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BIOTECHNOLOGIE
BESONDERE LEISTUNGEN VON PILZEN
Second Life – Verwertungswege für biogene Stoffströme, November 15th 2017, Bingen
Fungi are the loosers of the
evolution:
- no photosynthesis
- they can‘t run off
Prof. Dr. Meike Piepenbring, April 2017
Fungi are not the loosers of the evolution:
- apparently they are extremly versatile chemists
PENICILLIUM CHRYSOGENUM
(PENICILLIUM NOTATUM)
The discovery of penicillin by
Sir Alexander Fleming (1928)
"Synthetic Production of Penicillin TR1468" by Official photographer -
http://media.iwm.org.uk/iwm/mediaLib//32/media-32192/large.jpgThis is photograph TR 1468 from the
collections of the Imperial War Museums.. Licensed under Public Domain via Commons -
https://commons.wikimedia.org/wiki/File:Synthetic_Production_of_Penicillin_TR1468.jpg#/media/File:S
ynthetic_Production_of_Penicillin_TR1468.jpg
Von This image was created by user Tatiana Bulyonkova (ressaure) at Mushroom Observer, a source for mycological images.You can contact this user here. - This image is Image Number 214245 at Mushroom Observer, a source for mycological images.This tag does not indicate the copyright status of the attached work. A normal copyright tag is still required. See Commons:Licensing for more information., CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=20985352
Strobilurus tenacellus
Bitterer Kiefern-Zapfenrübling
Pinecone cap
7
From submerged cultures of S. tenacellus (3 mg/l); strong antifungal activity, non-toxic for mice; MoA: inhibition of electron transport in the respiratory chain (bc1).
G. Schramm, Dissertation 1980
O
O
O
OO
O
NOO
O
Strobilurin AO
O
O
OO
O
O
Kresoxim-Methyl (BASF)
Sauter, H.; W. Steglich & T. Anke: Strobilurine: Evolution einer neuen Wirkstoffklasse. Angew. Chem. 111, 1416-1438 (1999); Int. Ed. 39, 1328-1349 (1999).
B. Schwalge, Dissertation 1986
O
NO
O
O
FUNGAL SECONDARY METABOLITES AS LEAD
STRUCTURES FOR AGROCHEMICALS
8
Qo inhibitor fungicides (QoI): the Qo
inhibitor fungicides all act at the Quinone
‘outer’ (Qo) binding site of the cytochrome
bc1 complex.
FUNGAL SECONDARY METABOLITES AS LEAD
STRUCTURES FOR AGROCHEMICALS
By C31004 at English Wikipedia, CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=24133439
Crystal structure of mitochondrial cytochrome bc1 complex bound with ubiquinone
Fungicide „mode of action“
9 FUNGAL SECONDARY METABOLITES AS LEAD
STRUCTURES FOR AGROCHEMICALS
10
Fungal biotechnology at the IBWF: based on
secondary metabolite research in
fungi/microorganisms.
The IBWF gGmbH is a non-profit Research
Institution bridging the gap between basic
university research and applied/industrial
research.
The IBWF is a non-profit limited liability
company (gGmbH).
