PR8.1
Sandy Merino
Novozymes Inc.
The majority of commercial enzyme products for biomass hydrolysis are produced
by the saprophytic mesophilic fungus
Trichoderma reesei.
Trichoderma
produces two cellobiohydrolases (CBHI and CBHII), five endoglucanases (EGs), and
two β-glucosidases (BGs). This mix of enzymes is relatively efficient at
cellulose degradation and large quantities of these proteins are secreted from
the fungus. However, improvements in the total enzyme specific activity
and secretion yield may improve this enzyme mix. A primary factor in the high
cost of enzymes for biomass hydrolysis is the amount of enzyme that must be
applied for efficient cellulose conversion to glucose. Compared with
starch hydrolysis, 15-100fold more enzyme is required to produce an equivalent
amount of ethanol, depending on specific process conditions. It is well
known that efficient cellulose hydrolysis requires a complex, interacting mix of
cellulose degrading proteins. To significantly reduce the enzyme loading
required, one may replace
Trichoderma
components with more efficient candidates, or augment the enzyme system with
additional components to improve the overall enzyme performance. In this study,
identification of new genes that improve specific performance in hydrolysis of
pretreated corn stover, and their expression in
Trichoderma
will be discussed.
Tianhong Wang,
Zhong-Hai Li, Chun-Mei Du,Yao-Hua Zhong
State Key Laboratory of Microbial Technology,
wangtianhong@sdu.edu.cn
Penicillium decumbens
is an important industrial filamentous fungus and has been widely used in
biorefinery for its high production of cellulase and hemicellulase. However,
genetic engineering strategies are still rarely applied for strain improvement
in P. decumbens. It has been proven that the frequency of targeted gene
replacement in fungi can be increased by inactivation of the main components
required for non-homologous end joining (NHEJ) pathway.
To improve gene targeting efficiency in P. decumbens, the putative
pku70 encoding KU70 homolog involved in the NHEJ process was identified and
deleted. The △pku70 strain displayed wild-type phenotypes regarding
vegetative growth, conidiation and cellulase production, and appeared the
similar sensitivity to hygromycin,
Establishment of highly efficient gene targeting system opens the way to
large-scale functional genomics analysis in P. decumbens and contributes
to the study of the mechanism of lignocellulose degradation by P. decumbens.
PR8.3
Victor Garcia Tagua[1]
H Ramirez-Medina[1] A Idnurm[2] C Sanz[3] AP
Eslava[3] E Cerdá-Olmedo[1] LM Corrochano[1]
1Dep
Genet, Univ Sevilla, 2School Biol Sci, Univ
victor_tagua@us.es
The biosynthesis of beta-carotene and its regulation by environmental factors,
including blue light and sexual interaction, has been investigated in detail in
the Zygomycete fungus
Phycomyces blakesleeanus.
Mutations in the regulatory genes
carS,
carF,
or
carD
result in mycelia that accumulate beta-carotene.
The gene
carS
is genetically linked to the structural genes
carB
and
carRA.
From the
Phycomyces
genome sequence we have identified several candidate genes in the vicinity of
the
carRA/carB
cluster. One of them encodes a putative protein similar to beta-carotene
oxygenases, and we found that a
carS
strain had a single mutation that replaced a Ser at position 433 by Leu. We
sequenced the gene strains carrying different carS mutations. We have sequenced
7 carS alleles and in all cases we have found single point mutations in the
putative beta-carotene oxygenase gene. For this reason we now consider this
putative beta-carotene oxygenase gene as the
Phycomyces
carS
gene.
The gene
carS
encodes a 628 aminoacid protein. It contains two introns and the cDNA has been
expressed in
E. coli.
The oxygenase CARS breaks beta-carotene in two molecules of 25 and 15 atoms of
carbon. The
carS
gene is repressed by light and it is activated by sexual interaction.
The discovery that mutations in the gene of a beta-carotene oxygenase causes the
acumulation of beta-carotene suggests that a derivate of beta-carotene acts as
repressor of the biosynthesis or that the CARS enzyme itself could act as a
repressor of the pathway.
Claudia Sigl[2]
Hubertus Haas[1] Heiko Eichhorn[2] Hubert Kürnsteiner[2]
Ivo Zadra[2]
1Biocenter,
Division of Molecular Biology, Innsbruck Medical University, Fritz-Pregl Strasse
3, A-6020 Innsbruck, Austria, 2Sandoz GmbH
Claudia.Sigl@student.i-med.ac.at
Penicillium chrysogenum
is the main industrial producer of the β-lactam antibiotic penicillin. Asexual
conidiation is a key developmental process in this filamentous fungus. Evidence
in filamentous fungi suggests that secondary metabolism might be linked with
developmental processes such as conidiation.
We therefore generated deletion mutants of the transcription factors
brlA
and
stuA
using a
Pcku70
deletion strain with improved gene targeting efficiency.
brlA
is known as a central regulator of asexual sporulation and plays a crucial role
in the development of conidiophores. The APSES domain transcription factor
stuA
influences conidiation by modulating the central developmental pathway that
directs the differentiation of conidiophores and conidia from vegetative hyphae.
Both deletion mutants,
ΔbrlA
and
ΔstuA,
showed drastic impairment in conidiation and morphology under light and electron
microscope. Besides, micro array analysis disclosed altered regulation of
various genes involved in differentiation in both mutants compared to the
parental strain
Pcku70.
While conidiation was completely blocked in
ΔbrlA,
it was severely decreased in
ΔstuA.
Furthermore, HPLC-, qPCR- and array analysis revealed a dramatic regulation of
penicillin biosynthesis in the
ΔstuA
mutant.
PR8.5
Hans Visser[1]
Rob Joosten[1] Peter Punt[2] Sandra Hinz[1] Jan
Wery[1]
1
Dyadic
hvisser@dyadic.nl
Filamentous fungi, particularly species such as
Aspergillus
Rob Joosten,
Sandra Hinz, Hans Visser, Jan Wery
Dyadic Nederland, Wageningen, Netherlands
rjoosten@dyadic.nl
Plant cell wall polysaccharides (PCWP) are large interconnected biopolymers that
together form complex rigid structures. For efficient breakdown of PCWP, e.g.
in second generation biofuel production or in feed and food applications, a
variety of enzymes is needed to efficiently hydrolyze the cellulosic and
hemi-cellulosic fibers. The
Chrysosporium lucknowense
C1-genome contains over 115 genes that encode PCWP-degrading enzymes and each of
these is being over-expressed in specially designed low protein background C1
strains. This has now resulted in an enzyme library of over 60 specific
carbohydrate active enzymes. This growing C1-enzyme library is used for mixing
and matching experiments, e.g. to establish limitations in current biofuel
enzymes, as well as for specific enzyme characterization and application studies
in the area of feed, food and pulp & paper. We continue to expand the enzyme
library in C1, which now contains mainly cellulases, hemi-cellulases and other
corresponding accessory enzymes.
Vivi Joosten,
Theo Verwoerd, Hans Visser, Jan Wery
Dyadic Nederland, Wageningen, Netherlands
vjoosten@dyadic.nl
Filamentous fungi have proven to produce and secrete large quantities of
extracellular enzymes. Species such as
Aspergillus
Khomaizon Abdul Kadir Pahirulzaman,
Colin Lazarus
bzkakp@bristol.ac.uk
Vector pTAex3 was modified for use in characterising fungal polyketide synthases
(PKS) and hybrid polyketide synthase-non-ribosomal peptide synthases (PKS-NRPS)
in an arginine auxotroph of
Aspergillus oryzae.
In pTAex3GS the strong, starch-inducible
amyB
expression cassette was modified for GATEWAY LR recombination, and in further
derivatives the
argB
selectable marker was replaced with basta- (bar)
or bleomycin- (ble)
resistance genes to allow co- or sequential transformation. Tailoring enzymes
convert PKS/PKS-NRPS products to final secondary metabolites; tenellin
production in
Beauveria bassiana
requires an enoyl reductase and two cytochrome P450s in addition to a PKS-NRPS
encoded by
tenS.
We produced tenellin in
A. oryzae
by introducing all four genes in
amyB
expression cassettes on three separate plasmids. To simplify plasmid
construction for whole-pathway expression pTAex3GS was converted to a yeast-E.
coli
shuttle vector. The
A. nidulans
argB
gene functions well in
A. oryzae,
so promoters from three other arginine biosynthesis genes (carbomylphosphate
synthase (P1), arginosuccinate synthase (P2) and arginosuccinate lyase (P3) were
inserted by homologous recombination in yeast. The resultant pTAYAGSarg3P was
further modified, replacing
argB
with
bar
and
ble
markers. Yeast recombination simultaneously placed the three tenellin tailoring
genes downstream of the
arg
promoters, creating pTAYAGSarg3genes, and introduction of
tenS
by GATEWAY recombination reconstructed the whole tenellin synthesis pathway in
pTAYAargTENELLIN, which was introduced into
A. oryzae.
While the easy construction principle has been proven, tenellin synthesis is not
yet a likely outcome because in tests of the
arg
promoters only P2 gave an acceptable level of eGFP expression.
Dominik Wagner[1]
Anne Schmeinck[1] Magdalena Mos[2] Igor Y Morozov[2]
Mark X Caddick[2] Bettina Tudzynski[1]
1Westfälische
Wilhelms-Universität Münster, 2University of
d_wagn01@uni-muenster.de
The rice pathogen
Fusarium fujikuroi
produces a broad spectrum of secondary metabolites. The synthesis of two of
them, gibberellin and bikaverin, is subject to the nitrogen metabolite
repression. One of the key elements of this complex regulatory network is the
GATA transcription factor AreA which activates the expression of the gibberellin
biosynthesis genes. In
A. nidulans
the main antagonist of AreA is NMR. The expression of NMR is positively
controlled by the bZIP transcription factor MeaB which is therefore an indirect
antagonist of AreA. We examined the role of MeaB in the nitrogen metabolite
repression of
Fusarium fujikuroi.
Knockout and overexpression mutants of
meaB
were created, and the impact on the expression of nitrogen repressed genes was
studied by Northern analysis. It was shown that MeaB has also a mainly negative
effect on the expression of these genes in
F. fujikuroi,
but that this effect is not as strong as in
A. nidulans.
Interestingly the repressing effect of MeaB is not mediated via the expression
control of
nmr
as proved by promoter studies in the ∆meaB-background.
Additionally, we identified two transcript sizes of
meaB
that are part of a complex regulation system. Western analysis indicated that
only the large transcript is translated. This regulation system allows MeaB to
migrate into the nucleus only under high nitrogen concentrations as shown by
MeaB-GFP-Fusion. The results indicate the role of MeaB as a fine tuning
regulator in the nitrogen regulation network and highlight differences between
F. fujikuroi
and
A. nidulans
concerning this role.
Katina Andrea Kiep,
Yvonne Göcke, Prof. Dr. -Ing. Dietmar C. Hempel, Prof. Dr. Rainer Krull
TU Braunschweig, Institute of Biochemical Engineering
k.kiep@tu-braunschweig.de
Filamentous fungi like
Aspergillus
The contribution displays the influence of pH-value, volumetric power input and
inoculum concentration on the observed morphology and the formation of
homologous recombinant β-fructofuranosidase under a constitutive promoter as
model product. Batch cultivations are monitored starting with the germination of
A.
