Guanghua Li

Document Type

Thesis - University Access Only

Award Date

1997

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

The exopolysaccharide pullulan is synthesized by the polymorphic fungus Aureobasidium pullulans. This complex polysaccharide is primarily composed of crosslinked maltotriose units. It can be produced under industrial conditions using sucrose, glucose or corn syrup as the source of carbon and energy. Although pullulan has impo1iant economic potential as a biodegradable agent in plastic production and as a blood plasma partial substitute in medical applications, nothing is known about the enzymes and genes that control pullulan biosynthesis. This study was carried out to obtain a better understanding of pullulan biosynthesis and to create pullulan overproducers by gene engineering. Mutagenesis of A. pullulans using ethyl methane sulfonate (EMS) and yirradiation was completed to obtain pullulan deficient (pur) mutants and OMP decarboxylase deficient (ura3-) mutants. The Random Amplified Polymorphic DNA (RAPD) technique was used to detect DNA polymorphism between A. pullulans wild type strain and pullulan deficient mutants. The polymorphic DNAs, presumably pullulan biosynthesis genes, were ligated to a ura3+ plasmid (pRS316) and the products were used to transfonn a ura3- and pur A. pullulans recipient to restore pullulan production ( complementation). Three polymorphic DNAs were obtained in RAPD runs using the random 10 mer primers OP AB-04, OPE-15 and OPX-05 (Figures 8, 9 and 10). Nucleotide sequence analysis indicated no match for AB04-D A and X05-DNA to any genes listed in Genbank. However, E15-D A was partially homologous to the exon region of a P. falciparum glutamic acid-rich protein gene in a non-redundant nucleotide sequence database. S. cerevisiae chromosome XI reading frame ORF YKL202W was found to have the greatest similarity to El5-DNA in a yeast (S. cerevisiae) genomic nucleotide sequence database. Seven pullulan deficient and ura3- double mutants were isolated and one of them (B40203) was used as a recipient for AB04-DNA, E15-D A and X05-D TA transformation. The circular fonn of the PRS316 vector had a higher transfon11ation frequency than the linear fonn. The numbers of transfom1ants obtained were proportional to the amount of plasmid used. Southern hybridizations revealed integration of the vector into the genomic DNA: of the transformants. A test of pullulan production showed that AP AB04-02, the only transformant with a slimy appearance on a SM plate, had better growth and pullulan accumulation in Ueda sucrose liquid medium than the other three transformants and the recipient strain B40203. A transfon11ation system using pRS316 as the vector and a ura3- and pur A. pullulans mutant as the recipient was developed. Polymorphic DNAs wi11 be used as probes to screen, if available, a genomic D A library of A. pullulans carrying pullulan biosynthesis genes.

Library of Congress Subject Headings

Polysaccharides -- Synthesis

Mutegenesis

Genetic transformation

Format

application/pdf

Number of Pages

103

Publisher

South Dakota State University

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