NATURAL PRODUCT RESEARCH AT THE IBWF
GGMBH
11
Fungi
Strain Collection: Asco-, Basidio- and
Zygomycetes
Taxonomy
Fermentation
FungalGenetics Screening
BioactiveNatural
Products
MoA-Studies
Development of Assay-Systems
OO
O
COOH
Natural products
BioactiveCompounds
Enzymes
Productionand
PurificationAnalytics
NATURAL PRODUCT RESEARCH AT THE IBWF
GGMBH
12
OO
O
OO
O O
H
O
O
O
OH
OO
O
O
O
OH
OH
O
OH
OH
OO
OH
O
OH
OH
OH
H
H
H
Cl
OCl
Cl
Cl
OH
O
O OO
H
H
OH
OH
OH
OH
OH
O
O
O
O
Cl
ClOH
Cl
HH
ClOH
OO
HO
H
O
H OH
HH
O
O
O
O
OO
O
OH
OH
OH
OH
OH
OH
OH
OOH
OH
OH
O
O
O
O
O
O
O
OH
O
O
OHH
O
O
O
OOH
OH O
O
HH
O
OHOHOH
O
OH
Cl
Cl
Cl
OH
OH
OH
H
O
N
N
N
N
O
OH
OH
OH
OH
O
H
H
O
OH
O
O
O
OH
O
OH
O
OH
OH
OH
OH
H
H
O
O
OH
O
O
O
O
OOH
H
H
O O
O
OH
O
O
O O
O
OOH
OOH
H
H
H
O
O
O
OH OH
H
O
O
O
O
O
OH
OO
OOH
OH
O
O
O
O
O
OH
H
H
O
OO
OH
H
O
O
OH
OH
OH
N+
N+
C
C
O
O
O
OO
O
O
O
OO
O
OH
OH
OH
HHH
O
OO
O
OH
O
Cl
O
OH
O
Br
O
O
OO
O
N
N
N
N
NNH
N
N
N
N
NH
NH
NH
O
O
O
O
O
O
OO
O
O
O
O
OOH
OHOH
OH
O
H
NH
OH
OH
OO
OOHO
O
H
H
O O
OO
OO
O
OH
OH
N
OO
O
O
H
H
H
H
O
NH
O
O
O
OH
O
OH
O
O
O HH
OO
OO
OO
O
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O
O
O
OHOH
OH
OH
OH OHOH
OH
OH
OH
H
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O
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OH
OH OHOH OHOH
OH
OH
OH
OH
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OHN
OH
OH
O
O
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N
O
O
O
OH
O OO
OO
OO
OH
O
O
OH
H
H
HH
H
H
H
NH
NH
O
NH
O
NH2
O
OOH
Cl
OH
OH
O
OH
OHOH
OH
OHO
OH
O
NATURAL PRODUCT RESEARCH AT THE IBWF GGMBH:
LEAD STRUCTURE IDENTIFICATION
13
106 fungal species [Hawksworth (2001), Mycological Research]
The enormous diversity of fungal organisms is reflected by the diversity of the secondary
metabolites
NATURAL PRODUCT RESEARCH AT THE IBWF
GGMBH
14
Number of fungal strains: > 19.000
Basidiomycetes: 3.200
Ascomycetes (of fruiting bodies): 1.300
Deuteromycetes: 7.500
Zygomycetes (from soil samples): 150
Endophytes: 4.900
Coprophilic fungi: 180
Fungi from marine habitats: 80
Carotinoid-producing microorganisms: 150
Marine bacteria: 400
Strains from commercial collections: 1.200
The culture collection of the IBWF is one of the largest non-commercial collections
NATURAL PRODUCT RESEARCH AT THE IBWF
GGMBH: THE CULTURE COLLECTION
15
• One of the largest “non-profit” culture collections.
• The strains were collected according to the “Convention on Biological
Diversity” (CBD).
• The collection is of high quality, since most Basidio- and Ascomycetes are
identified.
• The collection was started in 1977.
NATURAL PRODUCT RESEARCH AT THE IBWF
GGMBH: THE CULTURE COLLECTION
17
Zellulose Lignin Chitin
Monomer: Cellobiose
Pflanzliche Zellwände
Monomere: Cumarylalkohol, Coniferylalkohol,Sinapylalkohol
Holz
Monomer: Acetylglucosamin
Glieder- und Weichtiere, Pilze
HÄUFIG VORKOMMENDE BIOPOLYMERE
18
Aus: Webster & Weber: Introduction to Fungi, 2007
BIOPOLYMERE IN HOLZ
19
Coriolus versicolor,
A common white rot fungus on wood
White rot fungi initially destroy the lignin
part of the wood and later the celluloses
component.
In contrast brown rot fungi mainly degrade
the celluloses.
WHITE- AND BROWN ROT
20
The tinder fungus penetrates its
host plants via wounds in branches
and/or the trunk. In the heartwood
it causes intensive white rot
resulting in a decreased stability of
the wood/of the entire tree.