In conclusion, the protein formation in batch processes is linked to defined
cultivation conditions to reveal bottlenecks within the complex production path
from gene to product. Therefore the shown results indicate targets for
improving, optimising and controlling industrial bioprocesses.
Katarina Kopke,
Birgit Hoff, Ulrich Kück
Christian Doppler Laboratory for “Fungal Biotechnology”, Lehrstuhl für
Allgemeine und Molekulare Botanik, Ruhr-Universität
katarina.kopke@rub.de
To overcome the limited availability of antibiotic resistance markers in
filamentous fungi we have established a system for marker recycling. For this
purpose we have chosen the FLP/FRT
recombinase system from the yeast
Saccharomyces cerevisiae
for application in the penicillin producer
Penicillium chrysogenum.
In a first step, we generated a nourseothricin resistance cassette flanked by
the
FRT
sequences in direct repeat orientation (FRTnat1
cassette) and ectopically integrated this construct into a
P. chrysogenum
recipient strain. In a second step the gene for the native yeast recombinase and
in parallel a codon optimized
Pcflp
recombinase gene were transferred into the
P. chrysogenum
strain, carrying the
FRTnat1
cassette. The corresponding transformants were analyzed by PCR, growth tests and
sequencing to verify successful recombination events. Our analysis of several
single and multicopy transformants showed that only with the codon-optimized
recombinase a fully functional recombination system was achievable in
P. chrysogenum.
To further extend the application of the FLP/FRT
recombinase system, we produced a ΔPcFRTku70PcFLP strain to establish a highly
efficient homologous recombination system for the construction of marker free
knockout strains. The applicability of the developed FLP/FRT
recombinase system was further demonstrated by marker recycling in the
ascomycete
Sordaria macrospora.
In summary we have developed a optimized FLP/FRT
recombinase system as a molecular tool for the genetic manipulation of
filamentous fungi.
Birgit Hoff[1]
Jens Kamerewerd[1] Sarah Milbredt[1] Ivo Zadra[2]
Hubert Kürnsteiner[2] Ulrich Kück[3]
1Christian
Doppler Laboratory for “Fungal Biotechnology“, Allgemeine und Molekulare
Botanik, Ruhr-Universität Bochum, Universitätsstraße 150, 44780 Bochum, 2Sandoz
GmbH, 6250 Kundl, Austria, 3Christian Doppler Laboratory for “Fungal
Biotechnology“, Allgemeine und Molekulare Botanik, Ruhr-Universität Bochum,
Universitätsstraße 150, 44780 Bochum, Germany
birgit.hoff@rub.de
The recent discovery of a
velvet
complex containing several regulators of secondary metabolism in the model
fungus
Aspergillus nidulans
raises the question whether similar type complexes direct fungal development in
genera other than
Aspergillus.
Penicillium chrysogenum
is the industrial producer of the beta-lactam antibiotic penicillin, whose
biosynthetic regulation is barely understood. Here we provide a functional
analysis of major homologues of the
velvet
complex in
P. chrysogenum.
Data from northern hybridizations, HPLC quantifications of penicillin titres as
well as detailed microscopic investigations clearly show that all regulators
play not only a major role in penicillin biosynthesis but are also involved in
different and distinct developmental processes. While for example deletion of
the
velvet
homologue PcvelA leads to light-independent conidial formation, dichotomous
branching of hyphae and pellet formation in shaking cultures, a ΔPclaeA strain
shows a severe impairment in conidiophore formation in both the light and dark.
Furthermore, detailed bimolecular fluorescence complementation assays together
with yeast two-hybrid screens led not only to the identification for a
velvet-like
complex in
P. chrysogenum
but also to the detection of new components of this complex. Our results extend
the current picture of regulatory networks controlling both fungal secondary
metabolism and morphogenesis which is significant for the genetic manipulation
of fungal metabolism as part of industrial strain improvement programs.
PR8.13
Astrid R. Mach-Aigner,
Matthias G. Steiger, Robert L. Mach
Gene Technology Group, Inst. of Chemical Engineering, Vienna University of
Technology, Getreidemarkt 9/166/5/2, A-1060 Vienna, Austria
aaigner@mail.zserv.tuwien.ac.at
Second to cellulose, chitin is a most abundant, renewable organic source in
nature with an estimated annual biosynthesis of 109 to 1011
tons. The polymer is composed of ß-(1,4)-linked units of the amino sugar
N-acetyl glucosamine (GlcNAc). For industrial applications this renewable
resource is mainly extracted from crustacean shells. However, thitherto only a
minute amount of this renewable resource is used in industrial and agricultural
applications.
N-acetyl neuraminic acid (NeuNAc), a C9 mono-saccharide, is the most prevalent
exponent of sialic acids. Currently, more than 50 derivatives of sialic acids
are known to exist in nature. NeuNAc is believed to serve as a precursor of all
these derivatives as all biochemical pathways precede via this substance.
In biological systems sialic acids are mostly terminal components of
glycoproteins presented at the respective cell surface. Because of its exposed
position in cellular systems they play an important role in infection cycles of
viral diseases, e.g. influenza viruses A and B. Therefore, sialic acid
derivatives are successfully applied in the therapy of such virus-born diseases.
Preparations on the market are e.g. „Relenza“ (GlaxoSmithKline), functioning as
neuraminidase inhibitor and produced from the precursor NeuNAc.
We will present an alternative strategy of NeuNAc production based on a
genetically engineered
Trichoderma
strain producing and excreting NeuNAc. For the first time, this poses the
introduction of a multi-step enzyme cascade into a filamentous fungus as
heterologous host. This strategy involves the application of
Trichoderma
as a whole-cell catalyst for direct synthesis of NeuNAc from the cheap,
renewable biopolymer chitin.
Development of a novel inducible expression system for the production of
heterologous proteins in
Aspergillus:
successful production of lignolytic enzymes.
Sandra
de Weert1, Nick van Biezen2, Dennis Lamers2,
Jeroen Schouwenberg2, Kees van den Hondel1,2, Arthur Ram1
and Christien Lokman1,2
1 Institute of Biology Leiden,
Leiden University, Sylviusweg 72, 2333 BE Leiden, The Netherlands
2HAN BioCentre, P.O. Box 6960, 6503 GL Nijmegen, the Netherlands
christien.lokman@han.nl
For industrial applications and (medical) scientific research efficient
(heterologous) production of compounds like, enzymes, antibodies, viral
epitopes, chemical compounds and antibiotics is of great importance. For these
purposes
Aspergillus
functions as a suitable production host. Over the years all kinds of
commercially available expression systems have been developed. A well
established expression system is the one based on the protein Glucoamylase
(GlaA). However, in the case of heterologous protein production the efficiency
of these systems is still very depending on the protein to be produced.
Recently, we identified the
inuE
gene, encoding for the exo-inulinase protein in
A.
Characteristics of the system were studied by placing
gfp
behind the
inuE
promoter. This reporter strain showed that the
inuE
gene is highly expressed when grown on inulin and sucrose. No expression was
observed when grown on glucose, fructose and xylose indicating a tight control
on different carbon sources. This tight control can be a benefit if the
heterologous protein to be produced can be a disadvantage for fungal growth.
Finally, different peroxidases and a laccase were successfully produced in
Aspergillus
using this novel inducible expression system.
Matthias Georg Steiger,
Robert L. Mach, Astrid R. Mach-Aigner
msteiger@mail.tuwien.ac.at
N-acetylneuraminic acid (NeuNAc) belongs to the structural class of sialic acids
and is an acidic α-keto sugar with a C9 backbone. Derivatives of NeuNAc are used
as neuraminidase inhibitors to treat viral infections like influenza. Therefore,
the pharmaceutical industry is interested in a cheap source for NeuNac, but its
synthesis is costly (current market price: 3000 to 4000
Instead of N-acetylglucosamine, we use the renewable source chitin as a
substrate. We developed a whole-cell-bio-catalysis process based on the
fungus Trichoderma. Trichoderma is known for its high secretory
capacity for hydrolytic enzymes. This filamentous fungus is able to utilize
polysaccharides like cellulose or chitin which commonly occur in nature. We use
this ability of the fungus to degrade chitin into its monomer
N-acetylglucosamine. From N-acetylglucosamine we designed an intracellular
enzyme-catalyzed pathway for the synthesis of NeuNac. Therefore, we
heterologously expressed two bacterial enzymes, N-acetylglucosamine-2-epimerase
and NeuNAc synthase, in Trichoderma. This is necessary because the
wild-type of Trichoderma itself is not able to produce NeuNac. We will
illustrate the properties of a recombinant Trichoderma strain, which
expresses the two bacterial enzymes, and we will show the ability of this strain
to form NeuNac. This work demonstrates the potential of using Trichoderma
as a whole-cell catalytic system.
Hironobu Ogasawara[1]
Tsutomu Satoh[1] Hiroshi Konno[1] Yoji Hata[2]
Saori Takahashi[3] Katsuya Gomi[4]
1Akita
Konno Co. Ltd., Akita, Japan, 2Research Institute, Gekkeikan Sake Co.
Ltd., Kyoto, Japan, 3Akita Res. Inst. Food and Brewing, Akita, Japan,
4Graduate School of Agricultural Science, Tohoku University, Sendai,
Japan
hironobu@arif.pref.akita.jp
An active DNA transposon Crawler isolated from the genome of industrially
important fungus Aspergillus oryzae transposes under extreme stress
conditions. The DNA sequencing surveys revealed that the Crawler
element is widely distributed among A. oryzae and A. sojae
strains, which are commonly used in Japanese traditional fermentation
manufacturing. In the present study,
we analyzed the relationship between various stress stimuli and inhibition of
cryptic splicing of the Crawler mRNA by qRT-PCR to enhance the frequency
of Crawler-mediated mutagenesis in an A. oryzae industrial strain,
AOK139. Under the optimized stress conditions, in which conidiospores were
treated for 6 hr in 20 mM CuSO4 or at 52℃, various phenotypic mutants
different from the parent strain were isolated. These mutants exhibited white
color in conidiospores, less number of spores formed, shortened aerial hyphae,
thin colony mat and so on. DNA sequencing analysis of a white conidia
mutant revealed that Crawler was newly inserted within a coding region of
wA gene encoding polyketide synthase, which resulted in wA
deficiency. The insertion occurred also at a TA site with duplication according
to the manner of Crawler transposition. These results suggested
that transposon mutagenesis using active Crawler is potentially valuable
to improve characteristics of A. oryzae industrial strains.
Bettina Tudzynski[1]
Philipp Wiemann[1] Karin Kleigrewe[2] Marita Beyer[2]
Hans-Ulrich Humpf[2] Bettina Tudzynski[3]
1Institut
für Botanik / Institut für Lebensmittelchemie, WWU Münster, 2Institut
für Lebensmittelchemie, WWU Münster, 3Institut für Botanik, WWU
Münster
tudzynsb@uni-muenster.de
Fusarium
secondary metabolites are structurally diverse, have a variety of activities,
and their biosynthesis and regulation are generally poorly understood. The
F. fujikuroi
polyketide synthase gene
bik1
was previously shown to be responsible for formation of the mycelial pigment
bikaverin. Here we present the characterization of five genes adjacent to
bik1
as encoding a putative FAD-dependent monooxygenase (bik2),
an
O‑methyltransferase
(bik3),
an NmrA‑like protein (bik4),
a Zn(II)2Cys6 transcription factor (bik5),
and an MFS transporter (bik6).
