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Studies of Engineered and Native Cyanobacterial Strains for Increased Growth Rate

Description

The production of sustainable biochemicals has been a major topic of discussion in recent years. Using microbial cells for their production through genetic engineering has been a major topic of research. Cyanobacteria have been considered as a viable candidate for such production. However, the slow growth rate of the cells…

The production of sustainable biochemicals has been a major topic of discussion in recent years. Using microbial cells for their production through genetic engineering has been a major topic of research. Cyanobacteria have been considered as a viable candidate for such production. However, the slow growth rate of the cells presents a challenge for the possibility of scaling for use in industrial settings. This project focuses on two different solutions for this problem. The first is using four different engineered strains of Synechocystis sp. PCC 6803 that overexpress the proteins in the b6f complex to improve photosynthetic efficiency. It was found that the strains PetB and PetD showed an increase in growth rate compared to wild type cells. This was especially true under mixotrophic conditions and with a light intensity of 100 µmol photons*m-2s-1 for 3 days. The second solution is by using a newly discovered marine strain of cyanobacteria, Synechococcus sp. PCC 11901, which has a higher reported growth rate. Higher growth rates were achieved for this strain when it was grown mixotrophically with glycerol, and when grown in bubble cultures with aeration.
ContributorsWinsor, Kira Varga (Author) / Varman, Arul Mohzy (Thesis director) / Vermaas, Wim (Committee member) / School of International Letters and Cultures (Contributor) / Chemical Engineering Program (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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Knocking out the cytochrome bc complex in Heliobacterium modesticaldum

Description
The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria may also grow chemotrophically via pyruvate metabolism in the absence of light. In Heliobacterium modesticaldum, the cytochrome bc complex is…

The heliobacteria, a family of anoxygenic phototrophs, are significant to photosynthesis evolution research, as they possess the simplest known photosynthetic apparatus. Although they are photoheterotrophs in the light, the heliobacteria may also grow chemotrophically via pyruvate metabolism in the absence of light. In Heliobacterium modesticaldum, the cytochrome bc complex is responsible for oxidizing menaquinol and reducing cytochrome c553 in the electron flow cycle used for phototrophy. However, there is no known electron acceptor for cytochrome c553 other than the photosynthetic reaction center. Therefore, it was hypothesized that the cytochrome bc complex is necessary for phototrophy, but unnecessary for chemotrophic growth in the dark. Under this hypothesis, a mutant of H. modesticaldum lacking the cytochrome bc complex was predicted to be viable, but non-phototrophic. In this project, a two-step method for CRISPR-based genome editing was used in H. modesticaldum to delete the genes encoding the cytochrome bc complex. Genotypic analysis verified the deletion of the petC, B, D, and A genes encoding the catalytic components of complex. Spectroscopic studies revealed that re-reduction of cytochrome c553 after flash-induced photo-oxidation was ~130 to 190 times slower in the ∆petCBDA mutant compared to wildtype, phenotypically confirming the removal of the cytochrome bc complex. The resulting ∆petCBDA mutant was unable to grow phototrophically, instead relying on pyruvate metabolism to grow chemotrophically as does wildtype in the dark.
ContributorsLeung, Sabrina (Author) / Redding, Kevin (Thesis director) / Liu, Wei (Committee member) / Vermaas, Wim (Committee member) / School of Molecular Sciences (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05