Subject: abstracts

From: "Peter J. Punt,MBL-TNO,Rijswijk,The Neth." <[email-masked]>
Date: 16 Sep 1994 09:37:21 +0100

STRAIN IMPROVEMENT STRATEGIES FOR THE OVERPRODUCTION OF FUNGAL
PROTEINS BY FILAMENTOUS FUNGI
Jan C.Verdoes, Peter J. Punt and Cees A.M.M.J. van den Hondel.

Applied Microbiology and Biotechnology (1994) in press

I. INTRODUCTION

     Filamentous fungi may be isolated from a large number of
different natural environments (e.g. from soil, organic waste
and as pathogens from plants and animals). These different
habitats and the competition with other micro-organisms in the
same place may explain the extraordinary metabolic diversity
of many filamentous fungi, as illustrated by the wide variety
of carbon and nitrogen sources they can use for growth. The
availability of a multitude of biosynthetic and catabolic
pathways necessary for this metabolic diversity makes
filamentous fungi potential production organisms for a wide
range of low molecular-weight primary and secondary
metabolites, and even more importantly, intra- and
extracellular hydrolytic enzymes. In effect, filamentous fungi
or the enzymes they produce have been used in food and
foodprocessing industry for many decades which resulted in a
so-called GRAS status (Generally Recognized As Safe; U.S Food
and Drug Administration) for some of the species (such as
Aspergillus niger and A. oryzae).=20
     The industrial potential of filamentous fungi has
stimulated research in the development of large scale
fermentation processes, down-stream processing and methods for
strain improvement (see below section II), the results of
which further contributed to the use of filamentous fungi in
today's industry. The development of molecular genetic
techniques (in particular gene transfer technology) for most
"industrial" species (for reviews see Finkelstein, 1991; May,
1992) has even further opened the way for industrial
exploitation of these organisms in particular for the
production of enzymes encoded by fungal, and even non-fungal,
genes. In this chapter we give an overview of research carried
out towards improved production of extracellular fungal
proteins. In general, the proteins of interest are hydrolytic
enzymes involved in the degradation of polysaccharides, which
degradation product is used by the fungi as carbon source.=20

=0CTHE EFFECT OF MULTIPLE COPIES OF THE UPSTREAM REGION ON
EXPRESSION OF THE ASPERGILLUS NIGER GLUCOAMYLASE-ENCODING GENE=20
Jan C. Verdoesa&, Peter J. Punta, Adriaan H. Stouthamerb and
Cees A.M.J.J. van den Hondela

Gene (1994) in press

SUMMARY
     The regulation of transcription of the glucoamylase-
encoding gene (glaA) of Aspergillus niger was studied. To
facilitate this study a reporter strain containing a fusion of
the glaA promoter (PglaA) of A. niger to the =FE-glucuronidase-
encoding gene (uidA) of Escherichia coli was constructed. To
analyze whether regulatory proteins are involved in the
regulation of glaA, multiple copies of PglaA were introduced
into this reporter strain. Analysis of the resulting strains
revealed that introduction of an increasing number of PglaA
copies resulted in lower expression of the uidA reporter gene
and the endogenous glaA gene in cultures cultivated on
different inducing carbon sources. However, repression by
xylose was not influenced by the copy number of PglaA. These
results indicate that the expression of genes under control of
PglaA are regulated by specific trans-acting regulatory
protein(s).  Deletion analysis of PglaA indicated that
regulatory proteins interact with DNA sequences within 0.5-kb
upstream from the ATG, whereas sequences between about 0.8 and
0.5-kb upstream from the ATG are required for high-level
expression of glaA.

=0CCHARACTERIZATION OF AN EFFICIENT GENE CLONING STRATEGY FOR
ASPERGILLUS NIGER BASED ON AN AUTONOMOUSLY REPLICATING
PLASMID: CLONING OF THE NICB GENE OF A. NIGER

Jan C. Verdoesa*, Peter J. Punta, Pieter van der Bergab, Fons
Debetsb, Adriaan H. Stouthamerc and Cees A.M.J.J. van den
Hondela=20

