MINI PROJECT

BED 350
6-PENTYL-α-PYRONE
Under the guidance of
Prof. Atul Narang

Chirag Mittal
2012BB50049
 INTRODUCTION
 Isolated from Trichoderma species

 Solubility in water ???

 Volatile ??

 Antifungal compounds (antagonistic ability)

 Used in food industry principally as a flavour enhancer in soft

drinks or yogurts

 Have coconut aroma

 TRICHODERMA SPECIES

 Fungus

 Ability to degrade a variety of polysaccharides (cellulose,

hemicelluloses) and related polymers such as chitin

 Ability to attack wood is particularly weak

 Obligate aerobes

 Oxygen supply regulate cellulase formation

 CULTURE CONDITIONS
 The most favourable pH ranges between 5.5 and 7.5

 Best grown at a temperature range of 25°C to 30°C

 Aeration by agitation: Greatest biomass was recorded at 300 rpm

 The best 6-PP production would be obtained when sucrose, potassium

dihydrogen phosphate, ammonium sulfate, sodium nitrate and ammonium
chloride concentrations were used at maximum levels (Ramos et al; 2008)

 Concentration of inoculum should be kept to minimum (Ramos et al; 2008)

(Singh et al; 2014)

Highest Producer

• The most efficient producer of 6-PAP on potato dextrose agar (PDA) was T.
atroviride AN35

• Grown at 20 degree Celsius

• 6-PAP was detected in the headspace of all T. atroviride and T. viridescens

isolates, as well as in individual cultures of T. citrinoviride (4 isolates), T.
hamatum (8 isolates), and T. viride (3 isolates).

(Henryk et al; 2014)

 Increase Yield

Upstream Fermenter
regulation

Through Genetic
Mutations Engineering

Screening
Method
 UPSTREAM REGULATION THROUGH GENETIC
ENGINEERING

The biosynthesis of 6PP, and indeed of all compounds in this group, is a matter for
conjecture.

• Incubation of growing cultures of Trichoderma harzianum and T. viride with

[U14C] linoleic acid revealed that both fungal strains were able to incorporate it
(50%)

• Results suggested that beta-oxidation of linoleic acid was a probable main step
in the biosynthetic pathway of 6-pentyl-a-pyrone in Trichoderma species.

(Serrano-Carreon et al., 1993)

  Linoleic acid was studied since this fatty acid is a natural component of
Trichoderma lipids and is supposed to be a precursor of different flavor
compounds.

 It confirms the involvement of lipid metabolism in the production of this

lactone.

 Lipoxygenase activity could be the limiting step in 6PP formation from

linoleic acid.

(Serrano-Carreon et al., 1993)

(Serrano-Carreon et al., 1993)
 CAN BE
DERIVED FROM
POLYKETIDE
PATHWAY

(K.SIVASITHAMPARAM and E.L.GHISALBERTI 1998)

GENE 1
 Heterotrimeric G proteins composed of α, β and γ subunits
are involved in transducing signals from transmembrane G
protein-coupled receptors to a variety of intracellular
targets

 G protein subunit Tga1 in Trichoderma is involved in the

production of 6-PP

 The deletion of the tag1 gene in T. atroviride resulted in

mutants with a reduced production of 6-PP and other
metabolites with a sesquiterpene structure
Reithner et al., 2005
 GENE 2
 tmk1 gene encodes a mitogen-activated protein kinase
(MAPK)

 Δtmk1 mutants exhibited overproduction of 6-pentyl-α-

pyrone and peptaibol antibiotics

 Quantitative analysis of 6-PP revealed 1.6-fold elevated

amounts secreted into PDA plates by the Δtmk1-12 mutant

 Therefore, a downregulation of 6-PP by an MAPK has been

observed
Reithner et al., 2007
GENE 3
 Transcription factor THCTF1 is involved in 6-PP production and in the
antifungal activity of T. harzianum

 THCTF1 might be a nuclear protein

 Disruption of the Thctf1 gene resulted in mutants that did not produce
two 6-PP derivatives and exerted a lower antimicrobial effect than the
wild-type strain

Rubio et al. (2009)

GENE 4
 Lipooxygenase gene Triat1: 33,350 unique to T. atroviride may be involved
in production of 6-PP

Kubicek et al., 2011

GENE 5
 For gpr1-silenced transformants reduced amount of
the antifungal secondary metabolite 6-pentyl-
alphapyrone (6-PP) was observed

