Date of Graduation

2018

Document Type

Thesis

Degree Type

MS

College

Davis College of Agriculture, Natural Resources and Design

Department

Biochemistry

Committee Chair

Daniel G Panaccione

Committee Co-Chair

Matthew Kasson

Committee Member

Nikola Kovinich

Abstract

Penicillium camemberti plays a major role in the ripening process of brie and camembert type cheeses. Investigation of the recently sequenced P. camemberti genome revealed the presence of a cluster of five genes previously shown to be required for ergot alkaloid synthesis in other fungi. Clustered with the five ergot alkaloid synthesis genes (aka eas genes) were two additional genes with that had the apparent capacity to encode enzymes involved in secondary metabolism. We analyzed samples of brie and camembert cheeses as well as cultures of P. camemberti grown under different conditions and were not able to detect any known ergot alkaloids, indicating the P. camemberti eas genes were either not expressed or encoded non-functional enzymes. We used a heterologous expression strategy to investigate the theoretical biosynthetic capacity of P. camemberti. Based on studies with the related ergot alkaloid-producing fungus Neosartorya fumigata (syn. Aspergillus fumigatus ), the five known eas genes found clustered in the P. camemberti genome should give the fungus the capacity to produce the ergot alkaloid chanoclavine-I aldehyde. We used a chanoclavine-I aldehyde-accumulating mutant of N. fumigata as a recipient strain in which to express the two uncharacterized P. camemberti eas cluster genes (named easH and easQ) to create a functioning facsimile of the P. camemberti cluster. Expression of easH and easQ in the chanoclavine-I accumulating N. fumigata resulted in the accumulation of a pair of compounds of m/z 269.1 in positive mode LC-MS. Since this m/z is consistent with the mass of the isomeric pair of [rugulovasine A/B + H], we analyzed a culture of the related rugulovasine producer Penicillium biforme and found the same isomeric pair of analytes. Fragmentation of the analytes yielded fragments typical of those resulting from fragmentation of rugulovasine A/B. The deduced activities of the products of easH and easQ catalyze theoretical reactions that provide a reasonable pathway from the precursor chanoclavine-I aldehyde to the products rugulovasine A/B. When individually studied, the chanoclavine-I aldehyde-accumulating mutant of N. fumigata transformed with the P. camemberti easQ gene yielded a ratio of analytes at m/z 271.1 vs. m/z 255.1 that support the theorized mechanisms of easH and easQ to produce rugulovasines. The P. biforme genome contains a homologous eas cluster with only 12 mutations relative to that of P. camemberti. Eleven of the 12 mutations were investigated via complementation studies of the mutated genes (dmaW, easC, easE) using knockout strains of N. fumigata. The data indicate that P. camemberti has the genes to produce the ergot alkaloid rugulovasine A/B but that during domestication isolates that failed to produce alkaloids were selected for.

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