TIBTECH - OCTOBER 1987 [Vol.

5]

EI BDDDB//
Enzymatic lysis and
disruption of microbial cells
B. A. Andrews and J. A. Asenjo
The development of expression systems for large recombinant
proteins which cannot be secreted by the host microbial cells
necessitates the development of novel techniques for cell disruption.
The use of enzyme systems which provide biological specificity to the
process of cell lysis and disruption shows tremendous promise as a
method of controlled lysis and selective product release.
Enzymatic lysis and disruption of
microbial cells has found a number
of important applications (Fig. 1).
These include the production of
specialty wall polysaccharides 1, production of intracellular, membrane
bound or wall enzymes 2, production
of yeast autolysates and products
from subcellular structures 3 and the
recovery of recombinant DNA products such as surface antigen particles manufactured in yeast 4.
At present, the only industrial
scale microbial cell breakage equipment is the high pressure homogenizer: other equipment, such as bead
mills are used mainly at bench and
pilot plant scale. However, these
mechanical methods have drawbacks. They have no biological
specificity, they generate high temperatures in the cell suspensions
which impose great demands on
cooling equipment, they generate
high shear stresses which can harm
the molecules to be recovered, and
they necessitate a large capital investment in specialized equipment.
Enzymes have none of these disadvantages. They can be used on
their own for the release of intracellular soluble proteins, particulate
inclusions and for wall and membrane associated materials. Or they
can be used in conjunction with

B. A. Andrewsand J. A. Asenjo are at the

Biochemical Engineering Laboratory,
University of Reading, PO Box 226,
Reading RG6 2AP, UK.

mechanical disruption to increase

the selectivity of product release, to
increase the rate and yield of
extraction, and to minimize product
damage. The industrial use of enzymes for the release of specific cell
proteins is coming of age with the
development of expression systems
for large recombinant proteins that
cannot be secreted by the cell. By
choosing an appropriate enzyme
system, selective and sequential
product release may be obtained.
This review summarizes the latest
developments and trends in the
production and utilization of enzyme
systems which lyse microbial cells.

The substrates for lysis- the cell

walls
The cell walls of yeast and bacteria
are distinctly different, hence, in
general, lytic systems are specific
for particular groups of microorganisms. Yeast cell walls (Fig. 2) have

two main layers, an outer layer

of protein-mannan complex and
an inner glucan layer 5. Enzyme
systems for yeast cell lysis are, therefore, usually a mixture of several
different enzymes which include
one or more [3(1-3)glucanase, protease, [5(1-6)glucanase, mannanase
or chitinase. They act synergistically
in the lysis of the cell wall but only
two are essential for whole cell
breakage; a specific wall-lytic protease to degrade the outer layer of
protein-mannan and a lytic [~(13)glucanase to degrade the inner
glucan layer.
In both Gram-positive bacteria and
Gram-negative bacteria, a peptidoglycan component is responsible for
the strength of the wall. In Grampositive bacteria peptidoglycans are
the major wall polymers and are
associated with teichoic acids and
polysaccharides. Gram-negative bacteria have a two layer wall structure:
the inner, rigid peptidoglycan component is covered by an outer layer or
outer membrane composed of proteins, phospholpids, lipoproteins
and lipopolysaccharides 6. Consequently, a single enzyme can lyse
Gram-positive bacteria but pretreatment with a detergent (e.g. Triton
X-100) is usually necessary to remove
the outer membrane of Gramnegative cells. Three types of bacteriolytic enzyme have been isolated;
glycosidases which split polysaccharide chains, acetylmuramyl-Lalanineamidases which cleave the
junction between polysaccharides
and peptides; and endopeptidases
which split polypeptide chains.

Sources and properties of cell lyric

enzymes
A large number of microorganisms

-Fig. 1

Recombinant proteins
Antibiotics

Lysed cell

Specialized lipids
Enzyme attack

Wall polysaccharides
Pigments
Enzymes

Productsthatcanbeisolated
~ ~'~-'~
after enzymatic lysis of microbial cells.