Deletion of each gene resulted in total loss or significant reduction of
bikaverin synthesis. Over-expression of genes involved in biosynthesis enabled
us to identify at least one intermediate of the bikaverin pathway. Expression
studies revealed that all
bik
genes are repressed by high amounts of nitrogen in an AreA‑independent manner
and are subject to a time- and pH‑dependent regulation. Deletion of the pH
regulatory gene
pacC
resulted in partial de-repression while complementation with a dominant active
allele resulted in repression of
bik
genes at acidic ambient pH. Furthermore, the global regulator Velvet represses
bik
gene expression and product formation most likely by interconnection with the
nitrogen regulation machinery. Thus, bikaverin synthesis is regulated by a
complex regulatory network. Understanding how different factors influence the
synthesis of this model secondary metabolite will aid understanding secondary
metabolism in general.
Sabine Albermann,Bettina
Tudzynski
sabine.albermann@uni-muenster.de
Each year about ten tons of gibberellins (GAs) are consumed by the agricultural
industry as plant growth regulators. GAs function as phytohormones in higher
plants and application of GAs induces flower bud formation and shoot elongation
as well as an increased fruit size and yield of crops such as seedless grapes
and corn.
The rice pathogen
Fusarium fujikuroi
is known to produce high amounts of this secundary metabolite and constitutes a
capable species for GA production by microorganisms. Therefore, we developed
different strategies for strain improvement. By providing a higher amount of the
GA precursor molecule geranyl-geranyl-pyrophosphate (GGPP) the yield of GAs
could be enhanced and costs of the production would be reduced. For that reason
expression of the key enzymes of the mevalonate pathway,
3-hydroxy-3-methyl-glutaryl-CoA-reductase (HMGR) and
farnesyl-pyrophosphate-synthase (FPPS), should be increased.
In former studies truncation and overexpression of HMGR in
Saccharomyces
cerevisiae
led to a higher accumulation of terpenoids. This is referred to the deletion of
the transmembrane domains of HMGR where the enzyme is regulated by feedback
inhibition.
Additionally, the ergosterol biosynthesis pathway, a branch of the mevalonate
pathway, shall be downregulated as it competes with the GGPP synthase for FPP.
Furthermore it would avoid formation of abnormal structures in the fungal
membrane.
Finally, localization studies with GFP fusion proteins of HMGR and FPPS shall
clearify subcellular organization of the mevalonate pathway and the role of the
two key enzymes in
F. fujikuroi.
Ronald de Vries[3]
Isabelle Benoit[1] Pedro Coutinho[2] Hala Al-Bushari[1]
Evy Battaglia[1] Birgit Gruben[1] Blanca Trejo-Aguilar[1]
Guillermo Aguilar-Osorio[1] Ad Wiebenga[3] Bernard
Henrissat[2]
1Microbiology,
Utrecht University, 2Architecture et Fonction des Macromolecules
Biologiques, UMR6098, CNRS, Univ. Aix-Marseille I & II 3CBS-KNAW
Funngal Biodiversity Centre
r.devries@cbs.knaw.nl
Plant polysaccharides are among the most prominent carbon sources for fungi,
which degrade these substrates through the production of diverse sets of
extracellular enzymes. This topic has been best studied in the Aspergilli, in
particular in
A.
CAZy-annotation of the genomes revealed significant differences in the sets of
hydrolytic enzymes and growth profiling of these fungi demonstrated strong
correlations between genome content and ability to degrade specific
polysaccharides. In addition, analysis of the secreted enzymes demonstrated
further differences that are likely caused by differences in transcriptional or
post-transcriptional regulation. Highlights from these results will be
presented.
These data will help with the further development of improved enzyme cocktails
as they enable a link between the composition of the enzyme set and the
efficiency with which different polysaccharides are degraded.
PR8.20
Habib Driouch,
Becky Sommer and Christoph Wittmann
h.driouch@tu-bs.de
Among filamentous fungi,
Aspergillus
In the present work, supplementation with microparticles was used as
novel approach to control the morphological development of
A.
The use of microparticles, allowing targeted engineering of cellular
morphology opens novel possibilities for future design and optimization of
recombinant enzyme production in
A.
[1] Driouch H., Sommer B., Wittmann C (2009) Morphology engineering of
Aspergillus
Becky Sommer,
René Stellmacher, Rainer Krull, D. C. Hempel
TU Braunschweig, Institute of
biochemical engineering
b.sommer@tu-bs.de
In many biotechnological processes filamentous fungi like
Aspergilli
are used for the production of organic acids, enzymes and antibiotics.
Furthermore, there is an increasing application of fungi as catalysts in
biotransformations. Therefore spores are used in inoculum development or
directly as starting culture in submerged cultivation processes. While the
influence of process conditions during cultivation is well-known, the influence
of the spore quality in seeding cultures has not been investigated in depth.
However the spore viability in seeding culture is an important criterion for
product quantity.
The contribution displays the investigation of the transferability of
fluoresence based rapid screening assays, developed for bacterial cells, to
study spore quality. The results of this method show the applicability to
determine the proportion of viable and defect spores in seeding cultures of
filamentous fungi. Additionally, good correlations to germination
characteristics in submerged cultivation are demonstrated and allow fast,
reliable and quantitative distinction between viable and dead spores.
Furthermore a biochemical method, based on enzyme activity, has been verified to
characterize the spore viability in seeding cultures. The assay allows the
characterization of the growth stadium of the filamentous fungi during the
sporulation process. Furthermore the enzyme based assay enables the
determination of biomass activity in the early phase of submerged cultivation
and reveals first information about the feasibility of the cultivation.
In conclusion the presented investigations display the possibility of assessing
the ratio of viable to defect spores in seeding cultures, whereby a routine
method prior to large scale cultivations could be applied.
An Li,
Nicole van Luijk, Martien Caspers, Marloes ter Beek, Jan Jore, Mariet van der
Werf, Peter J. Punt
TNO
Quality of life, Zeist, The
an.li@tno.nl
The black filamentous fungi
Aspergillus niger
has a long tradition of safe use in the production of enzymes and organic acids,
and is widely used in biotechnology as host for the production of food
ingredients, pharmaceuticals and industrial enzymes. Besides,
Aspergillus
Robbert Damveld,
Brenda Vonk, Cees Sagt, Hildegard Menke, Thibaut Wenzel
robbert.damveld@dsm.com
Modern biotechnology has generated an impressive set of molecular tools: for
instance the ability to generate large sets of error prone mutant libraries or
cDNA libraries. When these libraries are expressed in a host (e.g.
Aspergillus
Andreas Posch,
Christoph Herwig
Institute of Chemical Engineering, Division of Biochemical Engineering, Vienna
University of Technology, Getreidemarkt 9/166, A-1060, Vienna, Austria.
Andreas.Posch@tuwien.ac.at
Meeting QbD (Quality by Design) and PAT (Process Analytical Technology)
guidelines launched by the U.S. Food and Drug Administration, biopharmaceutical
manufacturers need to move away from traditional empirically based process
development towards optimized and science-based process design and control.
Within recent years, numerous PAT compliant tools allowing effective,
non-invasive in-situ monitoring of microbial production processes have been
successfully introduced. Moreover, several approaches for online process
characterization in bacterial and yeast systems have been suggested. Promising
procedures include applying metabolic modeling and online evaluation of
stoichiometric balances enabling to distinguish between different metabolic
states of cellular production systems.
For cultivation processes of filamentous fungi however, implementation of such
methods has been hampered by increased complexity of filamentous fungal systems
and additional factors affecting process performance, e.g. growth morphology and
extracellular proteolytic activity.
The aim of this work is to establish a methodology allowing generically
characterizing fungal filamentous systems by accurately quantifying fungal
response behavior to physiological stress under transient culturing conditions,
linking metabolic regulations to morphological changes. Dynamic experiments are
designed using DoE (Design of experiment) strategies, and carried out in fully
instrumented bioreactors coupled to a process management system performing
online data evaluation and reconciliation.
Combining process technology, morphological, and physiological analysis, allows
accurately quantifying filamentous fungal process behavior. Our approach will
thus facilitate process development and allow understanding biological
variability and, in turn, control those complex processes.
Terhi Puranen[1]
Leena Valtakari[2] Marika Alapuranen[1] Jarno Kallio[1]
Jari Vehmaanperä[1]
1Roal
Oy,
terhi.puranen@roal.fi
Cellulases are traditionally used in textile, detergent, pulp and paper, feed
and food industries, and are currently also extensively studied for cellulosic
biomass conversion to ethanol. Endoglucanases have been widely used in textile
industry for biostoning and biofinishing of cellulosic fibers. There has been
trend in the market, especially in
Pauline Krijgsheld[1]
Jeffrey H. Ringrose[2] A. F. Maarten Altelaar[2] Albert J.
R. Heck[2] Han A. B. Wösten[1]
1Microbiology
and Kluyver Centre for Genomics of Industrial Fermantation, Utrecht University,
Padualaan 8, 3584 CH Utrecht, 2Biomolecular Massspectrometry and
Kluyver Centre for Genomics of Industrial Fermantation, Utrecht University,
Padualaan 8, 3584 CH Utrecht
p.krijgsheld@uu.nl
In nature fungi degrade organic matter such as wood. To this end, they secrete
enzymes that convert the polymeric substrates into small molecules that can be
taken up to serve as nutrients. Hyphae at the periphery of the colony are
exposed to unexplored organic material, whereas the center of the colony
experiences a (partly) utilized substrate. This suggests that the enzymes that
are secreted by distinct zones in the colony are different. This hypothesis was
tested using the industrial important fungus
Aspergillus
DAVID VELA,
Antonio de Vicente, Alejandro Pérez
dvela@uma.es
Powdery mildew fungi (Erysiphales)
are probably the largest group of plant pathogens that remain uncharacterized
from the genetic and molecular points of view. Their lifestyle as obligate
biotrophic parasites and consequent inability to grow on culture media has
significantly hampered research. Appropriate disease management requires a good
understanding of the biology of the responsible pathogen. Regarding powdery
mildews research, a technologically important unsolved issue is their genetic
manipulation. Since the first reports of DNA-mediated transformation in model
filamentous fungi such as
Neurospora crassa
and
Emericella nidulans,
many commercially and agriculturally important fungal species have been
successfully transformed. However, the obligate biotrophs have, hitherto, proved
recalcitrant to this process.Most common methods such as protoplast fusion or
Agrobacterium-mediated
transformation are not suitable for such organisms like the first, or show
important methodological limitations like the second. In fact, to date particle
bombardment has been reported as the only valid method to transform this type of
parasites, although this technique has shown certain lack of reproducibility.
We report here on progress in developing a reliable and stable transformation
protocol for
Podosphaera fusca,
the causal agent of cucurbit powdery mildew, by the highly versatile
electrotransformation method. Two selectable markers have been used:the
hygromycin B resistance gene (hph)
and the enhanced green fluorescent protein gene (egfp).
