Current Genetics
Curr Genet (1992)22:377-383
9 Springer-Verlag 1992
Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants Gary D. Hunter 1,,, Christopher R. Bailey z,**, and Herbert N. Arst, Jr. 1 1 Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Du Cane Road, London WI2 0NN, England 2 Celltech Ltd., 216 Bath Road, Slough SL1 4EN, England Received March 6/May 15, 1992
Summary. The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.
gillus nidulans, the ease of selection and the phenotypes of mutations resulting in the loss of (anabolic) NADPlinked and (catabolic) NAD-linked glutamate dehydrogenases (Arst and MacDonald 1973; Kinghorn and Pateman 1973, 1976; Arst et al. 1975) and catabolic glutamate-oxalacetate aminotransferase (Kinghorn and Pateman 1977) are strongly consistent with the absence of aspartase as too is an [~SN]-ammonium assimilation study (Kusnan et al. 1989). Here we confirm the absence of aspartase in A. nidulans and report the expression of the aspartase gene from Escherichia coli K12 (Guest et al. 1984; Woods et al. 1986) in A. nidulans. When present in multiple copies and expressed using a powerful A. nidulans promoter, E. coli aspartase can circumvent the nutritional deficiencies resulting from mutational loss of (catabolic) NAD-linked glutamate dehydrogenase but apparently not (anabolic) NADP-linked glutamate dehydrogenase. The requirement for multiple copies of the aspartase gene for metabolic suppression of the NADglutamate dehydrogenaseless phenotype makes it a useful selective marker for obtaining multicopy transformants in A. nidulans.
Key words: Aspartase - Aspergillus nidulans - Physiological suppression - Transformation Materials and methods Introduction The reversible interconversion between L-aspartate and fumarate plus ammonium is catalysed by aspartase. In one direction this reaction could enable catabolism of aspartate (and metabolites capable of conversion to aspartate). In the other it could enable ammonium assimilation. The presence of aspartase has never, to our knowledge, been reported in a fungus. In the ascomycete AsperPresent addresses: * Department of Applied Biology, University of Hulll Hull HU6 7RX, England. ** Medeva Group Research, Langhurst, Horsham, Sussex RH12 4QD, England Correspondence to: H. N. Arst
Genetic techniques, growth testing and strains. Genetic techniques were modified after Pontecorvo et al. (1953), McCully and Forbes (1965) and Clutterbuck (1974). Growth testing of A. nidulans followed standard procedures (Arst et al. 1982). Media and nutritional supplements described by Cove (1966) were used. The principal strains of A. nidulans used in this work are listed in Table 1. The markers carried are in standard use and are listed by Clutterbuck (1990). Mutations important to this work are described in conjunction with their use. The aspA § transforming sequences in GH98 come from transformant G43 from which GH98 is derived by outcrossing. DNA manipulations. DNA from A. nidulans strains was prepared by the method of Raeder and Broda (1985). Southern blotting and other DNA manipulations followed standard procedures (Berger and Kimmel 1987; Perbal 1988; Sambrook et al. 1989). For DNA dot-blots the areA gene (Kudla et al. 1990) was used as a single-copy
378 Table 1. Principal A. nidulans strains used in this work
E Strain designation GH22 GH60 GH98 GH101 GH107 GH142 GH145 GH164 C56
Xb
Genotype
E
(amdI-18), amdS-320; pyro-A-4, gdhB-1; amdA-7, pantoB-lO0 paba-A-l; gdhA-lO; gdhB-1;fwA-1 pabaA-1; (amdI-18), amdS-320; pyroA-4, gdhB-1; amdA- 7, aspA +;fwA-1 argB-2; pyroA-4, gdhB-1; pantoB-lO0 areAr-1; gdhB-1; pantoB-lO0 pabaA-1; wA-4, ssuA-l, mauB-4 biA-1, yA2, pabaA-l; areA'-18; gdhB-1 yA-2; gdhB-1, inoB-2, mauA-2 suA-ladE-20, yA-2, adE-20; acrA-1; galA-l; pyroA-4; faeA-303; sB-3; nieB-8; riboB-2
Gene symbols are defined by Clutterbuck (1990). Brackets indicate mutations which are very likely to be present but have not been definitely shown to be present. Semi-colons separate mutations in different linkage groups, aspA + sequences in GH98 have been located to linkage group VII
Xb Xh
Xh h / ~ ) ~ i i ~ X K II EE
H
X H
Fig. 1. Features of pGH-1 and pGH-4 important in this work. gpdA promoter; I aspA coding sequence; ~ trpC terminator; argB gene;c223pUC-18; E - EcoRI; H - HindIII; K - KpnI; Xb - XbaI; Xh - XhoI
the addition of 50 gl of 300 mM sodium L-aspartate, pH 7.0. Fumarate production was monitored at 240 nm, as described by Williams and Lartigue (1969), in a Philips dual beam Pu 8820 UV/ VIS spectrophotometer. Soluble protein in extracts was determined with the protein assay kit supplied by Bio-Rad (Hemel Hempstead, Hertfordshire, UK) based on the method of Bradford (1976) using bovine serum albumin as a standard.
