Vol. 47, No. 2APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1984, p. 363-3680099-2240/84/020363-06$02.00/0Copyright C 1984, American Society for Microbiology

Protoplast Fusion of Trichoderma reesei, Using Immature ConidiaHIDEO TOYAMA, KOHTARO YAMAGUCHI, ATSUHIKO SHINMYO, AND HIROSUKE OKADA*

Department of Fermentation Technology, Faculty of Engineering, Osaka University, Yamada-oka, Suita-shi, Osaka 565,Japan

Received 11 July 1983/Accepted 15 November 1983

Protoplast fusion of strains derived from Trichoderma reesei QM9414 and QM9136 and the segregation ofthe resulting fusants were studied. Combinations of protoplasts prepared from young conidia with doubleamino acid requirements, one of which was a common requirement and the other uncommon, were fused inthe presence of polyethylene glycol 6000. Fusants were selected as regenerant colonies requiring only thecommonly deficient amino acid. The frequency of fusion was 0.9 x 10-4 to 4.0 x 10-4 for the startingconidia and 3.0 x 10-2 to 4.9 x 10-2 for the regenerated protoplasts, which was significantly higher than theexpected reversion frequencies by mutation. Conidia generated on the fusant colonies showed diversephenotypes, i.e., parental types (40 to 80%) and nonparental types (20 to 60%). Colonies developed fromsingle conidia of the nonparental phenotype contained special spots called "knobs" that have a higherdensity of mycelia. The phenotype of the knobs was again varied among prototrophs, parental types, andrecombinant types; and their traits were inherited stably. The phenotype of the mycelia in the nonknob partwas essentially the same as that of the original conidia and again formed knobs in colonies upon transfer of apiece of mycelia to a fresh medium. The conidial DNA content of the knob clone was almost the same asthat of the parents, but that of the fusants was 1.2 to 2.0 times higher than that of the parents. From these re-sults, we conclude that knobs are the segregants from the fusants. One knob clone showed twice thecarboxymethyl cellulose hydrolyzing activity of the parents, suggesting the possibility of breeding T. reeseicells by the protoplast fusion technique.

Trichoderma reesei is widely used for cellulase produc-tion. The technique of protoplast fusion in the presence ofpolyethylene glycol has been developed and applied tovarious kinds of cells, including cultured cells of plants andanimals, yeasts, Streptomyces sp., fungi, and bacteria. Ashas been reported, our establishment of a method for prepar-ing protoplasts of T. reesei from its immature conidia (9)containing a single nucleus provides us with a useful methodof breeding by protoplast fusion. Recently, Picataggio et al.(6) have reported the formation of spheroplasts from T.reesei mycelia by using a commercial enzyme and theregeneration of the spheroplasts at a high frequency. Thispaper describes the protoplast fusion of T. reesei, segrega-tion of gene markers among clones derived from the fusant,and the possibilities of applications for enhanced cellulaseproductivity.

MATERIALS AND METHODSOrganisms. T. reesei QM9414 (high cellulase producer)

and QM9136 (cellulase nonproducer) were strains obtainedby Mandels (3); auxotrophs derived from them were usedthroughout this study and are listed in Table 1. Theseauxotroph gene markers were stable and had reversionfrequencies of less than l0-5. Strains were inoculated on apotato-dextrose agar medium (Difco Laboratories, Detroit,Mich.) and incubated at 30°C for 7 days; after green conidiaformation they were stored at 4°C.

Culture media. The basal medium contained 20 g ofglucose, 15 g of agar, 80 mg of streptomycin, and 1 ml ofTriton X-100 in 1 liter of the salt solution defined by Mandels(3). If required, 80 mg of each appropriate amino acid wassupplemented. The salt solution contained 1.4 g of(NH4)2SO4, 0.3 g of urea, 2.0 g of KH2PO4, 0.3 g of CaC12,0.3 g of MgSO4 * 7H20, 5.0 mg of FeSO4 * 7H20, 1.6 mg of

* Corresponding author.