These markers were tested under the control of different promoter and terminator
sequences such as those of the
gpd
and
trpC
genes of
E. nidulans,
the
isl
gene of
N. crassa,
and the promoters of the b-tubulin and CYP51 genes of
P. fusca.
Transformants were obtained with most of the constructs used. Under selective
conditions the hygromycin B resistant transformants grew very slow. Similarly,
weak GFP signals were associated to fluorescent hyphae of transformants. In all
cases we detected sequences of marker genes by PCR and found copies of the
transforming plasmids integrated in the fungal genome. To our knowledge this is
the first case of genetic transformation of a powdery mildew fungus by
electroporation of conidia. An update of the results obtained so far will be
presented.
Barbara Oportus,
Nicole Lorenz, Anna Koppe, Sabine Giesbert, Paul Tudzynski
Institut für Botanik, Westfälische Wilhelms-Universitaet, Schlossgarten 3,
D-48149 Muenster,
oportusm@uni-muenster.de
Ergot alkaloids (EA) are secondary metabolites synthesized by the
phytopathogenic ascomycete
Claviceps purpurea.
The altogether 14 genes encoding the specific enzymes for the biosynthesis of EA
are clustered. To date the molecular mechanisms of cluster regulation in
C.
purpurea
are unknown. No transcription factor gene has been found within the cluster
region involved in the synthesis of EA. It is only known to date that the EA in
C.
purpurea
wild-type are produced during the ripening of the sclerotium and not in axenic
cultures. Mutant strains producing alkaloids in submersed cultures require under
specific conditions: (a) tryptophan as inducer and precursor, (b) a high osmotic
value, (c) a low phosphate level. The alkaloid biosynthesis was speculated to be
regulated by changes in the chromatin organization, a hypothesis checked by the
cultivation of
C. purpurea
in the presence of either inhibitors of histone deacetylases (HDACis) or histone
acetyltransferases (HATis). The production strain P1 of
C. purpurea
was cultivated under both alkaloid producing (T25N medium) and non-producing
(BII medium) conditions. Unexpectedly, the alkaloid biosynthesis is repressed
instead of induced by the addition of HDACis (euchromatin formation) and vice
versa, indicating a complex regulation system (1). Another global regulatory
system of secondary metabolism and development was discovered in
Aspergillus nidulans, velvet
(2). We are investigating if in
C.
purpurea
the biosynthesis of ergot alkaloids could be regulated through velvet. We have
identified and sequenced a velvet homologue in
C.
purpurea
(cpvel1)
and started a functional analysis.
(1)
Lorenz et al. (2009)
Phytochemistry.
70:1822-1832. (2) Kim et al. (2002)
Fungal Genet Biol.
37:72-80.
Jaewoo Yoon,
Tuerxun Aishan, Jun-ichi Maruyama, Katsuhiko Kitamoto
Department of Biotechnology, The
ayoon@mail.ecc.u-tokyo.ac.jp
Filamentous fungi have received attention as a host for heterologous protein
production because of their high secretion capability. However, a bottleneck in
post-transcriptional processing in the secretory pathway limits protein
production yields. The vacuolar protein sorting gene
VPS10
encodes a sorting receptor for the recognition and delivery of several yeast
vacuolar proteins. Although it can also target several recombinant and aberrant
proteins for the vacuolar degradation, there is limited knowledge of the effect
of its disruption on heterologous protein production. In this study, cDNA
encoding AoVps10 from the filamentous fungus
Aspergillus
oryzae
was isolated. Microscopic observation of the transformant expressing AoVps10
fused with enhanced green fluorescent protein showed that the fusion protein
localized at the Golgi and prevacuolar compartments. Moreover, disruption of the
Aovps10
gene resulted in missorting and secretion of vacuolar carboxypeptidase AoCpyA
into the medium, indicating that AoVps10 is required for sorting of AoCpyA to
vacuoles in
A. oryzae.
To investigate the extracellular production level of heterologous protein, we
constructed an ΔAovps10
mutant expressing bovine chymosin (CHY). Interestingly, ΔAovps10
increased the maximum extracellular production by three fold. Western blot
analysis of extracellular CHY also demonstrated an improvement in productivity.
These results suggest that the AoVps10 plays a role in the regulation of
heterologous protein secretion in
A. oryzae,
and may be involved in vacuolar protein degradation through the Golgi apparatus.
This is the first report demonstrating that disruption of a vacuolar protein
sorting gene in filamentous fungi leads to enhanced production levels of
heterologous protein.
Sorensen Annette,
Peter S. Lubeck, Mette Lubeck, Birgitte K Ahring
Section for Sustainable Biotechnology, Copenhagen Institute of Technology,
aso@bio.aau.dk
Through a broad cellulolytic activity screening of fungi isolated from various
locations, a very promising fungus, identified as a black
Aspergillus,
was selected for further studies. An enzyme extract of this fungus was obtained
through solid state fermentation and tested to profile its beta-glucosidase
activity. Four beta-glucosidase genes from this fungus have been cloned through
pcr with degenerate primers designed from conserved motif regions of known
beta-glucosidase genes from
Aspergilli
followed by genome walking strategies. The goal is to have these genes expressed
in
Trichoderma reesei
QM6a ΔpyrG. An expression system was constructed with the constitutive ribosomal
promoter RP27 for expression of the his-tagged beta-glucosidase genes and with
the pyrG gene to complement the mutation. Analysis of the different
transformants is currently on-going and subsequently the different
beta-glucosidase proteins will be purified for detailed analysis of specific
activity, Km, sugar tolerance, thermostability as well as their ability to break
down shorter chained oligosaccharides.
Kristiina Hilden[4]
Taina Lundell[1] Miia Mäkelä[1] Johanna Rytioja[1]
Paula Nousiainen[2] Jussi Sipilä[2] Alexey Chernykh[3]
Ludmila Golovleva[3] David Archer[4] Annele Hatakka[1]
1Department
of Applied Chemistry and Microbiology, University of Helsinki, Finland
2Department of Chemistry, University of Helsinki, Finland
3G.K. Skryabin Institute of Biochemistry and Physiology of
Microorganisms, Puschino, Russia
4School of Biology, University of Nottingham, UK
kristiina.hilden@nottingham.ac.uk
The white rot, softwood biopulping basidiomycetous fungus
Physisporinus rivulosus
isolate T241i produces two laccase isozymes with novel catalytic properties such
as thermotolerance and atypically low pH optima (pH 3.0-3.5) for the
methoxylated phenolic laccase substrate compounds syringaldazine,
2,6-dimethoxyphenol and guaiacol (1). The fungus was previously distinguished in
a large screening study as the most selective and efficient lignin decomposer of
spruce (Picea
abies)
wood (2, 3), and is shown to produce several manganese peroxidase and laccase
isozymes as well as oxalic acid when growing on spruce wood chips (4). Recently,
we have cloned and characterised at molecular level two
P. rivulosus
laccase genes. The translated mature Priv-lac1 and Priv-lac2 enzymes demonstrate
structural similarity with
Trametes versicolor
and
Lentinus tigrinus
laccases, respectively, as deduced by protein 3-D homology modelling. The
Priv-lac1
and
Priv-lac2
coding sequences are interrupted by 11 and 12 introns and they host the four
conserved His-containing Cu-binding regions typical for laccases. The protein
architectures are viewed at the modelled substrate-binding pocket to elucidate
the atypical pH optima for the phenolic compounds as electron donors. When
P. rivulosus
T241i is cultivated in spruce sawdust-charcoal containing liquid medium, higher
levels of laccase activity is produced. The activity is separated within over
five isoforms, and purified
P. rivulosus
laccases display catalytic efficiency for oxidation of the higher-redox
potential substrates Reactive Black 5 and veratryl alcohol when the reactions
are supplied with natural-type of oxidation-mediator compounds. These properties
well support the
P. rivulosus
laccases to be applied as robust biocatalysts for e.g. bleaching of dye
compounds.
(1) Hildén K., Hakala T.K., Maijala P., Lundell T.K., Hatakka A. (2007) Appl
Microbiol Biotechnol 77:301-309
(2) Hakala T., Maijala P., Konn J., Hatakka A. (2004) Enzyme Microb Technol
34:255-263
(3) Hatakka A., Maijala P., Hakala T.K., Hauhio L., Ellmén J. (2003) Int pat
appl WO03/080812
(4) Hakala T., Lundell T., Galkin S., Maijala P., Kalkkinen N., Hatakka A.
(2005) Enzyme Microb Technol 36:461-468
Ilse de Lange,
Jan-Metske van der Laan, Richard Kerkman, Wim Bijleveld, Mark Stor
DSM Biotechnology Center , P.O. Box 1, 2600 MA Delft, The Netherlands,
www.dsm.com
ilse.lange-de@dsm.com
The filamentous fungus
Aspergillus
Graham Whyteside,
Rohana Mat Nor, Marcos Alcocer, David Archer
graham.whyteside@nottingham.ac.uk
S. cerevisiae
and
P. pastoris
are hosts for the production of heterologous proteins. The secreted yield of a
heterologous protein varies according to the protein being expressed and we aim
to provide predictability based on the stability of protein folded states.
Evidence was gained from the secretion of human lysozyme variants that have
single amino acid changes. A direct relationship between decreasing protein
stability and secreted yield was demonstrated in
P. pastoris
(Kumita et al. 2006 FEBS J 273, 711-720) and in
S. cerevisiae
(unpublished). The reasons underlying the impact of stability on secreted yields
were investigated and the unfolded protein response (UPR) was the focus in terms
of its kinetics of induction and its severity. We showed that genes encoding
both BiP (chaperone) and PDI (foldase) were transcriptionally up-regulated more
quickly and to higher levels in response to secretion of less-stable lysozymes.
The transcript level of
HAC1
mRNA is not altered substantially in
S. cerevisiae
by ER stress but the transcript level of
HAC1
from
P. pastoris
was enhanced markedly and increasingly so with the more unstable variant
lysozymes. We conclude that the folded state of a protein is detected and
responded to in the ER lumen by the quality control system and the response is a
measured one and proportionate to the folded state stability of a secreted
protein. This analysis is being extended to other proteins, e.g. antibody
fragments, and the differences in the UPR between
S. cerevisiae
and
P. pastoris
are being explored further.
Junichiro Marui[1] Sumiko Ohashi[1] Marie Nishimura[2]
Hideaki Koike[1] Masayuki Machida[1]
1National
Institute of Advanced Industrial Science and Technology (AIST), 2Natl.
Inst. Agrobiol. Sci.
hi-koike@aist.go.jp
Aspergillus oryzae
is one of the most important organisms in Japanese fermented food industry.
Although it hardly produces secondary metabolites, related organisms are
producers of diverse metabolites. A major objective of our project is to develop
a system using
A. oryzae
to generate diverse metabolites. To reveal regulation of metabolic pathways in
A. oryzae
under various conditions, gene expression profiles under variety of conditions
and change depending on time were analyzed by DNA micro array and so on. The
factors affecting expression level of metabolic genes are being studied. To
develop the system, novel vectors and host strains of
A. oryzae
have been constructed. We have replaced promoter regions of some metabolic genes
and successfully altered the level of some metabolite productions. Although it
is well known that
A. oryzae
does not produce toxic metabolites, less attention has been paid to its
non-toxic secondary metabolites. Genomic analysis revealed that
A. oryzae
possessed the orthologous gene cluster for penicillin production. The penicillin
production was positively regulated by a global gene regulator required for
transcriptional expression of the penicillin biosynthetic genes. Overexpression
of the biosynthetic genes by a strong promoter yielded a greater than 100-fold
increase in penicillin production. Transcriptional repression of a wide range of
secondary metabolism genes in
A. oryzae
is a valuable characteristic for the production of a particular secondary
metabolite with higher purity and safety. It appears that genetically engineered
A. oryzae
should be extremely useful as a cell factory for industrial production of
beneficial secondary metabolites.