probe for calibrating concenctrations, and hybridisation intensity was measured using a Joyce Loebl Chromoscan 3 Densitometer.
Results Plasmids. pGH-1 was constructed by ligating the 2 kb AflIII fragment containing the open reading frame of aspA from pGS94 (Woods et al. 1986) into pAN52-3 (Punt et al. 1991) which had been digested with NcoI and treated with phosphatase (Fig. 1). This destroys both AfllII and NcoI sites and enables the initiation codon of the E. coli K-12 aspA gene to replace that of the A. nidulansgpdA gene, placing the aspA gene under the control of the strong, constitutive promoter of the gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase) and just upstream of the A. nidulans trpC transcription terminator region, pGH-4 was constructed by ligating the 3.3 kb XbaI fragment containing the entire A. nidulans argB gene from pMA2 (Parsons et al. 1987) into XbaI-digested, phosphatase-treated pGH-1 (Fig. 1). pAMD21 has been described by Hynes et al. (1988).
Transformation. A. nidulans transformation was carried out by the method of Tilburn et al. (1983) as modified by Tilburn et al. (1990). For selection of amdS + transformants, 1 M sucrose was present as an osmotic stabiliser (and inevitably as an additional carbon source) with 1% D-glucose as a carbon source and 10 mM of acetamide as a nitrogen source in the presence of 12.5 mM of CsC1 to reduce residual acetamide utilisation. Selection of argB+ transformants was identical except that acetamide and CsC1 were replaced by 10 mM of ammonium [as the (+)-tartrate]. For direct selection of aspA + transformants, 0.6 M KCI was present as an osmotic stabiliser and 50 mM of L-aspartate (Na + salt) served both as a carbon and a nitrogen source. The final resuspension of protoplasts for plating in this case was in 0.6 M KC1 plus 50 mM CaC1z thus avoiding possible carryover of sorbitol which can be utilised as a carbon source. Aspartase [L-aspartate ammonia lyase ( EC 4.3.1.1)] assays. Mycelia for enzyme assay were grown in shaken liquid glucose-minimal medium (Cove 1966), with the nitrogen sources indicated, for 15 h at 37~ Mycelia were harvested as described by Cove (1966) and used immediately for enzyme assay. Mycelium (500 rag) was ground in chilled mortar and pestle with 500 mg of acid-washed sand and 500 p,l of extraction/assay buffer (55 mM tris, pH 8.5; 2.2mM MgSO4; 11 gM EDTA) and spun for 10rain at 13000rpm in a microfuge. Then 500 gl buffer was added to the decanted supernatant for a further 30 min microfuge spin. Following this, 20 rtl of extract was mixed with 430 ~tl of buffer and the reaction started by
Selection of transformants containing aspA sequences and metabolic suppression o f the gdhB- phenotype T h e a b s e n c e o f d e t e c t a b l e a s p a r t a s e activity in A. nidulans was c o n f i r m e d for the t r a n s f o r m a t i o n recipient strain G H 2 2 (Table 2). I n i t i a l l y it was n o t k n o w n w h e t h e r expression o f the E. coli a s p a r t a s e gene aspA w o u l d overc o m e n u t r i t i o n a l deficiencies (i.e., the i n a b i l i t y to utilise L - g l u t a m a t e a n d c o m p o u n d s