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Description
The primary objective of this project is to further the knowledge about SCL26 family of anion transporters. The goals of the experiment were to find the lowest sulfate concentration where the yeast without Sulp1 and Sulp2 is able to grow, but it grows very slowly, and to find a higher sulfate concentration where the yeast grows quickly, with or without the sulfate transporters. The lowest sulfate concentration where the yeast without the sulfate transporters is able to grow was determined to be 2-4 mM, however, this range can likely be refined by more quantitative analytical methods. At a sulfate concentration of 20 mM sulfate or higher, the yeast is able to grow quickly without high-affinity sulfate transporters. The next step in the project is to re-introduce the Sulp1 and Sulp2 genes into the yeast, so that growth in low and high sulfate conditions can be compared with and without the Sulp1 and Sulp2 proteins. The long-term goals of the project are to bring experience with yeast to Dr. Nannenga’s structural discovery lab, to determine if yeast sulfate transporters respond in the same way to drug candidates as human sulfate transporters, and to determine the structure of the proteins using cryo-electron microscopy.
ContributorsCall, Nicolas I (Author) / Nannenga, Brent (Thesis director) / Wang, Xuan (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this study show that indeed CEF is enhanced by the high amount of PSI present in WT. On the other hand, mutants with less PSI and less cyclic electron flow appeared able to maintain healthy levels of ATP synthesis through other compensatory mechanisms. Reduction in PSI abundance is naturally associated with reduced chlorophyll content, and mutants with less PSI showed greater primary productivity as light intensity increased due to increased light penetration in the cultures. Another question addressed in this research project involved the effect of deletion of flavoprotein 3 (an electron sink for PSI-generated electrons) from mutant strains that produce and secrete a fatty acid (Chapter 3). Removing Flv3 increased fatty acid production, most likely due to increased abundance of reducing equivalents that are key to fatty acid biosynthesis. Additional components of my dissertation research included examination of alkane biosynthesis in Synechocystis (Chapter 4), and effects of attempting to overexpress fibrillin genes for enhancement of stored compounds (Chapter 5). Synechocystis is an excellent platform for metabolic engineering studies with its photosynthetic capability and ease of genetic alteration, and the presented research sheds light on multiple aspects of its fundamental biology.
ContributorsMoore, Vickie (Author) / Vermaas, Willem (Thesis advisor) / Wang, Xuan (Committee member) / Roberson, Robert (Committee member) / Gaxiola, Roberto (Committee member) / Bingham, Scott (Committee member) / Arizona State University (Publisher)
Created2017
Description
In this work, secretion of free fatty acids (FFAs) and ω-hydroxy FFAs wasachieved in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis), and
FFAs were detected by a novel fluorescence assay. Current methods of detecting FFA
concentrations, including HPLC-based and GC-based methods or enzyme-based kits,
have hindered research advancement due to their laborious and/or expensive nature. The
work herein establishes a novel, rapid, fluorescence-based assay for detecting total FFA
concentrations secreted by Synechocystis FFA secretion strains. The novel FFA-detection
assay demonstrates the efficacy of using Nile Red as a fluorescent reporter for laurate or
palmitate at concentrations up to 500 µM in the presence of cationic surfactants. Total
FFA concentrations in Synechocystis supernatants quantified by the novel, Nile Red fluorescence-based assay are demonstrated herein to be highly correlative to total FFA
concentrations quantified by LC-MS; this correlation was seen in supernatant samples of
wild type Synechocystis and Synechocystis FFA secretion strains, both in 96-well plates
and 30-mL, aerated culture tubes.
This work also establishes the expression of a cytochrome P450 fusion enzyme,
CYP153A-CPRmut, or a monooxygenase system from Pseudomonas putida GPo1,
AlkBGT, in FFA secretion strains of Synechocystis for the generation of ω-hydroxy
laurate from laurate. After finding greatly increased ω-hydroxylation activity of
CYP153A-CPRmut with concurrent superoxide dismutase and catalase overexpression, 55
or 1.5 µM of ω-hydroxy laurate were produced over five days by Synechocystis strains
expressing CYP153A-CPRmut or AlkBGT, respectively. As further indication of the
presence of reactive oxygen species affecting ω-hydroxy laurate production with
Synechocystis strains expressing CYP153A-CPRmut, concentrations of ω-hydroxy laurate
in the supernatant increased over two-fold in the presence of 250 µM of the anti-oxidant,
methionine, in bench-scale cultures and in 96-well plate cultures. Additionally, a
mutation at the 55th amino acid position in AlkB (tryptophan to cysteine; AlkBW55C),
resulted in a more than two-fold shift in AlkB’s substrate preference from decanoate
towards the desired substrate, laurate. As a result, Synechocystis expressing AlkBW55C
could produce 5.9 µM ω-hydroxy laurate and 2.0 µM dodecanedioic acid over five days
of growth.
ContributorsAshe, Christopher (Author) / Vermaas, Willem Fj (Thesis advisor, Committee member) / Wang, Xuan (Committee member) / Nielsen, David R (Committee member) / Misra, Rajeev (Committee member) / Arizona State University (Publisher)
Created2023
Description
Biomass synthesis is a competing factor in biological systems geared towards generation of commodity and specialty chemicals, ultimately limiting maximum titer and yield; in this thesis, a widely generalizable, modular approach focused on decoupling biomass synthesis from the production of the phenylalanine in a genetically modified strain of E. coli BW25113 was explored with the use of synthetic trans-encoded small RNA (sRNA) to achieve greater efficiency. The naturally occurring sRNA MicC was used as a scaffold, and combined on a plasmid with a promoter for anhydrous tetracycline (aTc) and a T1/TE terminator. The coding sequence corresponding to the target binding site for fourteen potentially growth-essential gene targets as well as non-essential lacZ was placed in the seed region of the of the sRNA scaffold and transformed into BW25113, effectively generating a unique strain for each gene target. The BW25113 strain corresponding to each gene target was screened in M9 minimal media; decreased optical density and elongated cell morphology changes were observed and quantified in all induced sRNA cases where growth-essential genes were targeted. Six of the strains targeting different aspects of cell division that effectively suppressed growth and resulted in increased cell size were then screened for viability and metabolic activity in a scaled-up shaker flask experiment; all six strains were shown to be viable during stationary phase, and a metabolite analysis showed increased specific glucose consumption rates in induced strains, with unaffected specific glucose consumption rates in uninduced strains. The growth suppression, morphology and metabolic activity of the induced strains in BW25113 was compared to the bacteriostatic additives chloramphenicol, tetracycline, and streptomycin. At this same scale, the sRNA plasmid targeting the gene murA was transformed into BW25113 pINT-GA, a phenylalanine overproducer with the feedback resistant genes aroG and pheA overexpressed. Two induction times were explored during exponential phase, and while the optimal induction time was found to increase titer and yield amongst the BW25113 pINT-GA murA sRNA variant, overall this did not have as great a titer or yield as the BW25113 pINT-GA strain without the sRNA plasmid; this may be a result of the cell filamentation.
ContributorsHerschel, Daniel Jordan (Author) / Nielsen, David R (Thesis advisor) / Torres, César I (Committee member) / Wang, Xuan (Committee member) / Arizona State University (Publisher)
Created2016