Intracellular polymers

~) 1987, Elsevier Publications, Cambridge 0166- 943087/$02.00

TIBTECH --Fig. 2
Opening in
mannoprotein layer

Openinginglucanlayer

Exposed
glucanlayer
have been found to produce microbial lytic enzyme systems 7-25 (Table
1). These organisms exhibit predatory activity against yeast and other
microbial cells and have been isolated from such diverse habitats as
decaying plant material (e.g. rice7),
brewery effluents 8, sewage plants 9,
estuaries 9, soil 1 and the human
mouth 11.
Virtually all microorganisms used
for production of lytic enzymes are
safe (class 1 classification in ATCC)
and non-pathogenic. The only strains
that do not fall into this category are
some strains of Staphylococcus
(class 2 in ATCC) and they are
considered only mildly pathogenic.
Conditions for use of microbial
cell-lytic enzyme systems have been
investigated by different authors 12.
The pH and temperature optima vary
considerably in the range pH 6-11
and 35-60C (Table 1).
For all the lytic systems, there is
considerable variation between the
molecular weights of the different
systems and their component enzymes. However, most are relatively
small (in the range of 10 to 30 kDa), a
property which is important in that it
makes them relatively easy to separate from large intracellular proteins
after cell lysis.
There is little information available on the effect of enzyme and
substrate concentration on microbial
cell-lyric enzymes. However, one
study 28 showed that the rate of protein release from whole yeast cells in
the presence of lytic enzymes was a
linear function of enzyme concentration and was used with substrate
concentrations up to 110 gl -~ dry
weight of yeast cells.

Lysis of yeast
The yeast-lytic enzymes produced
by different Arthrobacter sp. have
been extensively studied in batch
culture and found to be inducible (by
whole yeast cells and cell walls) and
subject to catabolite repression by
g l u c o s e 1 5 ' 2 6 ' 2 7 : [~(1-3)glucanase, protease and mannanase activities have
been detected. The cell wall degrading enzymes from different strains of
Oerskovia xanthineolytica have also
*Jeffries, T. W. (1976)PhD Dissertation,
Rutgers University, New Jersey, USA.

wall
ofOuter
yeast cell

OCTOBER1987[Vol.5]

f ~

" ~

Lysing yeast cell showing wall structure.

been purified and characterized 13'*.

Jeffries* found four different fi(1-3)glucanases with distinct action patterns in batch culture with autolysed
yeast cells as inducer: Scott and
Schekman z3, using a different strain,
found two synergistic activities in
batch culture with yeast glucan as the
carbon
source;
an
endo-lytic
glucanase and an alkaline protease.
In general, in the enzyme systems
which lyse yeast, the optimum pH
values for the constituent enzymes
are markedly different, for glucanase
it is usually neutral whereas for
protease it is alkaline in most cases.

Lysis of bacteria
Bacteriolytic enzymes tend to have
pH optima around 6 or 7. Optimum
temperature ranges from 30 to 60C
with most between 35 and 40C. At
temperatures 5-10C above optimum
temperature, the stability of the
enzyme is usually low and the rate of
denaturation is high. Most of the
enzymes are active, not on whole,
live cells but on pretreated cells (e.g.
lyophilised or heat killed) or on
isolated cell walls. Many enzymes
are specific for Gram-positive bacteria and few are active on Gramnegative cells. However, the lytic
protease of Micromonospora was
active against lyophilised cells
of Serratia marcescens, Pseudomonas aeruginosa, E. coli and Bacillus subtilis 23 and the enzyme produced by Streptomyces coelicolor
lyses cells of both Gram-positive and
Gram-negative bacteria 25. The lytic
protease from Bacillus subtilis can
lyse cells of E. coli apparently without the need for pretreatment 24. The
enzyme system from Cytophaga sp.
has been used for lysis of E. coli cells
in the presence of detergent (unpublished results from the authors'
laboratory).

Plasmamembrane
surface

(TakenfromHunterandAsenjo5.)

Methods of production
The production of bacteriolytic
enzymes has been studied mainly for
possible use in food preservation and
investigation of bacterial cell wall
structures. Almost all of the work has
been done in batch culture and early
studies indicated that enzyme synthesis was non-inducible 22'29. However, in these studies a complex
nitrogen and carbon source was used
(bean cake extract), a component of
which might have induced bacteriolyric activity.
The production of enzymes able to
lyse whole yeast cells has been
studied in batch and continuous
culture. The synthesis of the lyric
enzymes of Cytophaga 9497 in batch
culture appeared to be constitutive 3.
Recent work on the regulation of cell
lytic enzyme synthesis in Cytophaga
NCIB 9497 and Oerskovia xanthineoIflica included both batch and
continuous culture studies 19'*. For
both strains, the synthesis of lytic
enzyme systems is inducible and
subject to catabolite repression by
glucose. At low dilution rates in
carbon limited continuous culture,
high ~(1-3)glucanase activities and a
high glucanase/protease ratio are
obtained in both strains, at high
dilution rates all enzyme activities
are similar to batch values. For both
systems, continuous culture provides a several fold increase in
enzyme concentration and productivity over batch culture. This has
meant that a process for the production of protein using yeast lyric
enzymes can be designed in which an
enzyme production fermenter of only
1.5-2 m 3 is required to lyse the yeast
cells produced in a 100 m 3 continuous culture fermenter. The medium required for enzyme production

~Andrews, B. A. (1985) PhD Thesis,

University of London, UK.

