Selective Isolation of Derepressed Mutants of an a-Amylase Yeast by

the Use of 2-Deoxyglucose

INTRODUCTION

The production of a-amylase by yeasts and other microorganisms may be subject

to carbon catabolite repression.'** The same applies to many other microbial enzymes
including some of industrial interest such as invertase3 and p-gl~cosidase.~ Carbon
catabolite repression may be affected by nonmetabolizable analogs of glucose; in
Saccharomyces cerevisiae glucosamine and 2-deoxyglucose had the same repressing
effect as glucose on the formation of several enzymes of the gluconeogenesis path-
way.5 Taking advantage of this, Zimmermann and %heel3 used a rafinose-deoxy-
glucose medium for the selective isolation of spontaneous mutants of S. cerevisiae
derepressed for invertase and maltase. Using a similar technique we obtained a
number of mutants of the yeast Lipomyces kononenkoae derepressed for the for-
mation of a-amylase.

MATERIALS AND METHODS

The wild strain of Lipomyces kononenkoae IGC 4052 was originally received from
the Yeast Division of the Centraalbureau voor Schimmelcultures. Delft. The Neth-
erlands, No. CBS 5608. This strain converts starch into single-cell protein (SCP)
with a high yield and produces an extracellular amylolytic enzyme system capable
of total starch hydr~lysis.~,'
The wild strain was grown with shaking at 25°C in the following liquid medium:
2% (wiv) glucose; I% (wiv) peptone (Difco); 0.5% (wiv) yeast extract (Difco); and
distilled water. In the mid-exponential phase the cells were harvested by centrifu-
gation and resuspended in distilled water. Of this suspension 0. I ml samples con-
taining about 7 x lo6 viable cells were spread onto the surface of plates of the
following medium: 1.5% (wiv) commercial corn starch (Maizena Knorr); 0.01% (wi
v) 2-deoxyglucose (Sigma); I % (wiv) peptone (Difco); 0.5% (wiv) yeast extract
(Difco); 2% (wiv) agar (Difco). and distilled water. The plates were irradiated with
ultraviolet light during a period of time long enough (40 sec under our conditions) to
reduce the viability of the inoculum to about 30%. Incubation was at 25°C in the
dark.
After one to two weeks most plates displayed irregular islands of surface growth
(rather than neatly isolated colonies) interspersed and surrounded by areas free of
visible growth where the medium had become transparent due to starch hydrolysis.
Cells were plated on the same selective medium and a number of strains were
obtained from these secondary plates by picking well-isolated, tiny (less than 1 mm)
colonies surrounded by large ( 10- 15 mm) clearing zones.
a-Amylase activity was estimated by the use of starch and iodine, and one unit is
defined as the quantity of protein mediating AE:SCom,,,, of 0.1 under the assay condi-
tions.'
To test for derepression and hyperproductivity the strains were grown in a liquid
mineral medium with vitamins (MV medium)8 to which carbohydrates were added
as indicated in the text.

Biotechnology and Bioengineering, Vol. XXII, Pp. 651-654 (1980)

@ 1980 John Wiley & Sons, Inc. 0006-3592/80/0022-0651$01.OO
652 BIOTECHNOLOGY AND BIOENGINEERING VOL. XXII (1980)

RESULTS AND DISCUSSION

The wild strain of L. kononenkoue IGC 4052 was unable to grow on the starch
medium containing 0.01% 2-deoxyglucose. This was thought to be due to catabolite
repression of a-amylase formation by the deoxyglucose rather than to growth inhi-
bition. Indeed, 0.01% deoxyglucose only slightly inhibited growth on the same
medium when glucose was substituted for starch, and mutagenic treatment with
ultraviolet irradiation gave rise to populations that were able to grow on deoxyglu-
cose-starch medium with the formation of clearing zones.
It was not possible to calculate the mutation frequency since neatly isolated
colonies with clearing zones did not appear on the primary plates and growth
consisted of irregular confluent areas probably composed of several mutant popu-
lations mixed with opportunistic wild cells. By secondary plating on the selective
deoxyglucose-starch medium, 32 strains were isolated from the 10 irradiated plates.
To test for derepression the strains were first grown overnight on 2% (w/v)
glucose-peptone-yeast extract-agar. From each culture a small amount was trans-
ferred to a 100 ml Erlenmeyer flask containing 50 ml MV medium with 2% (wiv)
glucose and 0.4% (wiv) soluble starch (Difco). Incubation was at 25°C with shaking.
After 30 hr, while the glucose concentration was still high (about 1.7% in the culture
of the wild strain), the cultures were centrifuged and the supernatants tested for a-
amylase activity and residual starch (as blue color with iodine). In cultures of the
wild strain and four of the isolates the starch had not detectably decreased and a-
amylase activity was absent. In the cultures of 13 isolates some starch was still
detectable and the a-amylase activity was low, ranging from 1 to 4 unitdml. How-
ever, in the cultures of I5 strains the starch had disappeared from the medium and
a-amylase activity was much higher ranging from 5 to 17 unitshl. These 15 strains
were considered to be mutants with derepressed behavior.
To test for hyperproductivity five of the more active mutant strains and the wild
strain were grown in MV medium containing 0.5% (w/v) soluble starch (Difco). After
20.30, and 50 hr of growth a-amylase was assayed in the supernatants of centrifuged
samples. As is shown in Table I the activity was highest after 30 hr and the mutants
produced up to 3.7 times the amount of a-amylase produced by the wild strain.
Mutant strain IGC 4052 B and the wild strain were grown at 2 5 T , with aeration