Ken-Ichi Kusumoto,
Satoshi Suzuki
National Food Research Institute
kusumoto@affrc.go.jp
Overproduction and characterization of prolyl aminopeptidase from
Aspergillus oryzae
Ken-Ichi Kusumoto1, Mayumi Matsushita-Morita1,
Ikuyo Furukawa1, Youhei Yamagata2, Yoshinao Koide3,
Hiroki Ishida4, Michio Takeuchi2, Yutaka Kashiwagi1,
Satoshi Suzuki1 1National Food Research Institute,
Tsukuba, Ibaraki, Japan; 2Department of Agriscience and Bioscience,
Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan; 3Gifu
R & D Center, Amano Enzyme Inc., Kagamihara, Gifu, Japan; 4Research
Institute, Gekkeikan Sake Company Ltd., Fushimi-ku, Kyoto, Japan
Introduction:
Prolyl aminopeptidase (PAP) degrades only amino-terminal proline from peptides.
The food-grade fungus
Aspergillus oryzae
produces this enzyme only in small amounts. Therefore, we present here an
efficient production of recombinant PAP with an overexpression system of
A. oryzae
and characterization of its biochemical properties.
Methods and Results:
The gene encoding PAP was overexpressed as a His-tag fusion protein under a
amyB
promoter with a limited expressing condition in
A. oryzae.
The PAP activity in the mycelia grown in rich media containing glucose
(repressing condition) was twice that in starch (inducing condition). The
enzyme prepared as cell-free extract was partially purified through two-step
column chromatography.
A. oryzae
PAP was purified 1800-folds. The PAP was estimated to be a homo hexameric
protein and exhibited salt tolerance against NaCl of up to 4 mol l-1.
Discussion:
Overproduction of PAP under promoter inducing conditions led to an increase of
inactive PAP, possibly because of irregular folding. PAP with a high
specific activity and salt tolerance may be used effectively in the
manufacturing processes of fermented foods.
ANA MARÍA RINCON ROMERO[3]
Moreno-Mateos[1] Espeso, E.A.[2] Codón, A.C.[3]
Benítez, T.[3]
1Departamento
de Señalización Celular, Centro Andaluz de Biología Molecular y Medicina
Regenerativa (CABIMER), Av. Américo Vespucio, s/n, 41092 Sevilla, Spain
2Centro de Investigaciones Biológicas CSIC. Ramiro de Maeztu, 9,
28040 Madrid, Spain
3Universidad de Sevilla, Departamento de Genética, Facultad de
Biología, Avda.
Reina Mercedes, s/n, 41012
amrincon@us.es
Trichoderma
spp.
is a filamentous fungus used in biocontrol. Many attemps were carried out to
improve its antagonistic ability. However,
Trichoderma
adaptability to different soils might be affected under environmental stress
conditions, and finding resistance to adverse conditions would be a desirable
feature for the use of
Trichoderma
in agriculture. Recently, implication of Pac1, the homologue of the pH
transcriptional regulator PacC, in
Trichoderma
harzianum
parasitic relationships has been demonstrated. As its homologous PacC proteins
in other filamentous fungi, Pac1 is expected to be directly involved in fungal
stress response. Two mutants of
T. harzianum
CECT2413 have been obtained in Pac1: P2.32 expresses a constitutively active
Pac1, and R13 contains a null allele by gene silencing. Strain P2.32 shows
better growth than wild type in NaCl-, KCl- or sorbitol-containing media, while
strain R13 shows higher sensitivity in the same conditions. Phenotypes of P2.32
and R13 mutants in the presence of Lithium are opposed but contrary to those
described for PacC/Rim mutants in yeasts and other filamentous fungi.
Transcriptional regulation of
pac1
is altered under salt/osmotic stress. Pac1 mRNA levels are higher and the
protein produced able to bind DNA. However, Pac1 DNA binding ability did not
correlate to those levels of
pac1
expression in non stressing media. These results indicated that Pac1 is a key
factor for saline and osmotic stress resistance and might be subjected to a
complex combination of traductional and posttranslational regulation in
T. harzianum.
Satoshi Suzuki[1]
Sawaki Tada[1] Mari Fukuoka[1] Yoshiki Tsukakoshi[1]
Mayumi Matsushita-Morita[1] Yutaka Kashiwagi[1] Masanori
Sugiyama[2] Ken-Ichi Kusumoto[1]
1NFRI,
2Hiroshima Univ.
satosuz@affrc.go.jp
Introduction: Since
Aspergillus oryzae
produces large quantity of enzymes, it is significant to establish a host-vector
system to use this microorganism as a host for heterologous protein expression.
In various filamentous fungi, the genes, which confer resistance to hygromycin
B, aureobasidin, and G418, have been used as genetic markers for the gene
manipulation. However,
A. oryzae
is resistant to these antibiotics. We have recently developed a transformation
system for
A. oryzae
RIB40 by using bleomycin-resistance gene as a selective marker. In the present
study, we generated
ligD
knock out strain, by using bleomycin resistance selection. And we generated
exogenous or endogenous enzyme over-expresser by transformation with expression
vector carrying bleomycin resistant gene. Methods: The
ligD
disruption cassette consisted of bleomycin-resistance expression cassette and
2kb of 5’ and 3’ franking sequence of
ligD,
the
β-glucuronidase
encoding gene
uidA
from
E. coli
over-expression vector and a polygalacturonase encoding gene
pgaB
from
A. oryzae
over-expression vector were introduced into the protoplasts from
A. oryzae
RIB40 by the polyethylene glycol method, respectively. Results: The
disruption of the
ligD
locus in genomic DNA of transformants was confirmed by colony PCR and the
Southern blot analysis. Activity of each enzyme was detected from cell free
extract or culture filtrate of the each transformant selected by bleomycin
resistance. Discussion: In the present study, we successfully generated a gene
disruptant and over-expresser of two kinds of enzymes by using bleomycin
resistance transformant selection system of
A. oryzae.
Matias Pasquali,
Frederic Giraud, Emmanuelle Cocco, Lucien Hoffmann, Torsten Bohn
CRP Gabriel Lippmann
pasquali@lippmann.lu
Fusariotoxins present in wheat and barley are a major cause of economic loss,
especially for alimentary industries and breweries. Many predictive models based
on climatic and agronomic practices have been implemented in order to estimate
toxin content in grains. Here we show that genetic chemotyping of the Fusarium
population within a field may improve significantly the prediction of toxin
content on wheat. We analysed nivalenol (NIV, a trichothecene with higher
toxicity when compared to deoxynivalenol) content in wheat by LC/MS-MS analysis
from 17 different wheat fields sampled for two consecutive years. We determined
species and chemotype by using multiplex PCRs on a total of 1084 isolates. A
significant correlation (R= 0.75, p<0.00001) was found between the quantity of
Fusarium culmorum
isolates per 100 seeds with the NIV chemotype and the amount of NIV in grains. A
threshold, based on the amount of NIV isolates per 100 seeds for detecting NIV
in grains has been tested successfully on 9 independent additional fields
(P<0.001). Moreover we showed that the development of a real time PCR method for
the detection of
F. culmorum
with NIV chemotype from grains further improved the accuracy and speed of the
test. Here we demonstrated the reliability of Fusarium genetic chemotyping for
toxin prediction in the field. Furthermore, we verified the importance of
targeting genes linked to function (in this case trichothecene synthetic genes)
in PCR based screening for mycological application.
Jeffrey Shasky,
Wendy Yoder, Alfredo Lopez, Ron Kloberdantz, Michael McCulloch, Brian
Gore-Clancy, Dawn Lysaght
Novozymes Inc
jfys@novozymes.com
The industrially important QuornTM fungus
Fusarium venenatum
has previously been used as a host for heterologous protein expression.
Since the wild-type organimsm can grow on the toxic analogue 5-fluoroorotic acid
(FOA) despite having an intact
pyrG
gene, an alternative couter-selectable marker was needed for sequential gene
deletions to improve the host. A system was developed using two separate
gene cassettes, each controlled by a different heterologous promoter and
terminator, flanked by direct repeats. The
E. coli
hygromycin phosphotransferase gene
(hph),
conferring resistance to the herbicide hygromycin, was used for positive
selection and deletion of the gene of interest. The
herpes simplex virus
thymidine kinase (tk)
gene was used for counterselection on the uridine analogue 5-flourodeoxyuridine
(FdU), allowing selection for spontaneous excision of the region containing both
markers. Deletion constructs incorporating the two markers listed above,
flanking direct repeats, as well as flanking sequence for the gene of interest
were generated for two mycotoxin genes (trichodiene synthase gene (tri5)
and enniatin synthase gene (dps1)),
the orotidine- 5- phosphate decarboxylase gene (pyrG),
an alpha amylase gene (amyA)
and an alkaline protease (alpA)
gene.
Using deletion constructs above genes were sequentially deleted in
Fusarium venenatum
strain A3/5 in the following order:
tri5, pyrG, amyA, alpA, dps1.
The uridine auxotrophy caused by the
pyrG
deletion could be rescued by transformation with a
pyrG
fragment, allowing its use as a marker for heterologous protein expression. The
resulting strain also showed desired host characteristics as a result of the
gene deletions.
Marta Kolasa,
Birgitte K. Ahring, Peter S. Lübeck, Mette Lübeck
mk@bio.aau.dk
Lignocellulosic materials form a huge part of the plant biomass from
agricultural and forestry wastes. They consist of three major components:
cellulose, hemicellulose and lignin. Cellulose, the main constituent of plant
cell wall, is a polymer of D-glucopyranose units linked by β-1,4-glucosidic
bonds. Cellulose can be degraded to simple sugar components by means of
enzymatic hydrolysis. However, due to its complex, crystalline structure it is
difficult to break it down and the cooperative action of a variety of
cellulolytic enzymes is necessary. Fungi are known to have potential in
production of a variety of cellulolytic enzymes. The aim of this work is to
discover new thermostable and robust cellulolytic enzymes for improved enzymatic
hydrolysis of biomass. For this purpose two screening methods are applied in
different fungal strains with high cellulolytic activities: an expression-based
method using suppression subtractive hybridization and a targeted genomic
screening approach using degenerate PCR. Suppression subtractive hybridization
facilitates identification of genes encoding cellulolytic enzymes that are
expressed when cultivating a fungal strain in medium with cellulose as the
carbon source. By means of degenerate PCR, specific genes, homologous to the
genes of previously classified glycoside hydrolases from CAZY database, are
searched for in selected strains of
Aspergillus sp.,
Trichoderma sp.
and
Penicillium sp.
Both methods are anticipated to facilitate identification of target genes which
subsequently will be cloned and expressed in a relevant fungal host for further
characterization of the expressed enzymes. The goal is to introduce new enzymes
to industrial processes.
Thomas Haarmann,
Ute Knirsch, Jasmin Hack
AB Enzymes GmbH
thomas.haarmann@abenzymes.com
The aim of this project is to establish the QPix2 colony picker (Genetix) to
pick colonies from different filamentous fungi (e.g.
