Biotechnol. Appl. Biochem.
(2003) 37, 145–147 (Printed in Great Britain)
Enhanced production of verbenol, a highly valued food
flavourant, by an intergeneric fusant strain of Aspergillus niger
and Penicillium digitatum
Smitha C. V. Rao, Rati Rao and Renu Agrawal1
Department of Food Microbiology, Central Food Technological Research Institute, Mysore, India
An intergeneric hybrid, obtained from Aspergillus niger
and Penicillium digitatum, biotransformed (k)-cis-
α-pinene (5 mg/25 ml) into (k)-cis-verbenol (60 % ;
1.08 mg/g of biomass) in 6 h compared with 0.18 mg/g
of biomass for verbenol (10–15 % ; 0.18–0.27 mg/g of
biomass) in the initial parent cultures.
Introduction
Verbenol is a highly valued food-flavouring compound that is
widely used in soft drinks, soups, meats, sausages and ice
cream. Earlier work [1] with UV auxotrophs has shown an
enhanced yield of verbenol to 0.45 mg\g of biomass.
Recently, as there is now a great interest in natural flavours,
biotransformation of low-cost substrates has become im-
portant [2]. Biotransformation reactions are known in the
literature [3,4]. However, for commercial application higher
yields of the product are desirable to make processes
effective, efficient and economical.
Protoplast fusion is a well-known technique for inte-
grating advantageous traits. This has been well documented
in fungal cultures [5]. The transfer of genes by protoplast
fusion has been demonstrated as an efficient method for
improving industrially important micro-organisms.
In order to further enhance the yield of verbenol and to
make the process industrially viable, the present study on the
protoplast fusion of species from two different genera,
Aspergillus niger and Penicillium digitatum, was undertaken.
With the best traits of each parent culture strain, one
(A. niger) showing high yields of (k)-cis-verbenol and the
other (P. digitatum) with producing a high biomass (over
20 h), an improved fusant strain was produced that is a
hyper-performer.
Materials and methods
Protoplasts were isolated in citrate\phosphate buffer using
lysing enzymes and regenerated on minimal medium con-
taining K2HPO4 (6.3 g), KH2PO4 (1.82 g), (NH4)2NO3 (1 g),
MgSO4 (100 mg), CaCl2 : 2H2O (100 mg), FeSO4 : 7H2O
(100 mg), MnSO4 (0.6 mg) and Na2MoO4 (0.6 mg) per litre
with 2 % agar. Solvents, enzymes (cellulase, glucuronidase
145
and lysing enzymes) were obtained from Sigma (St. Louis,
MO, U.S.A.). The substrate (k)-cis-α-pinene (99 % pure) and
(k)-cis-verbenol were from the Aldrich Chemical Co.
(Milwaukee, WI, U.S.A.).
The protoplasts of the two genera were mixed in equal
numbers (106 cells\ml) and fused in the presence of poly-
(ethylene glycol) by the method of Kirimura et al. [6]. On
regeneration the fusant was characterized both morpholo-
gically and by studying the ploidy on haploidization by
benomyl treatment. The nuclei in the conidia were counted
after Geimsa staining and the structure of the fruiting body
was studied microscopically.
For biotransformation studies, the fusant was grown in
potato dextrose broth for 20 h in its exponential phase. A
biomass of 3 g\25 ml was taken in 0.05 M phosphate buffer
(pH 7.0), where it was incubated with (k)-cis-α-pinene
(5 mg\25 ml). At the end of the incubation period the
reaction mixture was extracted, with dichloromethane
(6 ml\25 ml) added twice for complete extraction. This was
dried over anhydrous Na2SO4. The extract was concen-
trated under nitrogen and an aliquot was taken for analysis.
The product was analysed by GC using a Shimadzu SE-30
column (3 m) with N2 at 30 ml\min with a temperature
increasing from 60 to 160 mC at 2 mC\min. The sample was
then characterized by GC-MS, using an SE-30 column (30 m)
with He at 1 ml\min (40 kPa). The product was calculated
on the basis of (k)-cis-verbenol formed against the amount
of total (k)-cis-α-pinene added to the reaction.
Results and discussion
Protoplasts were prepared for A. niger and P. digitatum using
a mycelial mat (40 mg\ml). Cell walls were digested using an
enzyme combination of cellulase, glucuronidase and lysing en-
zyme in a ratio of 1 : 1 : 1 and 1 : 2 : 1, respectively, in
Key words : α-pinene, biotransformation, food flavourant.
1
To whom correspondence should be addressed (e-mail
renuagrawal46!rediffmail.com).