363

MnSO4 * H20, 1.4 mg of ZnSO4 * H20. and 2.0 mg ofCoCl2 * 6H20 in 1 liter of deionized water (pH 5.0).For the regeneration of protoplasts or fused protoplasts,

the regeneration medium contained 20 g of glucose, 4 g ofagar, 200 g of sucrose, 80 mg of streptomycin, and 1 ml ofTriton X-100 in 1 liter of the salt solution, and if required, 80mg per liter of each appropriate amino acid was supplement-ed.

Protoplast formation from conidia. T. reesei was grown at30°C on the basal medium. After 3 or 4 days of culture, whiteconidia (immature conidia) were formed, which turned green(mature conidia) after 7 days. The immature conidial suspen-sion in a 0.1% Triton X-100 solution was filtered through asintered glass 3G-3 filter (Sibata Scientific Technology Ltd.,Tokyo) to remove mycelia. The filtrate was vigorouslyagitated to disperse conidial aggregates, and conidia werecollected by centrifugation at 6,000 x g for 5 min and washedonce with deionized water. Protoplast formation was carriedout by incubation of the conidial suspension (106 to 107/ml) in0.1 M acetate buffer (pH 5.0), containing 0.6 M sucrose (finalconcentrations) supplemented with 6% (wt/vol) of a lyticenzyme preparation from Trichoderma viride, on a recipro-cal shaker (100 strokes per min) at 45°C for 6 h under asepticconditions. The lytic enzyme preparation used was a lyophi-lized powder of an aqueous extract from a wheat bran, solidculture of T. viride B1A and was a gift from N. Toyama,Miyazaki University. It contained 35% less cellulase activity(substrate, filter paper) and 50 and 300% more activity of P-1,3-glucanase (substrate, laminarin) and chitinase (substrate,chitin), respectively, than did the commercial enzyme, Cel-lulase Onozuka (Kinki Yakult Co., Nishinomiya, Japan).The enzyme solution was used just after filtration through aMillipore HA filter (0.45 ,um; Millipore Corp., Bedford,Mass.).The protoplast yield was calculated from the numbers of

intact conidia observed under a microscope in the protoplast

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TABLE 1. Auxotrophic mutant strains of T. reesei used

Straina Phenotype frequency

T. reesei QM9414 (prototroph,cellulase hyper-producer)

85 Lys- 7.0 x 10-685-19 Lys- Leu- 2.0 x 10-6b85-84 Lys- Arg- 5.0 x 10-6b85-88 Lys- Arg- 5.0 x 10-6b85-90 Lys- Arg- 5.0 x 10-6b85-203 Lys- His- 5.0 x 10-6b46 Arg- <1 x 10-5b46-13 Arg- Leu- <1 x 10-5b46-25 Arg- Met- <1 x 10-5b46-26 Arg- Leu- <1 x 10-5b80 Met- <1 x 10-580-21 Met- Lys- <1 x 10-5b80-35 Met- Leu- <1 x 10-5b

T. reesei QM9136 (prototroph,cellulase nonproducer)

17 Lys- 7.0 x 10-617-11 Lys- Arg- 1.0 x 10-6b17-30 Lys- Arg- 1.0 x 10-6ba T. reesei QM9414 or QM9136 conidia were UV irradiated, and

single auxotrophic mutants were isolated; by a second UV irradia-tion, double auxotrophic mutants were obtained as shown by theseries of numbers.

b Reversion frequency of the second markers.

preparation samples appropriately diluted with distilled wa-ter containing 0.1% Triton X-100 and 0.1% bile acids or with0.1 M acetate buffer containing 0.6 M sucrose. The proto-plast population yield in all of the cells thus prepared wasdistributed from 60 to 80%, with a mean value of 70%, andthe recovery rate of protoplasts from the starting conidia was50 to 60%. In some experiments, protoplast preparation wasfractionated by discontinuous gradient centrifugation of 2 to3 ml of the reaction mixture on 7 ml of a 40% sucrose layer at400 x g for 20 min. Protoplasts were recovered at theinterface of 40% sucrose with a purity of about 95%.Protoplasts thus prepared showed a high regeneration fre-quency on the regeneration medium (0.3 to 1.0%).