m e t a b o l i s e d via g l u t a m a t e as n i t r o g e n a n d / o r c a r b o n sources) resulting f r o m a gdhB- ( N A D - g l u t a m a t e d e h y d r o g e n a s e l e s s ) m u t a t i o n in A. nidulans. T h e r e f o r e arndS + t r a n s f o r m a n t s o f G H 2 2 , w h i c h carries the large d e l e t i o n amdS-320 ( H y n e s et al. 1983) in the a c e t a m i d a s e s t r u c t u r a l gene, were selected after t r a n s f o r m a t i o n with p A M D 2 1 ( H y n e s et al. 1988) a n d c o - t r a n s f o r m a t i o n with p G H - l was m o n i t o r e d b y evidence for p h y s i o l o g i c a l s u p p r e s s i o n o f gdhB-1. O f 50 amdS + t r a n s f o r m a n t s nine s h o w e d stable s u p p r e s s i o n o f the gdhB- p h e n o t y p e . O f these, eight (G44, G45, G46, G47, G49, G51, G 5 2 a n d G54) were a b l e to utilise n i t r o gen sources n o r m a l l y m e t a b o l i s e d via g l u t a m a t e , such as L - a s p a r t a t e (Fig. 2), b u t c o u l d n o t utilise these s a m e c o m p o u n d s in the q u a n t i t a t i v e l y m o r e d e m a n d i n g c a p a c ity o f c a r b o n a n d n i t r o g e n sources. T h e n i n t h (G50) exhibits a s t r o n g e r degree o f p h y s i o l o g i c a l s u p p r e s s i o n o f the gdhB- p h e n o t y p e , utilising, for e x a m p l e , L - g l u t a m i n e a n d L - a s p a r t a t e as c a r b o n a n d n i t r o g e n sources. Prelimin a r y S o u t h e r n b l o t s ( d a t a n o t shown) i n d i c a t e d t h a t f o u r o f the c o - t r a n s f o r m a n t s , G44, G45, G 4 6 a n d G47, c o n tain at least two t a n d e m l y i n t e g r a t e d copies ofaspA. G48, a l t h o u g h p h e n o t y p i c a l l y u n s t a b l e (Fig. 2), also c o n t a i n s aspA-hybridising sequences; the recipient strain G H 2 2 c o n t a i n s no aspA-hybridising sequences. I n view o f the a p p a r e n t a b i l i t y o f aspA e x p r e s s i o n to o v e r c o m e the gdhB- p h e n o t y p e , aspA + t r a n s f o r m a n t s
379 stable transformant G40 has fewer copies of aspA and much lower activity. It is possible that aspartase activity, in G40 was diminished by loss of aspA sequences during growth of mycelia for enzyme determinations because, in a preliminary experiment using G40 mycelia grown with 5 m M of L-aspartate as a nitrogen source, an order of magnitude higher specific activity (89 units as average o f two determinations) was obtained. Under these conditions there would be selective pressure to maintain aspA sequences (but also the danger that genetic changes leading to increased aspartase levels would o c c u r - vide infra).
Fig. 2. Physiological suppression of gdhB-1 by aspA expression in transformants. Left to right: top row G39, G40, G41, G42; second row G43, G44, G45, G46; third row G47, G48, G49, G50; fourth row G51, G52, G53, G54; bottom row GH22 (recipient), wild-type (genotype biA-1). Growth medium contained 1% glucose as a carbon source and 5 mM L-aspartate (sodium salt) as a nitrogen source and was incubated for 2 days at 37 ~
Table 2. Aspartase activities of aspA transformants Strain no.