# 2003 Portland Press Ltd
146 S. C. V. Rao, R. Rao and R. Agrawal

Table 1 Morphological characteristics and biotransformation efficiency of (10 % ; 0.18 mg\g of biomass) and P. digitatum (15 % ;
α-pinene in parent and fusant cultures of A. niger and P. digitatum
0.27 mg\g of biomass). The α-pinene used in the present
PDA, potato dextrose agar ; YMGA, yeast extract, malt extract, glucose and work was 99 % pure and had an enantiospecificity of (1s)-
agar.
(k)-α-pinene with an optical rotation of 50.70m. The
Recombinant verbenol produced was enantiospecifically (S)-cis-verbenol
Characteristic A. niger P. digitatum fusant with an optical rotation of 9.0m and was 95 % pure. It was
Medium PDA YMGA PDA observed that incubating for longer periods or increasing the
Spore colour Black Grey Blackish-grey concentration of α-pinene did not enhance the conversion
Spore size ( µm) 3.7 5.6 3.9
Colony size (diameter in cm) 1.45 1.74 0.9 yield. Rather, a decrease in yield was seen, probably due to
Optimum growth temperature 30 37 35 substrate toxicity or, with longer incubation periods, other
(mC) metabolites were formed.
Sporulation time (h) 48 52 52
Citrate/phosphate buffer molarity 0.05 0.1 – The present work has brought about the formation of
(M) for protoplasting a stable recombinant fusant from two different filamentous
Enzyme combination ratio 1:1:1 1:2:1 – fungal genera which is a hyper-performer compared with
(cellulase/glucoronidase/lysing)
Biomass growth at 20 h (g/25 ml) 2.0 3.0 3.5 the parent strains. The efficiency of conversion has been
Verbenol conversion of α-pinene 15 10 60 maintained over the last 2 years and the improved fusant
(%) strain has been found to be stable. This enhanced product
Verbenol (mg/g biomass) 0.27 0.18 1.08
yield for (k)-cis-verbenol through microbial conversion of
(k)-cis-α-pinene is reported here for the first time. Inter-
citrate\phosphate buffer (0.05 and 0.1 M, respectively ; generic hybridization of A. niger and Trichoderma viride by
pH 5.8) with 0.7 M KCl as an osmotic stabilizer. The two protoplast fusion has been reported in the past [6]. Intra-
protoplasts were fused in a medium containing 20 % poly- and inter-strain crossing were also discussed previously in A.
(ethylene glycol) 4000. The fusion was conducted at 30 mC niger [7], as has biolistic nuclear transformation in S. cerevisiae
for 10 min. A regeneration frequency of 1.5 % was obtained. [8]. Intergeneric protoplast fusion between Monascus anka
The recombinant fusant was selected after haploidization and A. oryzae [9,10] was achieved in a study of strain
with benomyl. Although 4 % of the colonies were hetero- improvement by inter-specific protoplast fusion. In the
karyons, 96 % of the colonies obtained were recombinants. present work the fusant culture strain has been found to be
All the recombinant colonies were characterized, stable in terms of the product yield for subsequent genera-
screened and then studied for their biotransformation tions. On characterization, the fusant strain was found to
efficiency. One of the fusant colonies produced 6-fold more have a mix of characters from both parents. The fusant is a
verbenol (60 % conversion efficiency) with higher biomass hyper-performer converting 60 % of α-pinene added to
(3.5 g\25 ml) than the two parent strains (Table 1). This 100 % pure (k)-cis-verbenol.
fusant mycelium was more hyaline and white in colour. The
fruiting body was smaller in size and the structure was like
that of A. niger when observed under the microscope,
whereas the spores were blackish-grey in colour, which is a Acknowledgments
mixture of the two parental phenotypes, black for A. niger
and light grey for P. digitatum (Table 1). On staining the We thank Dr V. Prakash, Director of the Central Food
conidium, only a single nucleus was found to be present. Technological Research Institute, Mysore, India, for his
The temperature for growth was very critical (35 mC). When continuous support, and Dr M. S. Prasad, Head of the Food
a mixture of the protoplasts of the two genera were plated Microbiology Department, for his encouragement during
on minimal medium without poly(ethylene glycol) no fusant this work. We acknowledge the Department of Biotech-
colonies were developed. Mixtures of mycelial fragments of nology, Government of India, New Delhi, India, for funding
A. niger and P. digitatum were plated on minimal medium in the work. S. R. C. V. thanks the Department of Biotechnology
the presence of poly(ethylene glycol) but no viable colony for providing her project assistance during the work.
was seen. We have successfully produced an intergeneric
fusant by protoplast fusion.
The intergeneric fusant of A. niger and P. digitatum had a References
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Received 11 June 2002\7 November 2002; accepted 13 November 2002
# 2003 Portland Press Ltd