Eventually the tree breaks off.
The tinder fungus can survive for a
long time as saprophytic fungus on
the dead substrate. Bild: Wikipedia.de
TINDER – HOW TO START FIRES WITH FUNGI?
21
TINDER – HOW TO START FIRES WITH FUNGI?
Tinder is easily combustible
material used to start a fire.
Tinder is a finely divided, open
material which will begin to
glow under a shower of sparks.
Tinder is one of the greatest
inventions of mankind. Fungi
were used as tinder by
prehistoric man to produce fire.
Bild: Wikipedia.de
22
Etymology: Tinder (Zunder) –„Thunder“ (Donner)
Tinder: In general species of the genera Fomes
The tinder fungus: Fomes fomentarius (Fungus of the year 1995)
Highly ignitable tinder can be produced by soaking slices of the flesh of the
fruiting body in nitric acid.
Bilder: Wikipedia.de
TINDER – HOW TO PRODUCE FIRES WITH FUNGI?
23
Dashtban et al., 2010
MICROBIAL DEGRADATION OF LIGNIN
24
Vanilin p-Cumarinsäure Ferulasäure
Phenoloxidase:
•Laccase
Peroxidasen:
•Ligninperoxidase
•Manganperoxidase
•Versatile Peroxidase
MIKROBIELLER ABBAU VON LIGNIN
25 ANWENDUNGEN VON LACCASEN/PEROXIDASEN
• Bleichen von Pulpe in der Papierindustrie
• Bleichen von Textilfarben
• Entfernung von Polyphenolen in Lebensmitteln
• Bleichmittel in Zahnpasta
• Herstellung von Weinkorken
• Behandlung von industriellen Abwässern (Entfernung von
phenolischen Komponenten)
• TNT-Abbau
• …
26 LACCASEN/PEROXIDASEN AUS PILZEN: SCREENING
STRATEGIE
Incubation on basal medium plus:
Cultivation in submerged culture
(120 rpm)
83 Basidimycetes+ 53 Ascomycetes
> 2 tests positive
Interesting enzyme activities
Mn2+3-nitroaniline
Poly R-478 Azure B
Incubation on WA-agar plus guaiacol
347 strains518 strains
Cultivation in 20 l fermentor (120 rpm, soybean or YMG medium + 1mg/l CuSO4)
14 strains
soybean mediumbasal medium
All positive
27
Extract Strain LAC
[nkat/ml]
pH-opt.
DMP
Heat st.
[%]
Biob. 14C-Pulp
[% cpm]
HBT ox.
[nmol
O2/s]
Kap. red.
[%]
F2 87135 200 6 40 + 99
F3 95164 1500 5 60 - 31 0
F4 89010 1300 4 85 + 24 48
F5 95338 26000 5 75 + 34 11 21
F6 89009 4300 5 95 - 27 77 13
F7 95318 17000 5 100 + 34 68
F8 78029 3250 5 95 ++ 20 46
F9 89141 200 6 90 ng 6 13 9
F10 95290 2000 5 70 ++ 39 101 37
F14 82066 960 7 0 + 0 7 *
F15 85042 1800 8 0 + 6 2 *
F16 82020 9300 5 95 - 7
K6 86012 4500 5 80 + 28
K7 JB597 11500 5 80 - 10
T.h. 2000 33 49 38
CHARACTERIZATION OF ENZYME CRUDE EXTRACTS
(LACCASES)
T.h. = Trametes hirsuta laccase (VTT-D-443); pH-opt. = pH optimum with DMP as substrate;
LAC = laccase activity, ABTS pH 4.5; Heat st. = heat stabililty, activity after 30 min at 60°C;
Biob. = biobleaching (+ = bleaching effect, - = no bleaching effect); ng = not grown;
HBT (hydroxybenzotriazole ox. = oxidation of HBT pH 5, * = pH 7.2;
Kap. red. = TCF-delignification pH 5, kappa reduction;
28
Temperaturabhängigkeit der Laccase
Temperaturstabilität der Laccase
20 30 40 50 60 70
Temperatur [°C]
0
5000
10000
15000
20000