Protoplast fusion. Protoplast fusion was carried out basi-cally by the methods of Peberdy and Gibson (5) and Svoboda(8). Two protoplast suspensions derived from two strains,each having one common and one different gene marker, ofapproximately the same number (106 to 107/ml) were mixed,and polyethylene glycol 6000 and CaCl2 were added at finalconcentrations of 33% and 66 mM, respectively. The mix-ture was incubated at 35°C for 10 min followed by centrifuga-tion at 700 x g for 5 min. Thus treated, the protoplastsuspension was diluted at least 10 times with 0.1 M acetatebuffer (pH 5.0) containing 0.6 M sucrose and plated on theregeneration medium supplemented with the amino acidrequired by both parents and incubated at 30°C for 7 to 10days. The fusion products were picked up from the plate andinoculated on a basal medium slant.Rapid estimation of cellulase productivity. An agar plate of

medium contained 1 g of Difco Proteose Peptone, 15 g ofagar, and 1 ml of Triton X-100 in 1 liter of the salt solutionoverlaid with carboxymethyl cellulose (CMC)-agar contain-ing 10 g ofCMC (CMC-DS 0.7; Brown Co.), 15 g of agar, and1 ml of Triton X-100 in 1 liter of the salt solution. A mycelialmat (ca. 5 by 5 mm) picked up from a slant culture wasplaced on the double-layered plate and incubated at 30°C for6 days and then at 45°C for 16 h. The size of the resultingclear zone was not necessarily related quantitatively to the

CMC hydrolyzing activity produced in the submerged cul-ture, but it was used as a semiquantitative index of theenzyme productivity.

Determination of DNA content in conidia. About 20 mg ofconidia of each clone were suspended in 0.1% Triton X-100solution and passed through a sintered glass, 3G-3 filter toremove mycelia. Conidia were precipitated by centrifugationat 6,000 x g for 5 min, and the DNA fraction was preparedby a previously described method (2) and determined spec-trophotometrically by the method of Ceriotti (1).

Estimation of cellulase productivity in submerged culture.One loopful of mycelia (or conidia) was inoculated in 30 mlof the preculture medium consisting of 3% CMC-Na (WakoPure Chemical Co., Osaka, Japan) and 0.1% Difco ProteosePeptone (pH 5.0) in a 100-ml Erlenmeyer flask and incubatedat 30°C for 4 days on a rotary shaker. Precultured broth (3ml) containing pulpy mycelia was transferred to the produc-tion medium which was same as the preculture medium,except that the concentration of CMC-Na was reduced to1%. After incubation for 6 days, the culture broth wascentrifuged at 6,000 x g for 5 min in a graduated conicaltube, and the packed volume of mycelia was measured. Theresulting supernatant was assayed for CMC hydrolyzingactivity. A total of 1 ml of packed volume per 10 ml ofculture broth corresponded to 3.1 mg of dry mycelia per ml.Assay of CMC hydrolyzing activity. CMC-hydrolyzing ac-

tivity was measured by the method of Mandels et al. (4)except that 1% CMC-Na (DS 0.7; Nakarai Chemicals, Ltd.)was used in the acetate buffer system. One unit of activity isdefined as the amount of the enzyme producing reducingsugars equivalent to 1 ,umol of glucose in 1 min.

RESULTSProtoplast fusion. The numbers of fusants regenerated on

the regeneration medium supplemented with the amino acidrequired by both parents, such as lysine in the 85 series x 85series and 85 series x 17 series, leucine in 85-19 x 80-35,arginine in 85-88 x 46-26 and 85-84 x 46-13, or methionine in80-21 x 46-25, are summarized in Table 2. The commonamino acid requirement was used to distinguish the fusants