TIBTECH- OCTOBER1987 [Vol. 5]

--Table 1
M i c r o b i a l lytic enzyme systems

Source

Optimum
Optimum temperature
pH
(C)
Substrate

Oerskovia xanthineolytica/
Arthrobacter luteus
A rth ro bacter GJ M - 1
(Zymolyase/Lyticase)
g lucanase
protease (alkaline)
whole yeast cell activity
Oerskovia CK
glucanase with
proteolytic activity
whole yeast cell activity
Rhizoctonia sp.

glucanase
protease
whole yeast cell activity
Cytophaga NCIB 9497

Ref.

Saccharomyces, 13-15
Candida,
Hansenula,
Pichia and
other yeasts

5-6.5
9-10
7.5

45-50
35
30-35

S. cerevisiae

16

9.0

35-40
60
35-40

5.5
6.5
6.0
9.0

55-60
40
40
45-55

Candida,

18

Saccharomyces,
Hansenula
S. cerevisiae,

19

Bacillus,
Corynebacteria,
E. coli

Lysozyme
(hen egg-white)

6-7

35

E. coli
M. lysodecticus
and other

9.5

50

Staphilococcus

6-7

37

21

6.5

50

M~rococcus
luteus
M. lysodecficus

bacteria
Cytophaga B-30
(Lysopeptase)
Staphylococcus sp.
Streptomyces globisporius
(N-acetylmuramidase)
(Mutanolysin)
Micromonospora sp. Nr. 152
(lytic protease)

Bacillus subtilis 797

(lytic protease)

22

Streptococcus
11

7.8-8.5

60

E. coil

23

30

S. marcescens
P. aeroginosa
B. subtilis
E. coil

24

aMiles Technical Information (1984)lysopeptidase.

in this process is only 0.25% of the

medium necessary for yeast production 31. Cloning the genes for the lytic
enzymes could result in a several fold
increase in activity of the enzyme
systems in the producer strains.
H o w are lyric enzymes used?
The cells to be lysed are usually
harvested from the fermenter by
centrifugation,
ultrafiltration
or
microporous filtration. They are
mixed in a lysis reactor with the lytic
enzymes and buffer. Typically, the
enzyme will be added as a crude
supernatant at a concentration of 3 30% v/v (which corresponds to 0.121.2 g protein 1-1 in the enzyme
reaction) or as low as 0.4% v/v if the
enzyme is produced in continuous
culture 15. These values are for the

lysis of B. subtilis cells at a concentration of 9-13 g 1-1 using the enzymes produced by Cytophaga sp.
With yeast cells, concentrations of
up to 110 g 1-1 dry weight have been
used in disruption reactors. With
lysozyme it was estimated that using
4000-5000 U 1-1, good protein extraction could be obtained from
bacterial cells in one hour. Commercial enzyme preparations contain
14 000-22 000 U g-1.
The only lytic enzyme available on
a commercial scale for the industrial
disruption of cells is lysozyme
(active only on bacterial cells and
specific activity as stated above);
other bacteriolytic and yeast lytic
enzymes (e.g. Zymolyase) are only
available as laboratory reagents, so
with present data it is not possible to