Recpient strain
Selection a s p A Aspartase Nitrogen marker copy no. activity source
GH22 G39 G40 G41 G42 G43 G120 G120 G121 G121 G 122 G122 G123 G127 G 127
GH22 GH22 GH22 GH22 GH22 GH101 GH101 GH101 GH101 GH 101 GH101 GH101 GH101 GH 101
-
aspA aspA aspA aspA aspA argB argB argB argB argB argB argB argB argB
0 32 8 64 32 32 8 8 1 i 0 0 1 16 16
Curr Genet (1992)22:377-383
9 Springer-Verlag 1992
Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants Gary D. Hunter 1,,, Christopher R. Bailey z,**, and Herbert N. Arst, Jr. 1 1 Department of Infectious Diseases and Bacteriology, Royal Postgraduate Medical School, Du Cane Road, London WI2 0NN, England 2 Celltech Ltd., 216 Bath Road, Slough SL1 4EN, England Received March 6/May 15, 1992
Summary. The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.
gillus nidulans, the ease of selection and the phenotypes of mutations resulting in the loss of (anabolic) NADPlinked and (catabolic) NAD-linked glutamate dehydrogenases (Arst and MacDonald 1973; Kinghorn and Pateman 1973, 1976; Arst et al. 1975) and catabolic glutamate-oxalacetate aminotransferase (Kinghorn and Pateman 1977) are strongly consistent with the absence of aspartase as too is an [~SN]-ammonium assimilation study (Kusnan et al. 1989). Here we confirm the absence of aspartase in A. nidulans and report the expression of the aspartase gene from Escherichia coli K12 (Guest et al. 1984; Woods et al. 1986) in A. nidulans. When present in multiple copies and expressed using a powerful A. nidulans promoter, E. coli aspartase can circumvent the nutritional deficiencies resulting from mutational loss of (catabolic) NAD-linked glutamate dehydrogenase but apparently not (anabolic) NADP-linked glutamate dehydrogenase. The requirement for multiple copies of the aspartase gene for metabolic suppression of the NADglutamate dehydrogenaseless phenotype makes it a useful selective marker for obtaining multicopy transformants in A. nidulans.
Key words: Aspartase - Aspergillus nidulans - Physiological suppression - Transformation Materials and methods Introduction The reversible interconversion between L-aspartate and fumarate plus ammonium is catalysed by aspartase. In one direction this reaction could enable catabolism of aspartate (and metabolites capable of conversion to aspartate). In the other it could enable ammonium assimilation. The presence of aspartase has never, to our knowledge, been reported in a fungus. In the ascomycete AsperPresent addresses: * Department of Applied Biology, University of Hulll Hull HU6 7RX, England. ** Medeva Group Research, Langhurst, Horsham, Sussex RH12 4QD, England Correspondence to: H. N. Arst
Genetic techniques, growth testing and strains. Genetic techniques were modified after Pontecorvo et al. (1953), McCully and Forbes (1965) and Clutterbuck (1974). Growth testing of A. nidulans followed standard procedures (Arst et al. 1982). Media and nutritional supplements described by Cove (1966) were used. The principal strains of A. nidulans used in this work are listed in Table 1. The markers carried are in standard use and are listed by Clutterbuck (1990). Mutations important to this work are described in conjunction with their use. The aspA § transforming sequences in GH98 come from transformant G43 from which GH98 is derived by outcrossing. DNA manipulations. DNA from A. nidulans strains was prepared by the method of Raeder and Broda (1985). Southern blotting and other DNA manipulations followed standard procedures (Berger and Kimmel 1987; Perbal 1988; Sambrook et al. 1989). For DNA dot-blots the areA gene (Kudla et al. 1990) was used as a single-copy
378 Table 1. Principal A. nidulans strains used in this work
E Strain designation GH22 GH60 GH98 GH101 GH107 GH142 GH145 GH164 C56
Xb
Genotype
E
(amdI-18), amdS-320; pyro-A-4, gdhB-1; amdA-7, pantoB-lO0 paba-A-l; gdhA-lO; gdhB-1;fwA-1 pabaA-1; (amdI-18), amdS-320; pyroA-4, gdhB-1; amdA- 7, aspA +;fwA-1 argB-2; pyroA-4, gdhB-1; pantoB-lO0 areAr-1; gdhB-1; pantoB-lO0 pabaA-1; wA-4, ssuA-l, mauB-4 biA-1, yA2, pabaA-l; areA'-18; gdhB-1 yA-2; gdhB-1, inoB-2, mauA-2 suA-ladE-20, yA-2, adE-20; acrA-1; galA-l; pyroA-4; faeA-303; sB-3; nieB-8; riboB-2
Gene symbols are defined by Clutterbuck (1990). Brackets indicate mutations which are very likely to be present but have not been definitely shown to be present. Semi-colons separate mutations in different linkage groups, aspA + sequences in GH98 have been located to linkage group VII
Xb Xh
Xh h / ~ ) ~ i i ~ X K II EE
H
X H
Fig. 1. Features of pGH-1 and pGH-4 important in this work. gpdA promoter; I aspA coding sequence; ~ trpC terminator; argB gene;c223pUC-18; E - EcoRI; H - HindIII; K - KpnI; Xb - XbaI; Xh - XhoI
the addition of 50 gl of 300 mM sodium L-aspartate, pH 7.0. Fumarate production was monitored at 240 nm, as described by Williams and Lartigue (1969), in a Philips dual beam Pu 8820 UV/ VIS spectrophotometer. Soluble protein in extracts was determined with the protein assay kit supplied by Bio-Rad (Hemel Hempstead, Hertfordshire, UK) based on the method of Bradford (1976) using bovine serum albumin as a standard.
