Graduate Thesis Or Dissertation

 

Gadusol Production in Saccharomyces cerevisiae Public Deposited

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/w3763c76s

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  • Gadusol is a UV-B-absorbent compound found in fish and other marine organisms where it is presumed to play a role as a sunscreen and antioxidant. In light of commercial potential as a replacement for problematic synthetic sunscreens, a process to produce recombinant gadusol in the yeast Saccharomcyces cerevisiae was investigated. Gadusol is derived from the pentose phosphate pathway intermediate sedoheptulose 7-phosphate (S7P) in a two-step reaction catalyzed by 2-epi-5-epivaliolone synthase (EEVS) and methyl transferase-oxidoreductase (MT-Ox). Zebrafish derived EEVS and MT-Ox cDNAs were placed under the control of a constitutive yeast promoter on separate high-copy number plasmids and introduced into S. cerevisiae to generate strain G0 that produced about 12 mg/L. A number of genetic and cultural interventions were subsequently investigated in order to increase yields. Deletion of TAL1 that encodes a well-characterized transaldolase catalyzing conversion of S7P and glyceraldehyde 3-P to fructose 6-P and erythrose 4-P increased yields to 20 mg/L (G1). To assess the additive effect of deleting the less active of the two known yeast transaldolase genes (NQM1), strain G2 was constructed that lacked both TAL1 and NQM1 and which produced about 30 mg/L. Strain G3, the highest yielding strain, was constructed by replacing the two original EEVS and MT-Ox plasmids with a single construct containing both EEVS and MT-Ox genes integrated into a yeast chromosome. Gadusol levels reached about 64 mg/L in G3 which represents a 5-fold increase over G0. Various nutritional modifications of the G3 growth medium were not found to increase yields further. Similarly, interventions intended to force glycolytic intermediates towards S7P, namely overexpression of sedoheptulose 1,7-bisphosphatase and deletion of phosphoglucoisomerase did not increase yields.
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  • 2018-03-09 to 2019-04-23

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