TABLE I
Production of Extracellular a-Amylase (units/ml) in
MV Medium with 0.5% Soluble Starch by
Derepressed Mutants and the Wild Strain of L.
kononenkoue IGC 4052
Time of harvest (hr)
Strains 20 30 50
Wild strain 4052 4.8 7.8 2.4
Mutant strains
4052-A 11.9 19.9 6.3
4052-8 11.7 27.6 8.2
4052-D 11.0 18.5 3.5
4052-c 10.7 11.6 4.6
4052-d 10.2 10.8 4.2
 COMMUNICATIONS TO THE EDITOR 653

TIME (h)

Fig. 1. Growth of L . kononenkoae in liquid peptone-yeast extract medium with

glucose (0.2%) and soluble starch (0.4%). (A) Concentration of starch measured as
blue color with iodine; (0) growth measured as optical density at 640 nm; (0)a-
amylase in unitdml. (a) Wild strain IGC 4052; (b) mutant strain IGC 4052B.

and stirring, in 500 ml Erlenmeyer flasks containing 200 ml MV medium with 0.2%
glucose and 0.4% soluble starch (Difco). As is shown in Figure I the wild strain
displayed a diauxic growth curve, the starch remaining intact and a-amylase re-
pressed while the population was consuming the glucose. Growth of the mutant
strain, however, was no longer diauxic, a-amylase no longer repressed, and starch
disappeared rapidly from the medium in the presence of glucose.
When deoxyglucose is used to select for derepressed mutants, strains may arise
with derepressed behavior without being truly repression r e ~ i s t a n tIn. ~ such strains
defects in the phosphorylation of glucose and analogs may cause simulation of
repression r e s i s t a n ~ e . ~ *This
~ * ’ may
~ have practical importance depending on the
industrial use one has in mind for the microorganism. While both types of mutants
produce enzyme in a derepressed way, the mutant type with defects in glucose
phosphorylation may have a specific growth rate in glucose medium lower than that
of the wild type. We have not yet tested our mutant collection with respect to the
specific growth rate on glucose.
The use of deoxyglucose permitted us to selectively isolate a relatively large
number of derepressed and hyperproductive mutant strains with a relatively small
investment of time. work, and material. Our results with an a-amylase yeast. in
addition to the results obtained earlier with an invertase yeast.3 raise hopes that
654 BIOTECHNOLOGY AND BIOENGINEERING VOL. XXII (1980)

nonmetabolizable repressors may become generally useful in the selective isolation

of microbial mutants derepressed and hyperproductive with respect to repressible
enzymes of industrial interest.

References
1. G. Moulin and P. Galzy, Z. ANg. Mikrobiol., 18, 329 (1978).
2. P. Kaiser, Ann. Inst. Pasteur, 69, 153 (1971).
3. F. K. Zimmermann and I. Scheel, Mol. G e n . Genet., 154, 75 (1977).
4. M. H. Smith and M. H. Gold, Appl. Environ. Microbio/., 37, 938 (1979).
5. I. Witt, R. Kronau, and H. Holzer, Biochirn. Biophys. A c t a , 118, 522 (1966).
6. I. Spencer-Martins and N. van Uden, Eur. J . Appl. Microbiol., 4, 29 (1977).
7. I. Spencer-Martins and N. van Uden, Eur. J. Appl. Microbiol. Biotechnol., 6,
241 (1979).
8 . N. van Uden, Arch. Microbiol., 58, 155 (1967).
9. K. D. Entian, F. K. Zimmermann, and I. Scheel, Mol. G e n . Genet., 156, 99
( 1977).
10. Z. Lob0 and P. K. Maitra, Mol. G e n . G e n e t . , 157, 297 (1977).

N. VAN UDEN.
C. CABECA-SILVA
A. MADEIRA-LOPES
I. SPENCER-MARTINS

Laboratory of Microbiology
Gulbenkian Institute of Science
Oeiras, Portugal

Accepted for Publication September 24, 1979