probe for calibrating concenctrations, and hybridisation intensity was measured using a Joyce Loebl Chromoscan 3 Densitometer.
Results Plasmids. pGH-1 was constructed by ligating the 2 kb AflIII fragment containing the open reading frame of aspA from pGS94 (Woods et al. 1986) into pAN52-3 (Punt et al. 1991) which had been digested with NcoI and treated with phosphatase (Fig. 1). This destroys both AfllII and NcoI sites and enables the initiation codon of the E. coli K-12 aspA gene to replace that of the A. nidulansgpdA gene, placing the aspA gene under the control of the strong, constitutive promoter of the gpdA gene (encoding glyceraldehyde-3-phosphate dehydrogenase) and just upstream of the A. nidulans trpC transcription terminator region, pGH-4 was constructed by ligating the 3.3 kb XbaI fragment containing the entire A. nidulans argB gene from pMA2 (Parsons et al. 1987) into XbaI-digested, phosphatase-treated pGH-1 (Fig. 1). pAMD21 has been described by Hynes et al. (1988).
Transformation. A. nidulans transformation was carried out by the method of Tilburn et al. (1983) as modified by Tilburn et al. (1990). For selection of amdS + transformants, 1 M sucrose was present as an osmotic stabiliser (and inevitably as an additional carbon source) with 1% D-glucose as a carbon source and 10 mM of acetamide as a nitrogen source in the presence of 12.5 mM of CsC1 to reduce residual acetamide utilisation. Selection of argB+ transformants was identical except that acetamide and CsC1 were replaced by 10 mM of ammonium [as the (+)-tartrate]. For direct selection of aspA + transformants, 0.6 M KCI was present as an osmotic stabiliser and 50 mM of L-aspartate (Na + salt) served both as a carbon and a nitrogen source. The final resuspension of protoplasts for plating in this case was in 0.6 M KC1 plus 50 mM CaC1z thus avoiding possible carryover of sorbitol which can be utilised as a carbon source. Aspartase [L-aspartate ammonia lyase ( EC 4.3.1.1)] assays. Mycelia for enzyme assay were grown in shaken liquid glucose-minimal medium (Cove 1966), with the nitrogen sources indicated, for 15 h at 37~ Mycelia were harvested as described by Cove (1966) and used immediately for enzyme assay. Mycelium (500 rag) was ground in chilled mortar and pestle with 500 mg of acid-washed sand and 500 p,l of extraction/assay buffer (55 mM tris, pH 8.5; 2.2mM MgSO4; 11 gM EDTA) and spun for 10rain at 13000rpm in a microfuge. Then 500 gl buffer was added to the decanted supernatant for a further 30 min microfuge spin. Following this, 20 rtl of extract was mixed with 430 ~tl of buffer and the reaction started by
Selection of transformants containing aspA sequences and metabolic suppression o f the gdhB- phenotype T h e a b s e n c e o f d e t e c t a b l e a s p a r t a s e activity in A. nidulans was c o n f i r m e d for the t r a n s f o r m a t i o n recipient strain G H 2 2 (Table 2). I n i t i a l l y it was n o t k n o w n w h e t h e r expression o f the E. coli a s p a r t a s e gene aspA w o u l d overc o m e n u t r i t i o n a l deficiencies (i.e., the i n a b i l i t y to utilise L - g l u t a m a t e a n d c o m p o u n d s m e t a b o l i s e d via g l u t a m a t e as n i t r o g e n a n d / o r c a r b o n sources) resulting f r o m a gdhB- ( N A D - g l u t a m a t e d e h y d r o g e n