrially vital chemicals are currently produced from petroleum, using very well-refined processes and a large industrial infrastructure. However, petroleum processing has a number of hazardous and otherwise negative impacts on the environment, as well as on human health. The supply and price of oil into the future are uncertain as well, and supplementing oil with feedstocks from renewable sources can help extend the current supply of oil and eventually replace it. Succinate is a specialty chemical currently produced from maleic anhydride from petroleum processing. If bio-based succinate could compete with the cost of maleic anhydride, it could replace maleic anhydride in a $15 billion commodity chemical market, taking advantage of the existing chemical production infrastructure. A major potential feedstock source for conversion to succinate is lignocellulose from agricultural waste or from bioenergy crops. The bacterium Actinobacillus succinogenes is one of the best natural succinate producers and it grows on a wide variety of carbohydrates, including the major sugars in lignocellulose. A. succinogenes grows well on glucose, the most common sugar in lignocellulose, but does not grow as quickly on other lignocellulosic sugars.I have evolved strains of A. succinogenes to grow faster on xylose, the second most common lignocellulosic sugar, as well as on arabinose, galactose, and lignocellulose hydrolysates. Many of the evolved strains produce more succinate than the parental strain as well, even though the evolution process did not specifically select for succinate production. The evolved strains were resequenced to identify the mutations accumulated during evolution. RNA sequencing of the xylose-evolved strains helped identify changes in transcript levels and was used to refine our conclusions about the xylose-evolved (X) strains. I discovered that the genes that encode many glycolytic enzymes were upregulated in at least one X strain, several genes encoding succinate production enzymes were upregulated, while genes that encode enzymes that redirect fluxes from the succinate pathway to other fermentation products were downregulated. During the directed evolution process, I obtained a strain of A. succinogenes that can grow on galactose, a sugar that the base strain cannot use. The final evolved strain grew faster than the wild-type strain on xylose, arabinose, and lignocellulose hydrolysate, and could grow on galactose. I determined that A. succinogenes will co-consume glucose and xylose, but that xylose represses arabinose consumption. After directed evolution, though, arabinose represses xylose consumption. Finally, I used multiplex transformation to introduce mutations from the evolved strains into the wild-type strain. The first strain produced, using the xylose symporter mutation from a xylose-evolved strain, produced 40% more succinate than wild-type A. succinogenes, even though it grew at less than half the speed. In summary, I have evolved a set of A. succinogenes strains that grow faster on lignocellulose sugars and some have a higher succinate yield, I know the location and nature of their mutations and have RNA sequencing data for the xylose-evolved strains. I have conducted numerous additional experiments to characterize sugar consumption in A. succinogenes and what causes the evolved strains to be able to grow faster and produce more succinate. My results lay solid groundwork for future work with A. succinogenes and other bacteria being grown on sugars and synthesizing succinate.
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