Aspergillus,
Trichoderma)
in a fast and efficient way. This is important because classical mutagenesis and
screening projects require huge amounts of mutants to be picked and analysed
which is highly time consuming if done manually. One additional benefit is that
all the work is carried out in an enclosed chamber which is easily kept sterile
by exposure to UV-light.
Since the robot was originally designed for picking bacteria or yeast we had to
overcome different obstacles regarding e.g. the composition of the culture
media, the design of the picking-pins or the speed of movement of the picking
head. Other critical parameters are the density of plated spores, age of
cultures and size of the colonies. Several experiments will be shown in which we
try to define the optimal values for each of these parameters.
Additionally, the colony picker will be used to carry out simple plate assays to
measure e.g. enzymatic activities of generated mutants semi-automatically.
All of these improvements will substantially enhance the speed of classical
strain development by mutagenesis and screening approaches.
PR8.42
Violeta Díaz-Sánchez, Javier Avalos, M. Carmen Limón
Universidad de Sevilla
carmenlimon@us.es
Fusarium fujikuroi
is a rice pathogen which produces different secondary metabolites. Some of them
are mycotoxins, i.e. bikaverins and fusarins, and others have biotechnological
applications, i.e., gibberellins and carotenoids. Fusarins are near UV-absorbing
polyketides that, accumulated at high concentration, provide a yellowish
pigmentation to
F. fujikuroi
mycelia. Little information is available on the regulation of fusarin
biosynthesis by this fungus. As formerly described, the synthesis was enhanced
at high temperature (30°C
vs
22°C). However, in contrast to the nitrogen repression described for the
synthesis of other metabolites, such as gibberellins and bikaverins, we found
that fusarin production is repressed by nitrogen starvation. As other fungal
polyketides, the synthesis of fusarins starts through the activity of a
multidomain enzyme, termed type I polyketide synthase (PKS). The PKS gene
responsible for fusarin biosynthesis was formerly indentified in
Fusarium graminearum
and other
Fusarium
species. High sequence conservation between
Fusarium
genomes has allowed us to clone the orthologous gene of
F. fujikuroi,
which we named
fusA.
Deletion mutants for this gene showed a total absence of fusarin production,
confirming the role of
PKS
FusA in the biosynthesis of this metabolite. Real-time RT-PCR analyses of
fusA
mRNA were achieved using a 3’ gene segment, absent in the other known
Fusarium
PKS genes. The results showed that the two regulatory conditions tested,
temperature and nitrogen availability, act at least partially on
fusA
gene expression.
Taija Leinonen[1]
Kari Juntunen[1] Leena Valtakari[2] Tapani Suortti[3]
Sanni Leinonen[3] Kristiina Kruus[3] Marja Paloheimo[1]
1Roal
Oy, 2AB Enzymes Oy, 3VTT
taija.leinonen@roal.fi
The carbohydrate binding modules (CBMs) are non-catalytic domains of
carbohydrate-acting enzymes, and are known to promote the association of enzyme
with its insoluble substrates. We studied the effects of CBM attachment on the
properties of
Trichoderma reesei
α-amylase (ID 105956 in http://genome.jgi-psf.org/Trire2/Trire2.home.html). This
amylase does not, in its native form, contain a CBM. A sequence encoding a
family 20 CBM from another fungal α-amylase gene was fused 3’ to the
T. reesei
amylase gene. The
T. reesei
amylase gene and amylase-CBM fusion construction were overexpressed in
T. reesei
using the strong
cbh1
(cel7A)
promoter. The two enzymes were purified from the culture supernatants, and their
physicochemical properties and performance in stain removal were determined. The
amylase-CBM fusion protein was stable in
T. reesei
culture supernatant. Its temperature and pH profiles were similar to those of
the native
T. reesei
amylase. However, its specific activity on Azurine-crosslinked amylase
(AZCL-Amylose) was over three times higher when compared to the native amylase.
Addition of the CBM20 was shown to have a positive effect on starch hydrolysis
rate but the hydrolysis product profiles were similar with the two enzymes. The
hydrolysis products consisted mainly of glucose and maltose during the whole
time-course of the reaction, and only low amounts of maltotriose, -tetraose and
–pentaose were detected. Insertion of the CBM had no positive effect on stain
removal performance on standard starch stain, possibly due to attachment of the
amylase-CBM form to the stain and/or on fabric surface.
Matthias Gehder,
Jan-Hendrik Sachs, Dietmar Hempel, Rainer Krull
TU Braunschweig
M.Gehder@tu-bs.de
Filamentous fungi such as Aspergilli are widely used in numerous
biotechnological applications. Their use ranges from the large scale production
of citric acid to the expression of highly complex catalytic and therapeutic
proteins. Furthermore, they are used in biotransformations both, inside and
outside the bioreactor. The widespread industrial usage of filamentous fungi has
consequently triggered tremendous interest in the research and optimization of
the cultivation process which is now well understood. However, what has been
left out so far is the influence of spore quality. This is partially due to the
lack of adequate methods for spore quality assessments and hinders the
interpretation of cultivation outcomes.
The contribution shows the applicability of spore autofluorescence as an
indicator for spore quality. Autofluorescence allows high throughput,
non-invasive investigations of important cell compounds and can be correlated to
key metabolic substrates also present in spores. The results of the
autofluorescence assay show a good correlation to different standard viability
assays.
The findings of these investigations show the possibility of assessing spore
quality as a routine measure prior to large scale cultivations, thereby
improving the cultivation outcome and minimizing bioprocesses with filamentous
fungi with inferior yields. The presented results also offer a new and fast tool
for the optimization of sporulation media.
ln@bio.aau.dk
Section for Sustainable Biotechnology, Copenhagen Institute of Technology,
We are in the process of developing a biorefinery concept for the use of
selected plant biomasses for production of high value biochemicals aiming at
replacing chemicals produced from fossil fuels. One aspect will be the
engineering of efficient biomass converting fungal strains with the ability to
produce high amounts of specific organic acids.
Aspergillus
Mohammad Saeid Jami Foroushani[1]
Carlos García-Estrada[1] Carlos Barreiro[1] Rebeca
Domínguez-Santos[2] Zahra Salehi-Nagafabadi[2] Ricardo V.
Ullán[1] Fernando Teijeira[1] Marta Fernández-Aguado[2]
Juan-Francisco Martín[3]
1INBIOTEC,
Instituto de Biotecnología de León, Avda. Real nº. 1, Parque Científico de León,
24006 León, Spain
2Área de Microbiología, Departamento de Biología Molecular,
Universidad de León, Campus de Vegazana s/n; 24071 León, Spain
3Área de Microbiología, Departamento de Biología Molecular,
Universidad de León, Campus de Vegazana s/n; 24071 León, Spain. INBIOTEC,
Instituto de Biotecnología de León, Avda. Real nº. 1, Parque Cient&a
msaeedjami@yahoo.com
Penicillium chrysogenum
is well-known by its ability to synthesize penicillin and related b-lactam
antibiotics as well as several other secondary metabolites. In addition,
P. chrysogenum
might be consideered, like other filamentous fungi, as an excellent host for
secretion of extracellular proteins due to the high capacity of their protein
secretion machinery. In this work, we have analysed by 2-DE the extracellular
proteome reference map for
P. chrysogenum
Mari Häkkinen,
Markku Saloheimo, Merja Penttilä, Tiina Pakula
VTT Technical Research Centre of
travel2@vtt.fi
Transcriptional analysis of
Trichoderma reesei
cultivated in the presence of different lignocellulose substrates
Mari Häkkinen , Markku Saloheimo, Merja Penttilä and Tiina Pakula VTT Technical
Research Centre, P.O.Box 1000 (Tietotie 2, Espoo), Fin-02044 VTT, Finland;
mari.hakkinen@vtt.fi, tel. +358 20 722 111, fax. +358 20 722 7071
Trichoderma reesei
is a soft rot Ascomycete fungus able to secrete enzymes extremely efficiently
and which is therefore used for industrial production of cellulolytic and
hemicellulolytic enzymes and heterologous proteins for applications in pulp and
paper, detergent, food, textile and feed industries and in biorefinery
applications. Production strains have been traditionally improved by classical
mutagenesis as well as by specific genetic modifications. The availability of
the complete genome sequence of
T. reesei
has made it possible to utilise genome wide analysis methods to study physiology
and protein production by the fungus at different conditions and to utilise the
information obtained to develop new strains with better enzyme production
qualities, such as capability for enhanced protein production or production of
modified enzyme mixtures for degradation of specific types of lignocellulose
materials. In this study
Trichoderma reesei
Rut-C30 was cultivated in the presence of different lignocellulose substrates in
order to study the hydrolytic system of
T. reesei
in the presence of the substrates. The substrates included e.g. sophorose,
cellulose, pretreated wheat straw, pretreated spruce, xylan and bagasse. For
bagasse, different pre-treatments were used in order to get different
combinations of the inducing substances. The cultures were subjected to
transcriptional profiling using oligonucleotide microarrays. Differentially
expressed genes were indentified from the data by comparing the signal
intensities between the induced samples and un-induced controls, and expression
profiles of glycoside hydrolase genes and other genes encoding lignocellulase
degrading enzymes were compared in the presence of the different substrates to
identify co-regulated groups of genes
Senta Heiss-Blanquet[1]
Nicolas Lopes Ferreira[1] Gaëlle Brien[1]
Sarah Morais[2]
Ed Bayer[2] Henri-Pierre Fierobe[3] Antoine Margeot[1]
1IFP,
2Weizmann Institute, 3CNRS
Senta.Blanquet@ifp.fr
The enzymatic cocktail of
Trichoderma reesei
is widely used for the hydrolysis of lignocellulosic substrates to fermentable
sugars in the bioethanol production processes. Its efficiency has still to be
increased, however, to lower the cost of the hydrolysis step. In order to
increase the specific activity of the enzyme mixture, a fusion protein
comprising the entire CBH1 enzyme linked to the catalytic domain of EG1 was
constructed. The construct was transformed into a
T. reesei
strain lacking the
cbh1
gene (CL847Dcbh1)
where it was expressed under the control of the
cbh1
promoter. Two stable clones secreting the fusion protein were isolated and one
of them was cultivated in a larger scale for purification of the recombinant
protein by FPLC. A partially pure protein could be obtained and its activity on
cellulosic substrates determined. The initial hydrolysis rate and final sugar
yield of a
T. reesei
enzyme cocktail supplemented with the fusion enzyme were measured on pretreated
wheat straw and softwood and compared to the hydrolytic activity of the native
mixture. In addition, the degree of synergy was evaluated by comparing the
activity of the fusion protein with an equimolar mixture of CBH1 and EG1.