make an accurate cost comparison

with non-enzymic breakage methods
on an industrial scale.
The enzyme system from Cytophaga sp. has been efficiently used
for the lysis of bacterial cells. The
activity of the Cytophaga sp. system
was approximately one order of
magnitude higher than that of other
bacteriolytic strains. Process design
calculations similar to those carried
out for yeast cell lysis have shown
that the enzyme production fermenter w o u l d be 1.5-2% of the
volume of the cell production vessel
in batch enzyme production and
if continuous culture is used, only
0.4-0.5%; in other words, a 42 1
fermenter could produce sufficient
enzyme for a cell production unit of
10 m 3 (Ref. 20).
Products of lysis
Ideally, the cell debris would be
particulate and the protein product to
be recovered would be larger than the
lytic enzyme protein. Then cell
debris separation could be achieved
by using a centrifuge or microporous
membrane filter and product purification and separation from the lytic
enzyme would be achieved by a
'classic' chromatography sequence
(e.g. ion exchange followed by gel
filtration). Some lytic enzymes have a
strong affinity for yeast glucan and
wall debris 2a, which w o u l d allow a
substantial fraction of the lytic
enzymes to be separated along with
the cell debris.
Process design
Tailoring enzyme systems for a
particular use and manipulating
process conditions introduces a considerable degree of specificity to
cell disruption and product release.
Since the pH and temperature optima
of the crucial enzymes needed for cell
breakage can be very different, pH or
temperature changes could provide,
for example, high protease activity in
the early part of the lysis reaction and
high glucanase and low protease
activities at a later stage 32. Protease
inhibitors (e.g. mannan) could also be
used to control the process35~ Eventually, it might be possible to
improve control by using genetic
manipulation to develop an organism
to produc e its own inducible lytic

TIBTECH- OCTOBER1987[Vol. 5]
- Fig. 3
1

enzyme system: this option is being

explored.
Yeast cells have a double layer wall
and by choosing an enzyme system
with a higher glucanase or higher
lytic protease or a combination
of different glucanases (producing
glucan oligomers of different sizes),
the rate of release of final product and
byproducts as well as its quality and
composition can be engineered. Examples of this are the release of
invertase from cell walls, the production of protein-flee glucan and
the production of glucan oligomers
with pre-specified characteristics 12.
If the enzymes of the yeast lytic
systems are purified it should be
possible to first treat cells with lytic
protease then remove or inactivate
the protease and degrade the wall
with lytic glucanase 12. In osmotic
solution the intracellular osmotic
pressure would not break the periplasmic membrane thus allowing the
degradation and depolymerization of
glucan only. Gentle agitation, or
another means of protoplast breakage, would then allow release of
intracellular material (Fig. 3) 12. It has
been found that in most cases wall
lytic proteases are very specific with
little or no activity on intracellular
proteins 33.

Applications
Bacteriolytic enzymes are already
used commercially on a large scale
for the release of intracellular and
membrane bound enzymes and antibiotics 12. Some of the applications of
microbial lytic enzyme systems are

Lytic
enzymes

~-'~
,~,Cellsr~
,~,
I-erm' ~
/~ ~ ~ 1

Organelle
product
Protein particle

Ex.Prod.

Wall
enzymes

Cytoplasmic
enzymes

Process for the lysis of microbial cells including sequential disruption for
selective product release. A, wall lysis in osmotic support; B, protoplast
disruption; C, lysis of organelles or protein particles. RM, raw materials; I,
inoculu[;n; Ferm, fermenter; Ex. Prod., extracellular product; E, enzyme of
reagent used to lyse organelle or renaturation of protein. (Taken from Asenjo
and Andrews12).

shown in Table 2. These include a

several fold increase in yield in
preparation of a soluble glucan
polysaccharide (12-15 fold with
bakers yeast) 1, the extraction of
lipids a4 and the extraction of human
serum albumin made by genetically
engineered yeast cells 33. Other important applications of cell lytic
enzyme systems are cell killing for
microbial containment and the selective lysis of mixed microbial populations which cannot be achieved by
mechanical means.

Conclusions
Enzymatic methods of cell lysis
can be highly specific in terms of the
microorganism lysed and the product
released. These two variables will
determine the activity profile of a
lytic enzyme system to be used in a
particular application. Mechanical
techniques for cell disruption are
effective but highly non specific. The
concept of a biochemical cell refinery
where enzymatic, physico-chemical
and/or genetic techniques are used to

--Table 2

Present and potential applications o f microbial lyric enzymes

Ref.

