PNNL-13501 - Pacific Northwest National Laboratory

Fungal Molecular Biology: Promoter Isolation and Characterization
Study Control Number: PN99028/1356
Johnway Gao, Todd O. Stevens, Fred J. Brockman
The capability to discover, isolate, and characterize novel genetic regulatory elements and to develop transformation
systems for industrially viable fungus strains is a vital part of creating new processes for efficient conversion of plant
materials (cellulose, hemicellulose, starch, and sugars), and to useful products, such as biocatalysts (foreign protein
expression) and bio-based fuels and chemicals (pathway engineering).
Project Description
The genetic regulatory patterns of native R. oryzae
(ATCC9363) promoters associated with the
phosphoglycerate kinase (pgk) and glucoamylase genes
were characterized by Northern blot analysis in various
growth environments containing starch, hexoses, and
pentoses. Chromosomal integration vectors were
designed and constructed for genetic transformation of
R. oryzae. Chromosomal integration vectors were
constructed using the phosphoglycerate kinase 1 (pgk1)
promoter to direct the expression of an antibiotic
resistance gene and a marker gene coding for a green
fluorescent protein (gfp). R. oryzae spores were
transformed with the integrating vector using an
electroporation method. The project also tested the
possibility to convert multi-nucleate spores of R. oryzae
to single-nucleate lineages using laser micromanipulation
and microsurgery techniques.
Introduction
To date, there are only a limited number of fungal strains
and expression systems available in the public domain.
The available expression systems include various species
of Aspergillus and the yeasts Saccharomyces cerevisiae
and Pichia pastoris. However, these strains represent
only a small sample of this diverse microbial community,
and they are not necessarily optimal for converting plant
material into useful products. For example, neither
Saccharomyces cerevisiae nor Pichia pastoris can grow
directly on plant materials, but require refined substrates
such as glucose, galactose, and methanol. The ability to
genetically harness other fungi requires a fundamental
understanding of gene regulation and its application to
product biosynthesis. The objective of the project was to
develop this expertise and to create a new capability in
fungal molecular biology. This was accomplished by
elucidating gene regulatory mechanisms and developing
transformation vector system in a model filamentous
76 FY 2000 Laboratory Directed Research and Development Annual Report
fungus with known utility (R. oryzae, capable of
degrading plant materials and producing lactic acid)
(Soccol et al. 1995; Yin et al. 1997).
Approach
The current project focused on the development of a
transformation system for the model filamentous fungi, R.
oryzae, as a viable platform that can be used in
biotechnology applications. The development of a
transformation system of R. oryzae requires a
fundamental understanding of important gene regulatory
characteristics under various environmental conditions as
well as the development of transformation and expression
vector. In addition, the characterization of multi-nuclei in
different growth stages of R. oryzae is also essential to
determine the feasibility to convert multi-nucleate fungal
spores to single-nucleate spores.
Results and Accomplishments
This project involved four principal efforts: 1) promoter
regulatory pattern characterization, 2) design and
construction of chromosomal integrating vector,
3) system transformation experiments, and 4) feasibility
of converting multi-nucleate fungal spores to singlenucleate
spores.
Promoter Regulatory Pattern Characterization
To characterize the regulatory patterns of the pgk1
promoter and glucoamylase promoter, R. oryzae was
grown in different culture media containing glucose,
starch, xylose, mannose, galactose, and arabinose,
respectively. Total RNA samples were isolated from the
mycelia biomass, separated in formaldehyde-agarose gel,
and subsequently capillary-blotted onto Zeta-Probe
membrane. Northern blots were hybridized with an α 32 PdCTP
labeled pgk1 gene probe and glucoamylase gene
probe, respectively. The Northern-blot analysis results