Document Type

Dissertation - Open Access

Award Date


Degree Name

Doctor of Philosophy (PhD)


Biology and Microbiology

First Advisor

Ruanbao Zhou


biofuel, cyanobacteria, farnesene, linalool terpenoids


In the last few decades, concerns over global climate change, energy security, and environmental pollution have been rising. To overcome these challenges, the concept of “-nth generation” biofuels has emerged as a strategy to convert solar radiation into fuels and bulk industrial chemicals for societal use, while decreasing our consumption of nonrenewable energy sources. Nitrogen-fixing cyanobacteria hold a distinct advantage in biofuel production over plants, given their ability to convert sunlight, air (CO2 and N2), and mineralized water to energy-dense carbon molecules, as well as fix atmospheric nitrogen gas into ammonia for metabolism. Engineered cyanobacteria with re-wired metabolic pathways have recently been designed through synthetic biology, and they possess the ability to synthesize new chemicals and biofuels, which are secreted from their cells. Terpenoids constitute one of the largest classes of organic molecules on Earth, and are attractive candidates as a fourth generation biofuel and industrial chemical. In cyanobacteria, the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is responsible for building essential metabolites involved in photosynthesis, as well as precursors for terpenoid biosynthesis. This dissertation encompasses research focused on redirecting MEP flux in the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120 to engineered terpenoid sinks, namely, linalool (C10H18O) and farnesene (C15H24). Chapter 1 is a review of literature in the field of biofuels and cyanobacteria, and chapter 2 is an introduction/list of objectives for the research in this dissertation. In chapter 3, we present the genetic engineering of Anabaena to synthesize farnesene by expressing a plant farnesene synthase. In chapter 4, we present the genetic engineering of Anabaena to synthesize linalool during N2-fixation, and increased linalool production is accomplished by the over-expression of three ratelimiting enzymes in the MEP pathway. In chapter 5, we examine the feasibility of a blocking a native carbon reservoir in the cyanobacterium to increase metabolite and energy availability for terpenoid synthesis, as well as physiological aspects of glycogendeficiency in the cyanobacterium during diazotrophic growth. In chapter 6, we focus on introducing a synthetic photorespiratory bypass to reduce photorespiration and increase carbon partitioning towards linalool synthesis.

Library of Congress Subject Headings

Biomass energy
Nitrogen-fixing microorganisms
Synthetic biology


Includes bibliographical references (pages 228-259)



Number of Pages



South Dakota State University


Copyright © 2017 Charles Travis Halfmann