TABLE 2. Fusion frequencies in the various combinations ofauxotrophic mutants

Fusant strainsa No. of fusants! Fusionno. of starting frequenCyb

A B conidia

85-19 (Lys- Leu-) 85-84 (Lys- Arg-) 9/(1.0 x 105) 9 x 10-585-19 85-88 (Lys- Arg-) 6/(1.7 x 104) 4 x 10-485-19 85-90 (Lys- Arg-) 20/(8.5 x 104) 2 x 10-485-19 80-35 (Met- Leu-) 12/(6.8 x 104) 2 x 10-485-203 (Lys- His-) 17-30 (Lys- Arg-) 5/(2.0 x 104) 2 x 10-485-203 17-11 (Lys- Arg-) 12/(6.8 x 104) 2 x 10-485-88 (Lys- Arg-) 46-26 (Arg- Leu-) 34/(3.0 x 105) 2 x 10-485-84 (Lys- Arg-) 46-13 (Arg- Leu-) 2/(1.2 x 104) 2 x 10-480-21 (Met- Lys-) 46-25 (Arg- Met-) 1/(1.0 x 104) 1 X 10-4

a Protoplasts prepared from strains A and B were mixed in almostthe same numbers and subjected to fusion treatment as described inthe text. They were then placed on the regeneration mediumcontaining an amino acid required commonly by strains A and B.The number of colonies that appeared on the regeneration mediumwas counted as the number of fusants.

b Fusion frequency was calculated based on the number ofstarting conidia. Since the recovery of protoplasts from conidia wasabout 40% and the frequency of regeneration of protoplasts was 0.3to 1.0%, the fusion frequency based on the number of protoplastgenerated was on the order of 10-2.

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TABLE 3. Distribution of amino acid requirements of fusant conidiaa

Relative no. of colonies grown on minimum medium supplemented with:b

None Lys Arg Leu Met His Lys Lys Lys Leu Arg Lys Arg His Lys Lys LysArg Met Leu Arg Met His Leu Arg Leu Arg Arg Met Arg His

85-84 (Lys- Arg-) x 0 21 0 0 80 63 0 100 (133)85-19 (Lys- Leu-)

85-88 (Lys- Arg-) x 0 34 0 0 85 31 0 100 (368)85-19 (Lys- Leu-)

85-90 (Lys- Arg-) x 0 37 0 0 77 33 0 100 (830)85-19 (Lys- Leu-)

85-88 (Lys- Arg-) x 0 0 22 0 66 0 64 100 (526)46-26 (Arg- Leu-)

85-84 (Lys- Arg-) x 0 0 10 0 68 0 45 100 (95)46-13 (Arg- Leu-)

80-21 (Lys- Met-) x 0 0 0 18 0 78 25 10042-25 (Arg- Met-) (82)

85-203 (Lys- His-) x 0 24 0 0 60 25 0 10017-30 (Lys- Arg-) (300)

85-203 (Lys- His-) x 0 65 0 0 70 86 0 10017-11 (Lys- Arg-) (91)

a Fusant colonies formed on regeneration soft agar medium were conidiated on an agar slant and plated on the respective agar media asshown.

b Numbers of colonies on each medium were expressed relative to the numbers on medium supplemented with three amino acids. Numbersin parentheses indicate the total number of colonies grown on the medium with three amino acids.

from the contaminants. The fusion frequency was distribut-ed from 0.9 x 10-4 to 4 x 10-4 of the total starting conidia,depending on the combination of parental strains. The fusionfrequency would be 100 times higher if the regenerationfrequency of 0.3 to 1.0% were taken into account (data notshown). As a control, conidia of two parental strains incombinations in Table 2 were mixed and plated on theregeneration medium and gave no more colonies than ex-pected from the reverse mutation rate. The reversion fre-quency of protoplasts regenerated without fusion treatmentwas similar to that of intact conidia (data not shown).

Colonies of fusants grown on the regeneration mediumwere transferred to the basal medium containing a requiredamino acid and were incubated at 30°C to allow conidiaformation. The resulting conidia were subjected to singleconidium separation and grown on the basal medium with orwithout supplementation of amino acid(s) as shown in Table3. The major portion of conidia showed an amino acidrequirement of either one of the parental types, which wasexpected of segregants of heterocaryotic mycelia by conidiaformation. In addition to the parental types, a minor portion,but a high frequency, of the nonparental-type conidia, Lys-Arg+ Leu+ in 85-19 x 85-84, 85-19 x 85-88, or 85-19 x 85-90,Arg- Lys' Leu+ in 85-88 x 46-26 or 85-84 x 46-13, Met-Lys' Arg+ in 80-21 x 46-25, and Lys- His' Arg+ in 85-203x 17-30 or 85-203 x 17-11 fusants were observed. Othernonparental types such as Lys- Arg- Leu-, Arg- Lys-Met-, or Lys- His- Arg- may exist, but we could not detectthem.