 In T. atroviride silencing of the cAMP-receptor-like

class protein encoding gene gpr1, provoked slightly
reduced levels of intracellular cAMP and protein kinase
(PKA) activity

(Omann et al; 2012)

Dotted lines indicate
proposed effects
whereas solid lines
indicate confirmed
effects.
 IMPORTANCE OF ELICITATOR
 The production of secondary metabolites has been effectively increased by
the addition of fungal elicitors, such as mycelia, carbohydrate fragments,
proteins, oligosaccharides, or secretions derived from fungi in certain plant
cell cultures

 Nonviable R. solani is able to increase the accumulation of 6-pentyl-a-

pyrone (6PP) by T. harzianum

 6PP production by Trichoderma harzianum is elicitated by Rhizoctonia

solani mycelium as an antagonistic response that can be modulated by the
state of the host

(Serrano-Carreon et al; 2004)

 Elicitated cultures produced 6PP during the growth phase.

 6PP has been always considered as a secondary metabolite

 Elicitation not only increases the final product concentration but it also
induced a shift in the metabolism of Trichoderma harzianum.

 3 fold higher concentration can be achieved

 SUGGESTED APPROACH FOR GENETIC
ENGINEERING

 c-DNA library construction

 Subtractive hybridisation
 in silico study
 Find a pathway

 Proper Elicitator

But of all this requires time and may not be the best approach to
achieve the desired yield
 MUTATIONS
 In past it has been observed that random mutations has resulted in higher
yield (of about to 10 fold increase in case of Penicillin production)

 Need a better screening method to isolate the desired mutant

 Determination of 6PP using simple plate bioassay

 Antifungal activity can be evaluated by measuring the diameter of the

inhibition zone in the overlay disc containing testing organism
OVERLAY TECHNIQUE FOR SCREENING

Overlay disc
containing testing
fungus which will be
inhibited by 6PP
Mutant
Trichoderma
specie colony Agar plate
Why can’t achieve high yield ???
Several research has already been conducted for improving the yield but
there are several hurdles

 Inhibitory effect (at 400 mg/l)

 Transformation of the molecule

 Secondary metabolite

 Agitation
 FERMENTATION
 Maximum 6-PAP concentration increased by a factor of 2 to 40 mg/l with the
addition of 2%w/v iron (II, III) oxide (Etschmann et al; 2014)

 Culture morphology has a big impact on formation of 6-PAP (Etschmann et al; 2014)

 Microparticle-enhanced cultivation (MPEC) to tailor fungal morphology (Etschmann

et al; 2014)

 Use of microparticles enables free mycelium and precise engineering of

morphology (Etschmann et al; 2014)

 6-PP productivity is determined by hydrodynamic stress (Galindo et al; 2003)

 Growth-associated in the high volumetric power drawn cultures (above 0.25

kW/m3) (Galindo et al; 2003)
WAYS TO COUNTER INHIBITORY EFFECT
 Pervaporation

 Use of absorbents (use of hexadecane)

• Low polarity-high molecular weight solvents exhibit less microbial

toxicity(serrano et al; 2002)

 Two-phase extractive bioconversion (Shinobu et al; 2009)

• Polymeric micro-material, a ballooned

polyacrylonitrile microshpere (MS)

• Extractive organic solvent were MFL-80SDE (non-

coated type) and KF-96L-1CS (dimethyl silicone oil)
Can it work ???