LA
C-A
kti
vit
ät
[nka
t/m
g]
95°C75°C56°C
0 10 20 30 40 50 60
Inkubationzeit [min]
0
25
50
75
100
125
LA
C-A
kti
vit
ät
[%]
2 3 4 5 6 7 8 9
pH-Wert
0
1000
2000
3000
4000
5000
6000
7000
LA
C-A
kti
vit
ät
[nka
t/m
g]
2 3 4 5 6 7 8 9
0
1000
2000
3000
4000
5000
6000
LA
C-A
kti
vit
ät
[nka
t/m
g]
pH-Wert
LAC-Aktivität mit ABTS in Abhängigkeit vom pH-Wert
LAC-Aktivität mit 2,6-DMP in Abhängigkeit vom pH-Wert
CHARACTERIZATION OF LACCASE F10 - STRAIN 95290
29
Lignin-degrading basidiomycete
Modell organismus for investigations
concerning fungal development
Natural source for laccases
Expression of isoenzymes
COPRINUS CINEREUS AS SOURCE FOR LACCASES
• Copper-containing enzyme
• Complex structur
• Far distributed amongst plants and
microorganisms
• Degradation of phenolic
compounds (e.g. lignin)
Heterologous expression in Magnaporthe oryzae
30
a: Appressorium
c: Conidium
a
c
Magnaporthe oryzae
as ‚model-organism‘:
- Genome is sequenced
- Hemibiotrophic
- Established transformation
systems
- Genetically well
characterized
- Many mutants have been
generated and are available
THE RICE BLAST FUNGUS
MAGNAPORTHE ORYZAE (ANAMORPH: PYRICULARIA ORYZAE)
31
Cloning strategy
P TSP L
P = promoter
SP = signalpeptide
O = original protein
L = laccase (host foreign)
ST = Strep-tag
T = terminator
P TSP LO
ST
ST
Identified candidate from the secretome analysis: 13proS1
HETEROLOGOUS EXPRESSION SYSTEM IN MAGNAPORTHE
ORYZAE
32
Generation of transformants with up to 10-fold increased laccases-activity
within the culture filtrate
Isolation of a laccase via ion-exchange-chromatography (IEX) and gel filtration
(GF) (approx. 700 µg/l)
Identification of the laccase Lcc1 from Coprinus cinereus by mass
spectroscopy
HETEROLOGOUS EXPRESSION OF THE LACCASES LCC1 FROM
COPRINUS CINEREUS IN MAGNAPORTHE ORYZAE
Stamm
WT T1 T2 T3 T4 T5 T6
Lacc
ase
-Aktivität
[µkat/
ml]
0
100
200
300
400
500
600
700
800
33
Fermentationsdauer [d]0 2 4 6 8 10
Glu
cose
konze
ntr
ation [
g/l]
0
2
4
6
8
10
Standard
Fed-Batch
Beginn derZufütterung
Ende derZufütterung
APPLICATION OF „FED-BATCH“ CONDITIONS
Fermentationsdauer [d]
0 2 4 6 8 10
Lacc
ase
-Aktivität
[%]
bezo
gen a
uf
die
Sta
ndard
ferm
enta
tionsb
edin
gungen
0
100
200
300
400Standard
Fed-Batch
• Reduction of laccases-acivity after the free
glucoses within the medium is depleted
• Constant feeding of glucoses increases the
laccases-yield
• Up to day 10 the laccases activity was not
dependent on the availbility of the nitrogen
source
34
Overexpression of the transcription factor HAC1 involved in the
transcriptional regulation of helper-proteins appears more efficient
than manipulations of individual helper-proteins
OVEREXPRESSION OF HELPER-PROTEINS
Mo53Pro:Lcc1
MoEF1:BiP
MoEF1:CLX
MoEF1:PDI
MoEF1:H
AC1
Lacc
ase
-Aktivität
[µkat/
ml]
0
1000
2000
3000
4000
5000EF1
+53Pro:Lcc1
35
Promotor inducible by addition of
maltoses
Secretion of laccases after addition of