a s e l e s s ) m u t a t i o n in A. nidulans. T h e r e f o r e arndS + t r a n s f o r m a n t s o f G H 2 2 , w h i c h carries the large d e l e t i o n amdS-320 ( H y n e s et al. 1983) in the a c e t a m i d a s e s t r u c t u r a l gene, were selected after t r a n s f o r m a t i o n with p A M D 2 1 ( H y n e s et al. 1988) a n d c o - t r a n s f o r m a t i o n with p G H - l was m o n i t o r e d b y evidence for p h y s i o l o g i c a l s u p p r e s s i o n o f gdhB-1. O f 50 amdS + t r a n s f o r m a n t s nine s h o w e d stable s u p p r e s s i o n o f the gdhB- p h e n o t y p e . O f these, eight (G44, G45, G46, G47, G49, G51, G 5 2 a n d G54) were a b l e to utilise n i t r o gen sources n o r m a l l y m e t a b o l i s e d via g l u t a m a t e , such as L - a s p a r t a t e (Fig. 2), b u t c o u l d n o t utilise these s a m e c o m p o u n d s in the q u a n t i t a t i v e l y m o r e d e m a n d i n g c a p a c ity o f c a r b o n a n d n i t r o g e n sources. T h e n i n t h (G50) exhibits a s t r o n g e r degree o f p h y s i o l o g i c a l s u p p r e s s i o n o f the gdhB- p h e n o t y p e , utilising, for e x a m p l e , L - g l u t a m i n e a n d L - a s p a r t a t e as c a r b o n a n d n i t r o g e n sources. Prelimin a r y S o u t h e r n b l o t s ( d a t a n o t shown) i n d i c a t e d t h a t f o u r o f the c o - t r a n s f o r m a n t s , G44, G45, G 4 6 a n d G47, c o n tain at least two t a n d e m l y i n t e g r a t e d copies ofaspA. G48, a l t h o u g h p h e n o t y p i c a l l y u n s t a b l e (Fig. 2), also c o n t a i n s aspA-hybridising sequences; the recipient strain G H 2 2 c o n t a i n s no aspA-hybridising sequences. I n view o f the a p p a r e n t a b i l i t y o f aspA e x p r e s s i o n to o v e r c o m e the gdhB- p h e n o t y p e , aspA + t r a n s f o r m a n t s
379 stable transformant G40 has fewer copies of aspA and much lower activity. It is possible that aspartase activity, in G40 was diminished by loss of aspA sequences during growth of mycelia for enzyme determinations because, in a preliminary experiment using G40 mycelia grown with 5 m M of L-aspartate as a nitrogen source, an order of magnitude higher specific activity (89 units as average o f two determinations) was obtained. Under these conditions there would be selective pressure to maintain aspA sequences (but also the danger that genetic changes leading to increased aspartase levels would o c c u r - vide infra).
Fig. 2. Physiological suppression of gdhB-1 by aspA expression in transformants. Left to right: top row G39, G40, G41, G42; second row G43, G44, G45, G46; third row G47, G48, G49, G50; fourth row G51, G52, G53, G54; bottom row GH22 (recipient), wild-type (genotype biA-1). Growth medium contained 1% glucose as a carbon source and 5 mM L-aspartate (sodium salt) as a nitrogen source and was incubated for 2 days at 37 ~
Table 2. Aspartase activities of aspA transformants Strain no.
Recpient strain
Selection a s p A Aspartase Nitrogen marker copy no. activity source
GH22 G39 G40 G41 G42 G43 G120 G120 G121 G121 G 122 G122 G123 G127 G 127
GH22 GH22 GH22 GH22 GH22 GH101 GH101 GH101 GH101 GH 101 GH101 GH101 GH101 GH 101
-
aspA aspA aspA aspA aspA argB argB argB argB argB argB argB argB argB
0 32 8 64 32 32 8 8 1 i 0 0 1 16 16