Antonella Del Fiore[1] Massimo Reverberi[2] Patrizia De
Rossi[3] Valentina Tolaini[2] Corrado Fanelli[2]
1ENEA,
Centro Ricerche Casaccia, Roma, Italy, 2Dipartimento di Biologia
Vegetale, Università “La Sapienza”, Roma, Italy, 3ENEA, Centro
Ricerche Casaccia, Roma, Italy
The aim of this work is to set up a SYBR-green real time qPCR method,
based on the use of specie-specific primers for the early detection and
quantification of potential aflatoxigenic fungi Aspergillus flavus and
Aspergillus parasiticus on whole maize kernels. A primer pair was used for
amplifying a 352 bp fragment of aflR, gene regulator of the aflatoxin
biosynthesis gene cluster. DNA amplification was achieved only with DNA
extracted from fungal strains of A. parasiticus and A. flavus and from maize
kernels inoculated with A. flavus or A. parasiticus, never with DNA of the other
fungal species. Amplification was evident in maize artificially inoculated with
A. flavus 3357 starting from 6 hours of incubation after inoculation, when
mycelium was not visible by stereomicroscope analysis as yet. This real time
qPCR method could be a real, effective method for the early detection and
quantification of the most important aflatoxin-producing fungi in food
commodities. The method proposed in this work represents a useful tool to
evaluate the quality of raw material at different critical points of the food
chain. It could be used to predict potential risk for the presence of
potentially aflatoxigenic strains. The combination of this approach with the
more expensive and laborious conventional chemical analysis of toxin, could be a
real, effective alternative respect to traditional diagnostic methods for the
early detection and quantification of aflatoxigenic fungi in food commodities.
Jesús Fierro Risco,
Rincón, A.M., Codón, A.C., Benítez, T.
Universidad De Sevilla, Departamento de Genética, Facultad de Biología, Avda.
Reina Mercedes, s/n, 41012
jfierro@us.es
Although chemicals are usually used to preserve postharvest fruit quality, the
acceptability of these treatments by consumers is becoming increasingly limited.
Alternatively, biocontrol of postharvest fruit decay using antagonistic
microorganisms has been considered a desirable method.
Trichoderma
is a filamentous fungus widely used as a biocontrol agent against
phytopathogenic fungi. One of the mechanisms employed by
Trichoderma
during antagonism is the production of cell wall degrading enzymes (CWDEs) and
antibiotics (e.g. pyrones). In this study, treatments with conidia of
Trichoderma
in sterile distilled water were conducted to assess the effect on
Penicillium expansum
inoculated on apple and pear fruits. Application of
T. harzianum
CECT 2413 on apples was able to reduce to 60% the number of infected fruits.
However, treatment of pears with
T. harzianum
PF1 –a derived mutant overproducing CWDEs and secondary metabolites- was more
effective in disease reduction compared to the treatment with the wild type.
According to this, effect of enzymes and antibiotics excreted by
Trichoderma
was tested. Combinations of supernatants of PF1 growing either in
hydrolase-inducing (fungal mycelia) or pyrone-inducing media significantly
inhibited the growth
in vitro
of
P. expansum.
Moreover, the treatment with these combinations of supernatants drastically
reduced the number of apple fruits infected with
P. expansum.
These results indicated the importance of
T. harzianum
enzymes and antibiotics in protecting apple fruits from blue mold infection and
the suitability of these components in postharvest biocontrol.
Dominik Mojzita,
Marilyn Wiebe, Satu Hilditch, Harry Boer, Merja Penttilä, Peter Richard
VTT - Technical Research Centre of
dominik.mojzita@vtt.fi
Mucic acid is a dicarboxylic 6-carbons-containing organic acid, which has
applications in food and pharmaceutical industries and can also be used as a
platform chemical. Nowadays, mucic acid is produced either by oxidation of
D-galactose with nitric acid or by electrolytic oxidation of D-galacturonate.
These industrial processes are energy demanding and produce a significant amount
of toxic waste. Here we present an alternative route of synthesis of mucic acid
by means of biotechnology in Aspergillus
In nature, mucic acid is the first intermediate in the D-galacturonate catabolic
pathway in some bacteria, such as Agrobacterium or Pseudomonas. The reaction is
catalyzed by D-galacturonate dehydrogenase (udh), which is an NAD-dependent
enzyme. D-galacturonate is a main component of pectin, abundant and cheap raw
material derived from agricultural production. The bacterial udh gene was
introduced into the fungal strains with disrupted intrinsic D-galacturonate
metabolism in order to direct metabolic flux towards mucic acid. Both modified
strains converted D-galacturonate to mucic acid.
H. jecorina produced mucic acid in
high yield. A.
N.D.S.P. Carvalho,
T.R. Jørgensen,
n.carvalho@biology.leidenuniv.nl
The Unfolded Protein Response (UPR) regulates gene expression in response to
stress in the endoplasmic reticulum (ER). In
Aspergillus
clayw@usc.edu
Aspergillus
species are known to produce high value secondary metabolites such as lovastain.
Recently the genomes of several
Aspergillus
species have been sequenced. One of the most important findings from these
genome efforts is the realization that these organisms have the potential to
produce far more secondary metabolites than have ever been isolated and
identified. I will present our recent efforts in identifying new secondary
metabolites and their corresponding biosynthesis pathways from several different
Aspergillus
species. Finally we will present approaches to use natural products we have
isolated from
Aspergillus
as starting point for further drug discovery.
Michel Flipphi[3]
Pasqualina Magliano[1] Dominique Sanglard[2] Yves Poirier[1]
1Department
of Plant Molecular Biology, University of Lausanne, CH-1015 Lausanne,
Switzerland
2Department of Microbiology, University of Lausanne, CH-1015
Lausanne, Switzerland
3Co
nsejo Superior de Investigaciones Cientificas (CSIC)
flipphi@iata.csic.es
Biotin (vitamin H) is an essential cofactor for some (trans)carboxylation
reactions in carbon metabolism. It also plays roles in cell signaling,
epigenetic regulation, and chromatin structure. In fungi, biotin biosynthesis
genes are clustered. In
Aspergilli,
the central gene we termed
biDA,
encodes a protein that bares similarity to both
Escherichia coli
dethiobiotin synthetase (BioD) and 7,8-diaminopelargonic acid (DAPA)
aminotransferase (BioA). The divergently transcribed
biF
gene codes for a protein similar to 7-keto-8-aminopelargonic acid synthase
(BioF) while the
biB
gene, situated downstream of
biDA,
is similar to biotin synthase (BioB). E.
coli
mutants deleted for either of the structural genes
bioF,
bioA,
bioD
or
bioB,
could be complemented by the expression of
A. nidulans
biF,
biDA
or
biB
cDNAs. This confirmed that while seperate genes encode DAPA aminotransferase and
dethiobiotin synthetase in bacteria and yeast, both these activities are
performed by a single, bifunctional protein in
A. nidulans.
Three classical
A. nidulans
(biA1-3)
biotin-auxotroph mutants were found to have distinct mutations in the part of
the
biDA
gene that specifies the DAPA aminotransferase domain. Such mutants could be
complemented by transformation with the functional
biDA
gene from either
A. nidulans
or
Aspergillus fumigatus.
Approximately ten biotin autotroph colonies per 106 protoplasts per
microgram of plasmid, were routinely obtained. Co-transformation of
biDA
with a plasmid carrying sGFP under
A. nidulans
transcriptional control yielded co-transformants stably expressing sGFP over
several generations. These results showed that
biDA
orthologs can serve as the basis for a robust and convenient transformation
system in
Aspergilli.
Frank Kempken,
ElkBir Hihlal
Botanisches Institut, Christian-Albrechts-Universität, Olshausenstraße 40, 24098
fkempken@bot.uni-kiel.de
Transposons are mobile DNA sequences, which are found in all eukaryotic genomes
(1-3). We have analyzed the transposon content in two fungal genomes,
Aspergillus
Marika Vitikainen,
Mikko Arvas, Tiina Pakula, Merja Oja, Merja Penttilä, Markku Saloheimo
VTT Technical Research Centre of
marika.vitikainen@vtt.fi
Background:
Trichoderma reesei
is the main industrial producer of cellulases and hemicellulases used to
depolymerize biomass in many biotechnical applications. Many production strains
in use have been generated by classical mutagenesis. In this study we
characterized genomic alterations in hyperproducing mutants of
T. reesei
by high-resolution comparative genomic hybridisation tiling array (aCGH). Our
aim was to obtain genome-wide information which could be utilized for better
understanding of the mechanisms that underlie the efficient cellulase production
and enable targeted genetic engineering for improved production of proteins in
general. Results: We carried out aCGH analysis of four hyperproducing strains
(QM9123, QM9414, NG14 and RutC-30) using QM6a genome as a reference. In QM9123
and QM9414 we detected altogether 44 previously undocumented mutation sites
including deletions, chromosomal breakpoints and single nucleotide mutations. In
NG14 and RutC-30 we detected 126 mutation sites of which 17 were new mutation
sites not documented previously. Among these new sites there are first
chromosomal breakpoints found in NG14 and RutC-30. We studied the effect of two
deletions identified in RutC-30 (a deletion of 85 Kb in the scaffold 15 and a
deletion in the gene 72076 encoding a fungal transcription factor) on cellulase
production by constructing knock-out constructs in the QM6a background. Neither
of the deletions affected cellulase production. Conclusions: ArrayCGH
analysis identified dozens of mutation sites in each strain analyzed. The
resolution was at the level of single nucleotide mutation. High-density aCGH is
a useful tool for genome-wide analysis of organisms with small genomes e.g.
fungi, especially if large set of interesting strains is to be analyzed.
Masayuki Machida[1]
Hideaki Koike[1] Morimi Teruya[2] Masatoshi Tsukahara[3]
Yumi Imada[3] Youji Wachi[4] Yuki Satou[4]
Yukino Miwa[3] Shuichi Yano[3] Yutaka Kawarabayasi[1]
Osamu Yamada[5] Koji Jinno[6] Akira Hosoyama[6]
Takasumi Hattori[7] Motoaki Sano[8] Koichi Tamano[9]
Kazurou Fukuda[10] Takaomi Yasuhara[10] Kenichi Higa[11]
Shinichi Ohashi[5] Kotaro Kirimura[12] Masanori Arita[13]
Kiyoshi Asai[13] Keietsu Abe[14] Katsuya Gomi[14]
Shigeaki Mikami[5] Takeaki Ishikawa[15] Kaoru Nakasone[16]
Nobuyuki Fujita[6] Takashi Hirano[17] Ikuya Kikuzato[4]
Kazuhiro E. Fujimori[17] Hiroshi Horikawa[6]
1Natl.
Inst. Advanced Inst. Sci. Technol,
m.machida@aist.go.jp
Koji mold is the traditional name of
Aspergillus
species that are used for Japanese fermentation industries.
Aspergillus oryzae
has been widely used in Japanese fermentation industries, Japanese alcohol
beverage, soy sauce and so on for longer than a thousand of years. Comparison of
A. oryzae
genome with those of other
Aspergillus
species of smaller genome size revealed existence of non-syntenic blocks (NSBs)
specific to the
A. oryzae
genome.
Aspergillus awamori
is another industrial filamentous fungus, widely used for brewing Japanese
traditional spirits, Awamori, in
Nick van Biezen[1]
Jeroen E. Schouwenberg[1] Nico M.G. Oosterhuis[2] Anton
Tromper[2] Christien Lokman[1]
1HAN
BioCentre,
Nick.vanBiezen@han.nl
The application of single-use equipment becomes more and more common in the
biopharmaceutical industry. Disposable filters, bags for medium and product
storage are more and more used and the application of single-use process
equipment becomes more common nowadays. Compared to the traditional glass or
stainless steel stirred tanks, the single-use bioreactor offers clear
advantages: a shorter turnaround time (no SIP or CIP required), resulting in a
cost reduction; minimal utilities required; greatly reduced potential of cross
contamination (safety); greater operational flexibility; reduced validation
requirements.
