Graduate Thesis Or Dissertation
 

Evolutionary and Biochemical Studies of C₇-cyclitol Synthases and their Sunscreen Gene Clusters

Public Deposited

Downloadable Content

Download PDF
https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/wp988r02s

Descriptions

Attribute NameValues
Creator
Abstract
  • Sugar phosphate cyclases (SPCs) are enzymes involved in the biosynthesis of many important natural products. SPCs form a unique cyclitol during the early steps in the biosynthesis of many natural products. These natural products include rifamycin, which is chemically modified to rifampicin and used clinically as an antituberculosis drug, and acarbose, an antidiabetic. Other natural products formed by SPCs have functions including but not limited to antifungal, antitumor, antioxidant, sunscreen, and wound healing activities. Thus, SPCs are useful genome mining probes for the discovery of novel natural products. One subset of SPCs are the sedoheptulose 7-phosphate cyclases (SH7PCs), which consists of three enzymes that each form a distinct cyclitol product from the same substrate, sedoheptulose 7-phosphate (SH7P). The three SH7PCs are 2-O-desmethyl-4-deoxygadusol synthases (DDGS), 2-epi-5-epi-valiolone synthases (EEVS), and 2-epi-valiolone synthases (EVS). DDGS is only involved in the biosynthesis of mycosporine-like amino acids (MAAs), which are sunscreen natural products. In contrast, EEVS and EVS are found in the biosynthesis of a variety of pseudosugars and aminocyclitols. This dissertation describes the identification, distribution, and evolution of SH7PCs and their use as probes for forming natural and unnatural sunscreens. SH7PCs are thought to descend from the shikimate pathway enzyme 3-dehydroquinate synthase (DHQS), which all vertebrates lack. However, genes encoding putative EEVS enzymes were identified in the genomes of non-mammalian vertebrates. Using Danio rerio (zebrafish) as a model, the vertebrate EEVS and an adjacent gene encoding a methyltransferase-oxidoreductase (MT-Ox) were shown to make a sunscreen called gadusol. Due to its similarity to MAAs, gadusol was originally thought to have dietary origins in marine fishes. However, the gadusol genes were expressed in D. rerio embryos and gadusol was identified in embryo extracts. Furthermore, gadusol was produced enzymatically and by heterologous production in Streptomyces coelicolor and Saccharomyces cerevisiae, establishing sustainable production routes for further development. One of the setbacks in the field was that SPCs were often misannotated in public databases due to their sequence similarity. In this thesis, two conserved active site amino acid motifs were identified by bioinformatics, allowing SPCs to be distinguished based off primary amino acid sequence. These sequences are more conserved in DDGS than other SPCs, possibly due to stringent spatial requirements needed for performing a dehydration reaction that other SPCs do not. Each motif contains a catalytic amino acid verified by protein crystallography and site-directed mutagenesis. Point mutation either of these catalytic amino acids in DDGS or EEVS disrupted catalysis. The bioinformatic analysis highlighted the distribution and evolution for DDGS and EEVS, suggesting their broad roles in nature. While EEVS genes are mostly found in Streptomyces spp. and non-mammalian vertebrates, DDGS genes are mostly distributed in cyanobacteria, fungi, and non-Streptomyces Actinobacteria. Through a genome mining study, an MAA gene cluster was also found in the nursery plant pathogen Rhodococcus fascians D188. This gene cluster was expressed in S. coelicolor M1152 and shinorine was produced as the primary MAA, whereas porphyra-334 and mycosporine-alanine-glycine were identified as minor products. This is the first demonstration that a bacterial pathogen encodes functional MAA genes, and MAAs may provide R. fascians D188 an advantage in its native environment. With functional MAA genes on hand, a synthetic pathway was designed for expression in S. coelicolor M1152 using ValA, the model EEVS from the validamycin pathway, the D. rerio MT-Ox, and the last two R. fascians D188 MAA biosynthetic enzymes to form unnatural gadusol and MAA analogs. These new compounds, called gadusporines, show unique UV absorbance at 340 nm, which is red-shifted compared to their natural counterparts. Gadusporine A, a hydroxylated shinorine analog, was the major analog produced and gadusporine B, a mycosporine-alanine-glycine analog, was also purified. The expression of gadusporines establishes a platform for further engineering of nature-inspired sunscreens.
License
Resource Type
Date Issued
Degree Level
Degree Name
Degree Field
Degree Grantor
Commencement Year
Advisor
Academic Affiliation
Rights Statement
Publisher
Peer Reviewed
Language
Embargo date range
  • 2019-06-14 to 2020-07-14

Relationships

Parents:

This work has no parents.

In Collection:

Items

Thumbnail Title Date Uploaded Visibility Actions
file details OsbornAndrewR2019.pdf 2019-06-13 Public Download