Segregation of fusants. The genetic stability of Lys- Arg+Leu+ conidia obtained by 85-19 x 85-84, 85-19 x 85-88, or85-19 x 85-90 was studied. As the colony grew from theseLys- conidia on the basal medium supplemented with lysinein the presence of 0.1% Triton X-100, special parts with ahigh mycelial density appeared which were called "knobs"(Fig. 1A). The frequency of knob appearance was usually 5to 10 in one colony (diameter, 2 to 3 cm) with everycombination of parents. On the agar medium without TritonX-100, sectors instead of knobs were detected (Fig. 1B).

FIG. 1. Photographs of colonies generated from a single fusantconidium in the presence (A) or absence (B) of Triton X-100. Knobsand sectors are indicated by arrows. Bars, 1 cm.

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TABLE 4. Segregation pattern of phenotypes of mycelia generated from Lys- fusant conidiumNo. of No. of colonies with the following phenotypes

Fusants derived from: Mycelia type coloniestested Prototroph Lys- Lys- Arg- Lys- Leu- Lys- His- Arg- Leu- His-

85-84 (Lys- Arg-) x Knob 98 61 9 19 8 _a 1 085-19 (Lys- Leu-) Nonknob 25 0 25 0 0 0 0

85-88 (Lys- Arg-) x Knob 91 48 5 18 20 0 085-19 (Lys- Leu-) Nonknob 25 0 25 0 0 0 0

85-90 (Lys- Arg-) x Knob 78 66 6 4 2 0 085-19 (Lys- Leu-) Nonknob 25 0 25 0 0 - 0 0

85-203 (Lys- His-) x Knob 96 55 6 18 17 0 017-30 (Lys- Arg-) Nonknob 25 0 25 0 - 0 0 0

85-203 (Lys- His-) x Knob 99 56 7 27 9 0 017-11 (Lys- Arg-) Nonknob 25 0 25 0 0 0 0a , Not tested.

The distribution of amino acid requirements of myceliapicked up from knobs and nonknob parts are listed in Table4. The phenotypes of knobs were distributed among parentaltypes, Lys-, and prototrophs; the last phenotype appearedat a high frequency in spite of the fact that both parents hadthe same lysine deficiency gene in 85 series x 85 series. Theappearance of prototrophs at a high frequency was notspecific for fusants derived from Lys- parents, because 30to 50% of the knobs derived from Arg-, Arg-, and Met-fusants of 85-84 (Lys- Arg-) x 46-13 (Arg- Leu-), 85-88(Lys- Arg-) x 46-26 (Arg- Leu-), and 80-21 (Met- Lys-) x46-25 (Arg- Met-) were prototrophs. The genetic character-istics of clones isolated from knobs seemed to be stable; theyproduced no more knobs in the colony when a piece of knobmycelia was transferred to a fresh agar medium to allowcolony growth. The knob phenotype (Lys- or prototroph)was kept, with no change detected in the conidia borne onthe knob mycelia (data not shown). On the other hand, whenmycelia from nonknob parts were transferred onto a freshagar medium, the formation of knobs was observed.DNA content in conidia. The DNA content in the conidia of

the parents, fusants, and knob clones are summarized inTable 5. Parents and knob clones showed almost the sameDNA content in conidia, but conidia of the fusants containedDNA at a 1.2 to 2.0 times greater amount per conidia thanthose of the parents. The mean size of 50 conidial sampleswas measured from their photomicrographs. The mean sizeof fusant conidia was slightly larger than that of the parents

TABLE 5. DNA content in conidiaClone DNA content (Fmg/rg of conidia)a

85-203.5.2217-30.6.41Fusant 1.8.85Fusant 2.8.05Fusant 3.7.17Fusant 4.12.03Knob 1.5.41Knob 2.5.96Knob 3.5.50

a DNA content in conidia of parents (85-203 and 17-30). Thefusants (fusants 1 through 4) derived from the combination of theseparents, and the knobs (knobs 1 through 3) which appeared from thefusants were measured. The content in nonknob clones could not bedetermined because of poor conidiation.