Due to its two-dimensional movement profile, the CELL-tainer®
single-use bioreactor creates a superior gas-liquid mass-transfer compared to
other single-use bioreactors presently available. This offers the opportunity to
reach higher cell-densities and therefore higher volumetric productivities. With
the CELL-tainer kla values of 300 h-1 and above are
measured, making the system suitable for high density mammalian cell cultures,
but especially also for microbial, yeast, and mycelia cultures. Measurement of
the heat exchange capacity (cooling capacity) indicates that accurate
temperature control of such exothermic type of cultures is possible as well.
A comparison is made between the CELL-tainer® and the standard
stirred bioreactor during the heterologous production of a peroxidase in
Aspergillus
Christien Lokman, Dennis Lamers, Nick van Biezen, Andrea Thiele, Theo
Goosen, Kees van den Hondel
HAN BioCentre,
christien.lokman@han.nl
Filamentous fungi have a very efficient protein-production capacity which make
them suitable host organisms for the overproduction of commercially interesting
homologous and heterologous proteins. The overall efficiency of an enzyme
production process is influenced by the production yield (fermentation) and
purification yield (Down Stream Processing). Unfortunately, since every protein
is different, in many cases production and purification protocols and strategies
must be developed for each individual protein. In
E. coli
the fusion of proteins to oligohistidine tags followed by affinity
chromatography is a very common protein purification strategy. As far as we
know, the use of His-tags in the extracellular production and purification of
heterologous proteins in
Aspergillus
has not yet been demonstrated.
Recently, we successfully produced
Arthromyces ramososus
peroxidase (ARP) in
Aspergillus
Tiina Pakula,
Lise Bernard-Granger, Jaana Toikkanen, Merja Oja, Marilyn Wiebe, Markku
Saloheimo, Merja Penttilä
VTT Technical Research Centre,
tiina.pakula@vtt.fi
The work is part of a EUROSCOPE programme project Genophys, in which the aim is
to compare the physiology of heterologous protein production in different
microbial production hosts using a common model protein and applying genome-wide
analysis methods to study the cellular events at a selected set of cultivation
conditions.
We have carried out transcriptome and proteome analysis of a
Trichoderma reesei
strain producing antibody 3H6 Fab-fragment and a control strain expressing only
the selection marker gene
amdS,
and analysed the effects of production of the hetereologous protein as well as
cultivation temperature on the cellular responses. For the analyses, the
strains were cultivated in lactose-limited chemostats at 21.5°C, 24°C and 28°C.
The dilution rate used in the cultivations, 0.03/h, has been previously shown to
be optimal for protein production in similar type of chemostat cultures of
T. reesei.
The lower temperatures used in the study, favoured protein production by both
strains. In the Fab producing strain, both the total protein production into the
culture medium as well as Fab production were the highest at 21.5°C, the values
obtained at 24°C being close to the ones obtained at 21.5°C. In the control
strain the highest protein production was at 24°C. Comparison of differentially
expressed genes between the strains showed relatively few genes affected by the
heterologous gene expression, whereas comparison of the cultures at different
temperatures revealed a large number of genes with altered expression level.
However, the proteome analysis of the cultures using the 2D DIGE method
indicated also stress responses to the heterologous protein production. The
analysis showed differences especially in the intensity of protein spots
corresponding to different type of heat shock proteins (ER, mitochondrial,
cytoplasmic, ribosome-associated HSPs), actin and tubulin assembly factors, and
proteins related to ER-associated protein degradation.
Kenneth Bruno,
Kristen M. Meyer, Scott E. Baker
Pacific Northwest National Lab.
kenneth.bruno@pnl.gov
Mutation of the kusA gene in several fungi, including
Aspergillus
Rebeca Domínguez-Santos1[1] Carlos García-Estrada[2]
Mohammad-Saeid Jami[1] Ricardo V. Ullán[2] Fernando
Teijeira[2] Marta Fernández-Aguado[3] Juan-Francisco
Martín[4]
1Área
de Microbiología, Departamento de Biología Molecular, Universidad de León,
Campus de Vegazana s/n; 24071 León, Spain
2INBIOTEC, Instituto de Biotecnología de León, Avda. Real nº. 1,
Parque Científico de León, 24006 León, Spain
3Area de Microbiología, Departamento de Biología Molecular,
Universidad de León, Campus de Vegazana s/n; 24071 León, Spain
4Área de Microbiología, Departamento de Biología Molecular,
Universidad de León, Campus de Vegazana s/n; 24071 León, Spain INBIOTEC,
Instituto de Biotecnología de León, Avda.
Real nº. 1, Parque Cient&
msaeedjami@yahoo.com
Beta-lactam biosynthesis is subjected to complex regulatory processes. One of
the transcriptional factors recently identified in
Acremonium chrysogenum
is CPCR1, which acts as a regulator of the cephalosporin C biosynthesis and
shows a high similarity to transcriptional factors from the human family of
“winged helix” regulatory X factors (RFX).
The CPCR1 ortholog was characterized in
Penicillium chrysogenum.
The gene encoding this transcription factor (Pcrfx)
was identified in the
P. chrysogenum
genome (Pc20g01690). The protein encoded by this gene (PcRFX) shared a 47%
homology and 30% identity with the
A. chrysogenum
CPCR1 factor. Analysis of the promoter region of the penicillin biosynthetic
genes (pcbAB,
pcbC
and
penDE),
revealed the presence of one binding site (X-box) for this factor in each
promoter region. Gene silencing of
Pcrfx
gave rise to a decrease in the production of isopenicillin N and penicillin G in
the knock-down mutant after 48 and 72 h of culture. In this mutant, the
steady-state levels of the penicillin biosynthetic genes transcripts were
diminished, thus confirming the regulatory role of PcRFX in penicillin
biosynthesis.
Efficient gene targeting in Aspergillus
Homologous recombination frequencies in filamentous fungi, including
Aspergillus
To avoid any potential side effects of a
kusA loss-of-function mutation on growth and viability of
A.
Improved tools for fungal strain development
Margreet Heerikhuisen,
Cora van Zeijl, Peter Jan Punt
TNO Quality of Life, Dept. Microbiology,
margreet.heerikhuisen@tno.nl
Already from the very start of the molecular era for filamentous fungi, TNO has
been involved in developing molecular tools for gene expression and protein
secretion for filamentous fungi. In this poster we present a number of improved
tools for fungal strain development, like novel promoters and versatile
selection markers.
From the development of fungal gene transfer systems, the isolation of efficient
expression signals is one of the research lines to obtain efficient protein
production. As a result of gene expression studies we have discovered a new and
very strong promoter from A.
Versatile genetic modification of filamentous fungi requires the possibility to
modify, disrupt and express a number of different genes in a single fungal
strain. Therefore, the availability of a (series) of different selection markers
is essential. However, the possibility of repeated use of the same marker in
subsequent experiments can circumvent the requirement for multiple selection
markers. The usage of counter selectable markers like
pyrG and
amdS is very suitable for this
purpose, because these allow selection of the mutant and the transformant. Also
a synthetic amdS selection marker was
constructed which is devoid of most restriction sites that are present in the
native amdS gene.
Single cell analysis as a way to study hyphal heterogeneity
Charissa de Bekker,
Arman Vinck, Han A.B. Wösten
Microbiology and Kluyver Centre
for Genomics of Industrial Fermentations, Institute of Biomembranes, Utrecht
University, Padualaan 8, 3584 CH Utrecht, The Netherlands
c.debekker@uu.nl
Mycelial fungi use the hyphae growing at their apices to colonize a substrate.
These hyphae secrete enzymes that convert complex polymers into small molecules
that can be taken up by the fungus to serve as nutrients. Using GFP as a
reporter it has been shown that exploring hyphae of
Aspergillus
We here demonstrate that the expression of other genes encoding secretion
enzymes in A.
To unravel the mechanisms underlying heterogeneity we aim to perform a
genome-wide expression analysis of highly and lowly expressing hyphae. To this
end, protocols have been set up to collect individual hyphae using laser
micro-dissection and pressure catapulting (LMPC) and to isolate RNA from these
samples. QPCR has shown that we are able to extract RNA from a single hypha
only.
This research is supported by the Dutch Technology Foundation STW, applied
science division of NWO and the Technology Program of the Ministry of Economic
Affairs.
Oligosaccharide transport by Neurospora
crassa during cellulolytic growth
Jonathan M. Galazka1, Chaoguang Tian2, William T. Beeson1,
N. Louise Glass1 and Jamie H. D. Cate1
1University
of
The filamentous fungus, Neurospora
crassa, is capable of depolymerizing and metabolizing plant cell walls, but
this process is not understood in great detail.
Our group recently reported a systematic study of plant cell wall
degradation by N. crassa, utilizing
transcriptomics, secretome analysis, and the phenotypic characterization of gene
knockouts (1). Whole genome
microarrays identified 114 genes that show overlapping expression differences
when N. crassa is grown on either
ground Miscanthus stems, or pure
cellulose as carbon sources. Within
this set were 10 predicted major facilitator superfamily transporters.
We hypothesized that at least one of these proteins may transport
oligosaccharides, an activity that has been reported in
H. jecorina (2), but never attributed
to a particular gene. Mutations in
three of these transporters significantly affected growth of
N. crassa on crystalline cellulose.
We developed a method by which to rapidly assay the substrates of these
transporters, and report the identity of three oligosaccharide transporters in
N. crassa.
We believe these transporters may be useful in downstream processing of
depolymerized plant cell walls.
1. Tian & Beeson et al. (2009) PNAS 2009 106: 22157-22162.
2. Kubicek et al. (1993) J. Biol.
Chem. 268: 19364-19368.
Stefan Weber1
, Jeroen G. Nijland1, Andriy Kovalchuk1, Marco van den
Berg2, Roel Bovenberg2 and Arnold J.M.
Driessen1
1University
of Groningen, Department of Molecular Microbiology, Groningen Biomolecular
Sciences and Biotechnology Institute, and the Kluyver Centre for Genomics of
Industrial Fermentation, Kerklaan 30, 9751 NN Haren, The Netherlands, and
2DSM
anti-infectives, PO Box 425, 2600 AK Delft, The Netherlands
Industrial production of β-lactam antibiotics using the filamentous fungus
Penicillium chrysogenum is based on
successive microbial strain improvement cycles. Analysis of these strains has
led to the identification of several important mutations in high-producing
strains. These are amongst others the amplification of the penicillin
biosynthesis genes, the elevated transcription of genes involved in biosynthesis
of the amino acid precursors, and genes encoding microbody proteins. However,
many of the key (intra)cellular transport processes have remained obscure.
To identify ABC-transporters involved in β-lactam production,
P. chrysogenum was grown in the
presence and the absence of phenylacetic acid (PAA), a side chain precursor of
penicillin G. Expression of all ABC transporters was determined by Quantitative
Real Time PCR. This revealed a significantly increased expression level of ABC40
when grown with PAA. In another experiment, expression of all ABC transporters
was determined upon a penicillin G challenge, resulting in elevated levels of
the expression of ABC30, ABC42 & ABC48. Independent knock-outs strains of ABC30,
ABC40, ABC42 and ABC48 in a high producing strain were constructed, but none of
these strains showed a detectable phenotype. In another approach, the same genes
were inactivated in an early (low) production strain. The further analysis of
these strains is in progress.