ISSN 10227954, Russian Journal of Genetics, 2012, Vol. 48, No. 8, pp. 778–784. © Pleiades Publishing, Inc., 2012.
Original Russian Text © M.V. Dumina, A.A. Zhgun, A.G. Domracheva, M.I. Novak, M.A. El’darov, 2012, published in Genetika, 2012, Vol. 48, No. 8, pp. 918–925.
Intraspecies chromosomal polymorphism is a
common occurence in many species of yeasts and
fungi [1, 2]. A specific form of mutageninduced chro
mosomal polymorphism is typical for industrial fungal
strains. It is associated with amplification of genes
responsible for biosynthesis of specific target biologi
cally active metabolite . For instance, it is known
that in industrial
copy number of penicillin biosynthesis genes is
increased more than hundredfold .
At the same time the data on association of the
chromosomal rearrangements with the levels of CPC
production by the filamentous fungus
are contradictory. A negligible increase in the copy
numbers of CPC biosynthetic genes was observed in
several industrial strains [4, 5]. Translocations of
“early” CPC biosynthesis genes were detected in the
number of industrial
strains, but no
correlation with the levels of CPC production was
Previously using multisptep chemical mutagene
sisselection strategy we have obtained CPCoverpro
strain CB26/8 capable of mak
ing CPC at levels about 30 g/L . The most impor
tant industrial characteristics of this strain are (1) a full
prototroph and secondary prototroph related to amino
acid methionine, whose biosynthesis is often sup
pressed concomitantly during
producer strain generation and (2) a low level of the
intermediate metabolic forms of CPC (deacetyl and
This strain has some distinctive physiological and
morphological traits compared to the wild type strain,
such as a decrease in growth rate, reduction of air
mycelium and conidium formation, specific changes
in morphology and cell wall proteins , the
amounts of various fractions of inorganic polyphos
phates . In the present study, we investigate chro
mosomal polymorphism in this strain and potential
chromosomal rearrangements affecting the already
known genes of CPC synthesis and transport.
MATERIALS AND METHODS
Strains and culture conditions.
ATCC 11550 (wild type) and its derivative, a
CB26/8 were used in
the study. Strains
YPH857 were used as
markers of molecular mass in pulse electrophoresis.
strains were grown in a shaker at
200 rpm in 50 mL of liquid medium containing 30 g/L
sucrose, 15 g/L meat extract, and 5 g/L corn steep
liquor during 24–44 h at 28°C.
972h were grown in 50 mL of YPD
medium using the same conditions.
A. chrysogenum protoplast isolation.
For this pur
pose, mycelium was filtered, washed in 5 volumes of
0.9% NaCl, and preincubated in 50 mM citratephos
phate buffer, pH 7.6 (containing 10 mM DTT and
20 mM MgSO
). DTT was washed out from treated
mycelium by five volumes of 0.9% NaCl and lytic
enzyme cocktail: Zymolase 20T (ICM, United
States), 3 mg/mL and Lytic enzyme LI (MP Biomed
Chromosomal Polymorphism of
Strains Producing Cephalosporin C
M. V. Dumina, A. A. Zhgun, A. G. Domracheva, M. I. Novak, and M. A. El’darov
Bioengineering Center, Russian Academy of Sciences, Moscow, 117312 Russia
Received October 18, 2011
—Using pulse electrophoresis in controlled homogenous electric field we performed molecular
karyotyping of cephalosporin Cproducing industrial and laboratory strains of
Differences in size of several chromosomes of highproducing strain CB26/8 compared to the wildtype
strain ATCC 11550 were revealed. It was shown that chromosomal polymorphism in the highproducing
strain was not associated with alteration of localization and copy number of cephalosporin C (CPC) biosynthe
sis and transport genes. A cluster of “early” CPC biosynthesis genes is located on chromosome VI (4.4 Mb); a
cluster of the “late genes”, on chromosome II (2.3 Mb). Both clusters are presented as a single copy per
genome in the wildtype and in CB26/8 highproducing strains. Based on comparative anal
ysis of laboratory and industrial CPC producers, a karyotype scheme for
strains of various ori
gins was designed.
GENETICS OF MICROORGANISMS