and knob clones (5.5% longer in the major axis).Distribution of cellulase productivity. Qualitative cellulase

productivity of each clone was studied by the size of theclear zone that appeared around the colony on a CMC-agarplate. As mentioned above, the size of the clear zone (or theratio of diameters of the clear zone and the colony) did notexactly correlate to the production of CMC hydrolyzingactivity (CMCase) determined in submerged culture. Quali-tative expression of cellulase production is summarized inTable 6. All fusants obtained by fusion of cellulase producers(85-88) x producers (85-19) or producer (85-203) x nonpro-ducers (17-30) produced the enzyme, but knob clones gener-ated from 85-203 x 17-30 fusion fell into two groups,producers and nonproducers, in an almost equal ratio. Evenin the knob clone from a fusant of 85-88 x 85-19, both ofwhich are enzyme producers, 4 of 25 clones were noncellu-lase producers. On the other hand, all nonknob clones werecellulase producers, suggesting that the knob clones are thesegregants of nonknob mycelia.Among knob clones generated from 85-203 x 17-30, one

clone, 20s, which produced a large clear zone on a CMC-agar plate, was studied for its CMCase productivity in asubmerged culture. Clone 20s produced twice as muchCMCase as its cellulase-producing parent, 85-203 (Table 7).Knob mycelia were subjected to conidiation, and the

cellulase productivity of single, isolated conidia was tested.The results indicated the stable inheritance of high cellulaseproductivity of clone 20s to its descendants.

DISCUSSIONT. reesei QM9414, a hypercellulolytic mutant, is one of the

most promising candidates for microbial conversion of cellu-losic biomass. Although much effort has gone into theenzymology and production of cellulase of this fungus, theknowledge of its genetic background is still poor. This is thefirst report on the protoplast fusion and its segregation of T.reesei.

Establishment of protoplast formation from conidia con-taining a single nucleus within 5 to 6 h by the cell wall lyticenzyme of T. viride BlA (9) enabled us to overcome the firstbarrier in this study. Picataggio et al. (6) have shown that ittook 18 to 24 h for spheroplast formation from T. reeseimycelia by using Driselase (Kyowa Hakko Kogyo Co.,Tokyo) at 28°C. Rosen et al. (7) have observed under theelectron microscope that the doubling of the T. viride

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TABLE 6. Distribution of cellulase producer and nonproduceramong fusants, knobs, and nonknob clonesa

Clones No. of cellulase producers/total no. of clones tested

Expt. 1b85-203.............................. 10/1017-30.............................. 0/10Fusants ............................. 10/10Knobs .............................. 15/28Nonknobs ........................... 9/9

Expt. 2C85-88.............................. 8/885-19............................... 7/7Fusants ............................. 10/10Knobs .............................. 21/25Nonknobs ........................... 10/10a Mycelia of each clone were grown on CMC agar medium, and

cellulase producers were distinguished by clear zone formation asdessribed in the text.

b Experiment 1 shows populations of cellulase producers in bothparents (85-203 and 17-30), in fusants derived from the parents, andin knob and nonknob clones derived from several fusants randomlyselected.

c Experiment 2 shows the same populations as in experiment 1except that the parents were 85-88 and 85-19.

conidial nucleus occurred at 8 to 13 h in germination mediumat 25°C, so the protoplasts prepared under our conditionswere estimated to have a single nucleus (9), which ispreferable for the following genetic analysis.The conidia generated from the fusant mycelia were

classified into three phenotype groups, both parental typesand a nonparental type requiring a single amino acid forgrowth which was required by both parents. The conidia ofthe parental phenotypes could be the monocaryotizationproducts of the heterocaryon by conidiation. The inequalitybetween the frequencies of the two parental phenotypes(Table 3) suggests unequal proliferation rates of the twoparental nuclei in the heterocaryons. The appearance ofnonparental-type clones by conidiation is not a result ofcontamination or back mutation, because in those clones,the common genetic marker of both parents was conserved,and the frequency of the appearance of nonparental type (21to 65%) could not be explained by the back mutationfrequencies (<10-5; Table 1). This back mutation rate wasnot changed, even during protoplast regeneration, accordingto our control experiments (data not shown).The nonparental-type conidia can be formed either by

mechanisms of heterocaryotic conidiation or by diploid (ormero-diploid) formation, but the latter could be the case forthe following reasons. (i) The conidia of T. viride have beenreported to contain a single nucleus (7), and those of the