maltoses is delayed
Expression of laccases within 10
hours after addition of maltoses
USE OF INDUCIBLE PROMOTERS
MGG8-
number
Size of
the
protein
[AS]
Conserved protein domains
[PFAM 22.0]
Putativ
function
MGG_01096
.8655
Glycosyl hydrolases family 15
(PF00723)
Starch binding domain (PF00686)
Glucoamyl
ase
precursor
Fermentationsdauer [d]
0 2 4 6 8 10
Lacc
ase
-Aktivität
[µkat/
ml]
0
200
400
600
800
1000WT 70-15
Mo96iPro:Lcc1
Mo96iPro:Lcc1 nicht induziert
Induktion mit 2% Maltose
36
Zellulose Lignin Chitin
Monomer: Cellobiose
Pflanzliche Zellwände
Monomere: Cumarylalkohol, Coniferylalkohol,Sinapylalkohol
Holz
Monomer: Acetylglucosamin
Glieder- und Weichtiere, Pilze
HÄUFIG VORKOMMENDE BIOPOLYMERE
37
Schematic structure of cellulose with cellulolytic enzymes. BGL β-
glucosidase, CBH cellobiohydrolase, EGL β-1,4-endoglucanase
MIKROBIELLER ABBAU VON ZELLULOSE
Van den Brink & de Vries: Appl Microbiol Biotechnol (2011) 91:1477–1492
38 SCREENING STRATEGIE:
ENZYME ZUM ABBAU VON ZELLULOSE
Mikroorganismen/Pilze werden
kultiviert auf Medien, die
Carboxymethylcellulose (CMC)
enthalten.
Nachdem die Kulturen
gewachsen sind, werden sie mit
Gram’s Jodlösung überschichtet.
Kasana, R.C., Sarwan, R., Khar, H. Dutt, S. and Gulati, A. (2008) Curr Microbiol 57:503-507
39 COPROPHILE PILZE ALS QUELLE FÜR ZELLULOSE-
ABBAUENDE ENZYME
In Dung ist die weitgehend unverdaute Zellulose Kohlenstoffquelle für Pilze.
Coprophile Pilze müssen das Substrat erschließen können.
40
Pinen und Caren sind toxische Naturstoffe Abbau durch Pilze (?)
TERPENABBAU MIT PILZEN
41 TERPENABBAU - KONZEPT
α-Pinen Pinandiol Pinonsäure Pinsäure Pinsäureanhydrid
JGU Mainz (Waldvogel)
Elektrochemisch
IBWF (Thines/Jacob)
Biotechnologisch
Pinsäureanhydrid
+ Epoxidharz + Naturfaserverstärkung = Verbundwerkstoff
Transferstelle Bingen Entwicklung des Harzsystems und Verbundwerkstoffe
TH Bingen Analysen und Messtechnik
42
IBWF fungal culture collection: >18.000 strains
Preselection of candidates based on literature and IBWF-knowledge
Screening and identification of turpentine-tolerant strains
Identification and isolation of turpentine derivatives
Identification of enzymes involved
Heterologous /homologous expression of (modified) enzymes
Upscaling and Optimization of the biotransformation-process
43
(-)-α-pinene
(+)-α-pinene
(-)-β-pinene
3-carene
R-(+)-limonene
INITIAL RESULTS:
BIOTRANSFORMATION OF TURPENTINE COMPONENTS
44
(-)-α-pinene
45
(+)-α-pinene
46
(-)-β-pinene
47
3-carene
48
R(+)-limonene
49
Protein identification
and molecular genetics
Example 1:
The inactivation (knock-
out) of specific enzymes
will result in the
accumulation of
limonene-1,2diol
50
Protein identification
and molecular genetics
Example 2:
Heterologous expression of
limonene 1,2-
monooxygenase
[EC:1.14.13.107] and
limonene-1,2-epoxide
hydrolase [EC:3.3.2.8] from
Rhodococcus erythropolis in
E. coli or yeast we could
increase yields and
efficency.