Gene (1994) in press

SUMMARY

     The development of an improved gene cloning strategy by
complementation of mutant alleles in Aspergillus niger is
described. The strategy is based on the use of a fungal
autonomously replicating vector, pAB4-ARp1. This vector was
constructed by the introduction of a previously described
sequence (AMA1), involved in autonomous replication, into a
pyrG integrative vector, pAB4-1. With vector pAB4-ARp1, a 10
to 100-fold increase in transformation frequency was obtained,
as compared to pAB4-1. Furthermore, the transformation
frequency of a cotransformed plasmid is also increased using
pAB4-ARp1. A. niger transformants containing pAB4-ARp1 are
mitotically unstable. Cotransformed plasmids strictly
cosegregated with the autonomously replicating vector, as a
result of recombination between both vectors. The use of pAB4-
ARp1 in gene cloning was demonstrated by the complementation
of two linkage group VII-specific A. niger mutants.
Complementation of a lysF mutant was achieved by
cotransformation of pAB4-ARp1 with total genomic A. niger DNA
("instant bank"). A nicB-deficient A. niger was complemented
by cotransformation with pAB4-ARp1 and an A. niger cosmid
library. The complementing DNA was re-isolated from a Nic+
transformant by transforming E. coli with total genomic DNA of
this transformant. Gene disruption and genetic analysis was
carried out to prove that the previous unknown A. niger nicB
gene has been cloned.=20

=0CTHE COMPLETE KARYOTYPE OF ASPERGILLUS NIGER: THE USE OF
INTRODUCED ELECTROPHORETIC MOBILITY VARIATION OF CHROMOSOMES
FOR GENE ASSIGNMENT STUDIES.=20

Jan C. Verdoes*, M. Regina Calila, Peter J. Punt, Fons Debetsa,
Klaas Swarta, Adriaan H. Stouthamerb and Cees A.M.J.J. van den
Hondel.

Molecular General Genetics (1994) 244:75-80

Summary
A method is described for unambiguous assignment of cloned
genes to Aspergillus niger chromosomes by CHEF-Southern
analysis. All of the 8 linkage groups (LGs) with the exception
of LG VII have previously been assigned to specific
chromosomal bands in the electrophoretic karyotype of A. niger
(Debets et al. (1990) MGG 224: 264-268). Using a LG VII
specific probe (nicB gene of A. niger) we have shown that LG
VII corresponds to  a chromosome of about 4.1 Mb. Furthermore,
genetic localization of three unassigned genes (glaA, aglA and
pepA) in strains in which these genes were replaced using a
selectable marker gene led to a revised karyotype with respect
for the chromosomes corresponding LG VIII and VI. The revised
electrophoretic karyotype reveals only 5 distinct bands. The
presence of 3 pairs of equally sized chromosomes precluded
assignment of genes to one specific chromosome in the wild-
type strain. However, unambiguous chromosome assignment of
cloned genes using CHEF-Southern analysis was demonstrated
using a set of A. niger strains with introduced chromosomal
size variation. The availability of these tester strains
obviates the need of isolating or constructing mutant strains
for the purpose of chromosome assignment. =0CEVALUATION OF MOLECULAR =
AND GENETIC APPROACHES TO GENERATE
GLUCOAMYLASE OVERPRODUCING STRAINS OF ASPERGILLUS NIGER=20
=20
Jan C. Verdoes1 *, Anne D. van Diepeningen2, Peter J. Punt1,
Alfons J.M. Debets2, Adriaan H. Stouthamer3 and Cees A.M.J.J.
van den Hondel1

Journal of Biotechnology 36 (1994) 165-175

Summary
To evaluate the possibility to improve glucoamylase (GLA)
production in A. niger strains, carrying multiple copies of
the GLA encoding gene (glaA), additional glaA copies were
introduced either by genetic recombination or
retransformation. For strains to be used in such experiments a
genetic analysis was first carried out. The results of this
analysis clearly revealed that in each transformant
integration had occurred at a chromosome corresponding to a
single linkage group. The GLA production per gene copy showed
considerable variation in these strains, indicating a clear
effect of the site of integration on gene expression.
Introduction of additional gene copies by genetic
recombination experiments was carried out for different
combinations of strains, carrying glaA copies in different
chromosomes. The introduction of additional glaA gene copies
by genetic recombination did not result in a considerable
increase in GLA production compared to the parental strains.
In some strains recombination resulted in genetic instability,
observed by the frequent loss of glaA copies. Also
retransformation of multi-copy glaA strains did not result in
increase in GLA production. In several strains even a decrease
in GLA production was found after retransformation. Southern
analysis of these transformants suggested that newly
introduced gene copies were heavily rearranged which partly
explains why GLA production was not increased. Further
analysis of one such transformant provided evidence that the
overexpression of the glaA gene is limited by the amount of
trans-acting regulatory protein(s) available.