Preparation of protoplasts, cell fusion and transformation of yeast

Production of intracellular enzymes
Pretreatment to increase yeast digestibility
Preparation of soluble glucan polysaccharide
Alkali extraction of yeast protein
Pretreatment for Dyno-mill mechanical breakage of cells
Extraction of specialized lipids from yeast
Production of yeast extracts
Food preservation
Extraction of pigments from red yeast
Release of recombinant proteins e.g. human serum albumin
Ethanol recovery from spent brewers yeast a
Lysis of caries inducing microorganisms
aKrauss (1985) pers. commun.

r ~ - [ ,~,
r~
,~,
~ ~ , 1 . ) - ~ 1 ~-~ ~

26
2
18
1
18
18
34
18
35
17
33
36

selectively release products from the

cell is important.
It appears that continuous culture
is highly advantageous for the production of lytic enzyme systems both
to overcome catabolite repression
and to design systems with desired
enzyme composition: enzyme concentrations can be increased many
fold and the profile of enzyme
components can be manipulated.
Lytic systems with extremely high
activities can thus be obtained making large scale application of this
technology feasible in the near
future. Specific enzyme activities of
lytic systems are high and thus
should mean that the cost of large
scale enzymatic lysis is very reasonable. Cloning of the genes for lytic
enzymes in producer strains should
further decrease the cost of this
technology. Lytic enzyme technology
shows great promise for the isolation
of high value subcellular fractions of
some large intracellular recombinant
proteins that cannot be secreted by
the cell, and for other specialized
applications.
New and future developments
should focus on understanding the
mechanism by which whole microbial cells and subcellular fractions
are enzymatically cleaved. This will
allow the optimization of product
extraction particularly in those cases
where protein secretion cannot be
obtained. It will also result in the
design of new processes for the
specific extraction of intracellular
proteins.

References
1 Jamas, S., Rha, C. K. and Sinskey, A. J.
(1986) Biotechnol. Bioeng. 28, 769784
2 Er-A1, Z., Klein, D., Buttat, E. and

TIBTECH - O C T O B E R 1987 [Vol. 5]

3
4

6
7
8
9

10

11

12
13
14
15
16
17
18
19
20
21
22
23

Zorner, E. (1984) Third Eur. Cong.

Biotechnol. (Vol. I), 1-629
D'Souza, S. F. (1983) Biotechnol.
Bioeng. 25, 1661-1664
Valenzuela, P., Colt, D., MedinaSelby, M. A., Kuo, C. H., Van Nest, G.,
Burke, R. L., Bull, P., Urdea, M. S. and
Graves, P. V. (1985) Bio/Technology,
3, 323-326
Hunter, J. B. and Asenjo, J. A. (1986)
in Separation, Recovery and Purification in Biotechnology: Recent Advances and Mathematical Modelling,
(Asenjo, J. A. and Hong, J., eds), pp. 931, American Chemical Society
Schnaitman, C. A. (1971) J. Bacteriol.
108, 553-563
Kobayashi, R., Miwa, T., Yamamoto,
S. and Nagasaki, S. (1980) J. Ferment.
Technol. 58, 311-317
Kaneko, T., Kitamura, K. and
Yamamoto, Y. (1969) J. Gen. Microbiol. 15,317-326
Reichenbach, H. and Dworkin, M.
(1981) in The Prokaryotes (Starr,
M. P., Stolp, H., Truper, H. G., Balows,
A. and Schlegel, H. G., eds), pp. 356379, Springer-Verlag
Lechevalier, H. A. and Lechevalier,
M. P. (1981)in TheProkaryotes (Starr,
M. P., Stolp, H., Truper, H. G., Balows,
A. and Schlegel, H. G., eds), pp. 21202123, Springer-Verlag
Newman, M. G., Socransky, S. S.,
Savitt, E. D., Propas, D. A. and
Crawford, A. (1976) J. Periodontology,
47, 373-379
Asenjo, J. A. and Andrews, B. A. Adv.
Bioehem. Eng./Biotechnol. (in press)
Scott, J. H. and Schekman, R. (1980) J.
Bacteriol. 142, 414-423
Kitamura, K. (1982) Agric. Biol.
Chem. 46, 963-969
Vrsanska, M., Kratky, Z. and Biely, P.
(1977) Z. Allg. Mikrobiol. 17,391--402
Obata, T., Iwata, H. and Namba, Y.
(1977) Agric. Biol. Chem. 41, 23872394
Okagbue, R. N. and Lewis, M. J. (1983)
Biotech. Lett. 5,731-736
Kobayashi, R., Miwa, T., Yamamoto,
S. and Nagasaki, S. (1982) Eur. J. Appl.
Microbiol. Biotechnol. 15, 14-19
Andrews, B. A. and Asenjo, J. A.
(1986) Biotechnol. Bioeng. 28, 13661375
LeCorre, S., Andrews, B. A. and
Asenjo, J. A. (1985) Enzyme Microb.
Technol. 7, 73-77
Valisena, S., Varaldo, P. E. and Satta,
G. (1982) J. Bacteriol. 51,636-647
Hayashi, K., Kasumi, T., Kubo, N. and
Tsumura, N. (1981) Agric. Biol. Chem.
45, 2289-2300
Suzuki, K., Uyeda, M. and Shibata, M.
(1985) Agric. Biol. Chem. 49, 17191727