Organic solvent 6PP

Fungus
immobilised
on the sponge

CONCEPT
OF AIR LIFT
REACTOR
References
• Metabolism of linoleic acid or mevalonate and 6-pentyl-α-pyrone biosynthesis by trichoderna species; L. Serrano-carreon, Y.
Hathout, M. Bensoussan, and J.-M. BELIN american society for microbiology; vol. 59, no. 9; p. 2945-2950; 1993
• Secondary metabolism in Trichoderma and Gliocladium; K.SIVASITHAMPARAM AND E.L.GHISALBERTI; Trichoderma and
Gliocladium–vol. 1; 1998
• Thctf1 transcription factor of Trichoderma harzianum is involved in 6-pentyl-2H-pyran-2-one production and antifungal
activity; M. Belén Rubio, Rosa Hermosa, José Luis Reino, Isidro G. Collado, Enrique Monte; Fungal Genetics and Biology;
46;17–27; 2009
• Formation of 6-n-pentyl-2H-pyran-2-one (6-PAP) and other volatiles by different Trichoderma species; Henryk Jeleń, Lidia
Błaszczyk, Jerzy Chełkowski, Katarzyna Rogowicz and Judyta Strakowska; Mycol Progress 13; 589–600; 2014
• Genetic engineering of filamentous fungi — Progress, obstacles and future trends; Vera Meyer; Biotechnology Advances 26;
177–185; 2008
• Formation of 6-n-pentyl-2H-pyran-2-one (6-PAP) and other volatiles by different Trichoderma species; Henryk Jeleń, Lidia
Błaszczyk, Jerzy Chełkowski, Katarzyna Rogowicz and Judyta Strakowska; Mycol Progres; 13; 589–600; 2014
• The G protein subunit Tga1 of Trichoderma atroviride is involved in chitinase formation and diVerential production of
antifungal metabolites; Barbara Reithner, Kurt Brunner, Rainer Schuhmacher; Fungal Genetics and Biology; 42; 749–760; 2005
• Signaling via the Trichoderma atroviride mitogen-activated protein kinase Tmk1 differentially affects mycoparasitism and
plant protection; Barbara Reithner, Rainer Schuhmacher, Norbert Stoppacher; Fungal Genetics and Biology 44; 1123–1133;
2007
• Production of 6-pentyl-a-pyrone with Trichoderma atroviride and its mutant in a novel extractive liquid-surface
immobilization (Ext-LSI) system; Shinobu Oda, Kunio Isshiki, Shinichi Ohashi; Process Biochemistry 44; 625–630; 2009
• Production of 6-pentyl-α-pyrone by trichoderma harzianum in solid-state fermentation; Aline de Souza Ramos, Sorele Batista
Fiaux, Selma Gomes Ferreira Leite; Brazilian Journal of Microbiology39; 712-717; 2008
• Study on the production of 6-pentyl-a-pyrone using two methods of fermentation; A. Kalyani, S. G. Prapulla and N. G.
Karanth; Appl Microbiol Biotechnol 53; 610-612; 2000
• Thctf1 transcription factor of Trichoderma harzianum is involved in 6-pentyl-2H-pyran-2-one production and
antifungal activity; M. Belén Rubio, Rosa Hermosa, José Luis Reino; Fungal Genetics and Biology; 46;17–27; 2009
• Rhizoctonia solani, an elicitor of 6-pentyl-α-pyrone production by Trichoderma harzianum in a two liquid phases,
extractive fermentation System; Leobardo Serrano-Carre´on, Celia Flores, Blanca Rodrıguez & Enrique Galindo;
Biotechnology Letters 26: 1403–1406, 2004
• The Elicitation Effect of Pathogenic Fungi on Trichodermin Production by Trichoderma brevicompactum; Xu-Ping
Shentu, Wei-Ping Liu, Xiao-Huan Zhan, Xiao-Ping Yu and Chuan-Xi Zhang; The ScientificWorld Journal; Volume 2013
• Biotechnology and biology of trichoderma; Vijai K. Gupta, Monika Schmoll, Alfredo Herrera-Estrella, R. S.
Upadhyay, Irina Druzhinina, Maria G. Tuohy
• Quantitative determination of the antifungal compound 6 pentyl pyrone using a simple plate bioassay; J.M.
Conney, J.L. Vanneste, R.A. Hill; Letters in applied Microbiology; 24; 47-50; 1997
• Optimal Physical Parameters for Growth of Trichoderma Species at Varying pH, Temperature and Agitation;
Anuradha Singh, Mohammad Shahid, Mukesh Srivastava, Sonika Pandey, Antima Sharma and Vipul Kumar; Virol
Mycol; 3:1; 2014
• Improving 2-phenylethanol and 6-pentyl-α-pyrone production with fungi by microparticle-enhanced cultivation
(MPEC); M. M. W. Etschmann, I. Huth, R. Walisko, J. Schuster, R. Krull, D Holtmann, C. Wittmann and J. Schrader;
Yeast; 2014
• Production of 6-pentyl--pyrone by Trichoderma harzianum cultured in unbaffled and baffled shake flasks; Enrique
Galindo, Celia Flores, Patricia Larralde-Corona, Gabriel Corkidi-Blanco; Biochemical Engineering Journal 18; 1–8;
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