TABLE 7. Production of CMC hydrolyzing activity in submergedculture

Mycelial concn CMC hydrolyzing activityaStrain (mg of dry

weight/ml) Units/ml Units/mg of mycelia

85-203 16.5 + 0.9 0.164 ± 0.002 (0.10 ± 0.003) x 10-220sb 13.9 ± 0.3 0.285 ± 0.051 (0.20 ± 0.003) x 10-2

a Five parallel cultures were carried out, and mean values andstandard deviations are given. Experimental conditions are asdescribed in the text.

b Strain 20s was a knob clone generated from 85-203 x 17-30.

parental strains were observed to be singly nucleated at ahigh probability (>99%) which cannot explain the highfrequency (65%) of obtaining Lys- Arg+ Leu+ clones from85-203 x 17-11 fusants. (ii) The appearance of knobs whichare considered to be segregants of diploids or mero-diploids(as will be discussed later) in colonies developed fromnonparental-type conidia suggests the diploid or mero-dip-loid nature of the nucleus. (iii) The DNA content of nonpa-rental conidia was 1.2 to 2.0 times higher than that of theparental strains. If we assume single nucleated conidia, weconclude from this value that they are diploid or mero-diploid. Nevertheless, we cannot exclude the possibility thatsmall portions of heterocaryotic conidia exist in the popula-tion.

In the colonies grown from nonparental type fusant conid-ia, knobs were observed (Fig. 1), and the distribution ofphenotypes of mycelia in the knobs was diverse, but thephenotype of the nonknob parts was uniform and the sameas its original conidia.The knob clones were considered to be the haploid

segregants produced from nonknob clones for the followingreasons. (i) The DNA content of the knob clone conidia isthe same as that of the parental conidia and 1/1.2 to 1/2 thatof the fusants. (ii) The phenotypes of knob clones werestable, including cellulase productivity and nutritional re-quirements. (iii) In contrast to the stability of knob clones,nonknob clones segregate again into knob and nonknobclones when a small mycelial mat taken from a nonknobclone was placed on an agar plate to allow colony formation.(iv) The cellulase-negative marker in experiment 1 (Table 6)was distributed widely among knob clones that have anytype of nutritional requirement, which shows that recombi-nation occurred in a former stage.

It was a strange phenomenon that prototrophic cloneswere observed at a high frequency (50 to 66%) among knobclones, in spite of the fact that the Lys- phenotype in the 85series was derived from the same parent, 85 (Lys-). Thisfrequency was almost same as that observed in 85 series x17 series fusion, both of which were Lys- but of differentorigin. This phenomenon was not specific for the restorationof the Lys- phenotype, but similar restorations were alsoobserved with Arg- and Met- phenotypes.

In addition to the unexplained phenomenon above, theappearance of cellulase-negative knob clones from cellulase-positive parents suggests the existence of some unknownmechanisms of this fungus for which we need more geneticbackground data.One clone, 20s, derived from 85-203 x 17-30, the latter of

which is a cellulase-negative mutant, produced stably abouttwice the CMCase activity of its cellulase-producing parent,85-203. This result supports the possibility of breeding T.reesei strains by the protoplast fusion technique. It is knownthat cellulase of T. reesei strains consists of C1, Cx, and 1B-glucosidase. In our fusion and segregation system, it ispossible to elucidate whether these cellulase genes areclosely linked.

Recently, we obtained fusants between species of Tricho-derma, such as T. reesei x T. viride and T. reesei xTrichoderma koningii by the same method, and again, knobswere observed in the colonies that arose from the conidia offusants. These results will soon be published elsewhere.

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