24 Borovikova, V. P., Arsenovskaya,

V. E., Laurenova, G. I., Kislukhina,
O.V., Kalunyants, K. A. and Strepanov, V.M. (1980) Biokhimiya, 45,
1524-1533
25 Wohner, G., Voeiskow, H., Prave, P.,
Luck, E. and von Rymon Lipinski, G.
(1986) European Patent Application
EPO 181 562 A2
26 Kitamura, K. (1982) J. Ferment.
Technol. 60, 253-256
27 Rowley, B. I. and Bull, A. T. (1977)
Biotechnol. Bioeng. 19, 879-899
28 Asenjo, J. A. (1981) Advances in
Biotechnol., 3, (Vezina, C. and Singh,
K., eds), pp. 295-300, Pergamon Press
29 Yoshimoto, T. and Tsuru, S. (1972) J.
Biochem. 72,379-390
30 Asenjo, J. A., Dnnnill, P. and Lilly,
M.D. (1981) Biotechnol. Bioeng. 23,
97-109
31 Asenjo, J. A., Andrews, B. A., Hunter,
J.B. and LeCorre, S. (1985) Process

[]

[]

[]

[]

[]

[]

Riochem. 20, 158-164

32 Liu, L. C., Prokopakis, G. J. and
Asenjo, J.A. (1986) AIChE 1986
Annual Meeting, T203, FL, USA
33 Asenjo, J. A., Andrews, B. A. and
Pitts, J. M. (1987) Proc. 4th European
Congress of Biotechnology, Vol. 2,
(Neijssel, O. M., van der Meet, B. R.
and Lyben, K. Ch. A. M., eds), p. 497,
Elsevier
34 Hammond, E. G., Glatz, B. A., Choi, Y.
and Teasdale, M.T. (1981) in New
Sources of Fats and Oils (Pryde, E. H.,
Princess, L. H. and Mukherjee, K. D.,
eds), pp. 171-187, American Oil
Chemistry Society
35 Scott, H., Hammer, F. E. and Szalkucki, T.J. (1987) in Food Biotechnology (Knorr, D., ed.), pp. 413-440,
Marcel Dekker
36 Johnson, J. C. (1977) in Industrial
Enzymes, Recent Advances, p. 203,
Noyes Data Corporation
[]

[]

[]

[]

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Expression, glycosylation
and secretion of fungal
hydrolases in yeast
Hajirne Yoshizumi and Toshihiko Ashikari
Although there are now some systems for transfer and expression of
fungal genes, none gives expression at a level useful for production.
There are useful expression systems in yeast, however, and these have
been used to express genes coding for fungal extracellular hydrolases.
This review examines how properties of the genes and gene product
affect production and secretion of the enzyme in yeast. Similar
considerations apply to expression of other heterologous genes in
yeast and in other hosts.
Various h y d r o l a s e s used in i n d u s t r y
p r o d u c e d b y filamentous fungi offer
attractive targets for industrial gene
technologists. R e c o m b i n a n t DNA
t e c h n o l o g y is e x p e c t e d to i m p r o v e
the p r o d u c t i v i t y , stability and substrate specificity of such enzymes.
A l t h o u g h the m o l e c u l a r genetics of

H. Yoshizumi and T. Ashikari are at

the Laboratories of Applied Microbiology, Research Center, Suntory Ltd.,
1-1-1 Wakayama-dai, Shimamoto-eho,
Mishima-gun, Osaka, Japan.

f i l a m e n t o u s fungi has r e c e n t l y progressed and some gene transfer

systems for filamentous fungi have
b e e n r e p o r t e d 1-3. These have not yet
r e a c h e d the stage of practical applications. On the other h a n d m a n y
foreign proteins of various origins
have already b e e n e x p r e s s e d in
the yeast, Saccharomyces cerevisiae,
some of t h e m at a high level 4-7. Genes
of fungal extracellular proteins have,
therefore, b e e n i n t r o d u c e d into yeast
and their e x p r e s s i o n and secretion
studied. In this article, w e r e v i e w the

(~) 1987, Elsevier Publications, Cambridge 0166-9430/87/$02.00